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„training […] at his father’s hands and at those of the musical community that surrounded these families“: Musikerfamilien als Ort der Ausbildung und Professionalisierung
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This chapter is part of: 1
Roz Southey, Music-Making in North-East England during the Eighteenth Century, Al
dershot und Burlington 2006, S. 165. Music and the Arts
in England, c. 1670–1750 Edited by
Ina Knoth Published by musiconn.publish, Dresden 2020
DOI: https://doi.org/10.25366/2020.112 Published by musiconn.publish, Dresden 2020
DOI: https://doi.org/10.25366/2020.112 musiconn.publish has no responsibility for the persistence or accuracy of URLs for
external or third-party internet websites referred to in this publication and does
not guarantee that any content on such websites is, or will remain, accurate or
appropriate. The digital reproduction rights for all illustrations were either obtained by the
authors or granted by public domain licences. However, in case of suspected copy
right infringement, please contact the editor or the publisher. This chapter is available under a Creative Commons Attribution-NonCommercial-
ShareAlike 4.0 International (CC BY-NC-SA 4.0) licence. Melanie Unseld 2
Andreas Gestrich, Geschichte der Familie im 19. und 20. Jahrhundert, München 32013,
S. 5–6. Vgl. zum bürgerlichen Familienbild im deutschsprachigen Diskurs des 19. Jahrhunderts
v. a. Wilhelm Heinrich Riehl, Die Naturgeschichte des Volkes als Grundlage einer deutschen
Social-Politik, Bd. 3, Die Familie, Stuttgart 1855. S. a. Melanie Unseld, „Argumentieren in Wis
senschaft und Musik: Wilhelm Heinrich Riehls Hausmusik (1855)“, in: Zum Selbstverständnis
der Gender Studies. Methoden – Methodologien – theoretische Diskussionen und empirische
Übersetzungen (L’AGENda 1), hrsg. von Corinna Onnen und Susanne Rode-Breymann, Opla
den u. a. 2017, S. 121–128. Musikerfamilien als Ort der Ausbildung
und Professionalisierung „Music was frequently a family affair – sons followed fathers into the profes
sion.“1 Man mag diesen Satz für einen Gemeinplatz halten, immerhin ist die
Musikgeschichte voller mehrgenerationeller Musikerfamilien – und doch ist
bezeichnend, wie Roz Southey in ihrer Studie über Music-Making in North-
East England during the Eighteenth Century ein lange bekanntes Phänomen
beschreibt: Dass Musikerfamilien in England im 18. Jahrhundert existierten
und hier über Generationen hinweg ihre Profession patrilinear weitergege
ben wurde, liegt in der Aussage auf oberer Ebene zutage. So weit, so allge
meingültig (übrigens weit über die Grenzen Nordost-Englands hinaus). Was
aber im Satz verborgen liegt, ist die Frage nach dem Verhältnis zwischen
Musik als „family affair“ und Musik als „profession“: Ist mit „affair“ und „pro
fession“ das gleiche gemeint und wenn nicht, worin liegen die Unterschiede,
die (mögliche) Grenze? Weiters wäre zu fragen, ob im Feld der Musik von
einer „profession“ (im Singular) die Rede sein kann, und nicht zuletzt liegt
die Vermutung auf der Hand, dass sich hinter den beiden Begriffen (auch)
die Frage nach der weiblichen Teilhabe verbirgt: „family“ als gemischtge
schlechtliche Gruppe, „profession“ als patrilinineare Tradition. Damit wer This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 Melanie Unseld 34 den Fragen nach dem Konzept des Familialen gestellt, allerdings ohne die
Spezifik des Sozialgefüges ‚Familie‘ im 18. Jahrhundert explizit zu machen. den Fragen nach dem Konzept des Familialen gestellt, allerdings ohne die
Spezifik des Sozialgefüges ‚Familie‘ im 18. Jahrhundert explizit zu machen. Über Musikerfamilien auf diese Weise zu schreiben, ist keinesfalls unüb
lich. Dennoch stellt sich die Frage, ob die Art und Weise, wie also über Musik
als „family affair“ nachgedacht, gesprochen bzw. geschrieben wird, den Si
tuationen von Musikerfamilien im 18. Jahrhundert entspricht. Denn deutlich
wird beim oben zitierten Beispiel, dass die Tatsache, wie über Familie nach
gedacht und geschrieben wird, maßgeblich geprägt ist durch das dahinter
verborgene Familien-Bild: Von einer ‚father-to-son-profession‘ zu schreiben
etwa heißt, eine unilineare (genauer: patrilineare) Deszenz als Grundlage
von Professionalität anzunehmen, was nicht nur die Frage aufwirft, wie Vir
tuosinnen, Sängerinnen etc. zu Professionalität gelangen konnten, sondern
auch, welche Rolle Deszenz – mithin blutsverwandtschaftliche Abstammung
– in Musikerfamilien überhaupt spielte. I. Über ‚Familie‘ nachdenken und schreiben: Kritik am impliziten Modell
der Kleinfamilie Wenn sich im Sprechen über Musikerfamilien Grundannahmen über das
Familiale verbergen, sind diese bis heute häufig geprägt vom Familienbild
des bürgerlichen 19. Jahrhunderts, das auf „Privatisierung und Emotiona
lisierung der Familie“ sowie auf „einer verschärften Betonung der unter
schiedlichen Geschlechtscharaktere von Mann und Frau“ setzte.2 Das ein
gangs zitierte Beispiel ist nur eines von vielen, dass und wie (vor allem: wie
unsichtbar) sich diese Grundannahmen auch dort halten, wo sie – da aus
dem 19. Jahrhundert stammend – ahistorisch sind. Dass es sich dabei nicht
nur um eine sprachliche Ungenauigkeit handelt, sondern dass sich daran
grundlegende ‚blinde Flecken‘ historischer Forschung erkennen lassen,
zeigt ein jüngeres Beispiel aus der europäischen Frühgeschichte: 1878 wurde
im schwedischen Birka ein Wikingergrab entdeckt, das typische Grabbeiga This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 Musikerfamilien als Ort der Ausbildung und Professionalisierung 35 ben eines Kriegers beinhaltete, darunter mehrere Speere, Axt und Schwert,
zwei Pferde und ein Brettspiel. Die Entdeckung geschah zu einem Zeitpunkt,
als der Familiendiskurs weithin durch das Modell der bürgerlichen Klein
familie geprägt war, und so war es zunächst klar, dass – da es sich nicht um
ein Familiengrab handelte (keine Gruppe, keine Kinder), noch dazu die ent
sprechenden Grabbeigaben gefunden wurden – es sich um das Grab eines
Wikingerkriegers handelt. Diese Überzeugung hielt annähernd 140 Jahre. Erst 2017 kamen Wissenschaftler_innen auf den Gedanken, nach dem Ge
schlecht des Skeletts zu forschen: Es war weiblich.3 Offensichtlich aber hatte
das im 19. Jahrhundert weitverbreitete Familienbild als Denkgrundlage für
Forschung der Frühgeschichte gedient, so dass die These ausgeschlossen zu
sein schien, dass es sich bei dem Skelett um eine Frau handeln könne. Die
Frau im bürgerlichen Familienbild war nur innerhalb der Familie (und damit
in einem Familiengrab zu bestatten) denkbar.4 Als epistemologische Grund
lage frühgeschichtlicher Forschung wird hier der Diskurs um die bürgerliche
Kleinfamilie sichtbar, ein Diskurs, der sich ab dem mittleren 19. Jahrhundert
beobachten lässt und dessen Sedimentierung in Gesellschaft, Gesetzge
bung und Identitätskonzepte eine derart dominante Denkform abgab, dass
er sich nicht zuletzt auch in historisch arbeitenden Disziplinen festsetzte,
so dass ein kritisch-differenzierender Blick auf das normierende Potenti
al dieser Denkform lange kaum möglich schien. Die Denkform ‚Familie als
Klein- oder Kernfamilie‘ war seit dem bürgerlichen 19. Jahrhundert so stark
verankert, dass selbst hier mit diesem Modell im Sinne einer tendenziell
unsichtbaren, gerade daher aber umso wirkmächtigeren Annahme gearbei
tet wurde. 3
Charlotte Hedenstierna-Jonson u. a., „A Female Viking Warrior Confirmed by Geno
mics“, in: American Journal of Physical Anthropology 164 / 4 (2017), S. 853–860, https://doi.
org/10.1002/ajpa.23308 (abgerufen am 25.03.2020).
4
„Already in the early middle ages, there were narratives about fierce female Vikings
fighting alongside men. Although, continuously reoccurring in art as well as in poetry, the
women warriors have generally been dismissed as mythological phenomena.“ Ebd., S. 853.
5
Vgl. Dorett Funcke und Bruno Hildenbrand, Ursprünge und Kontinuität der Kernfamilie.
Einführung in die Familiensoziologie, Wiesbaden 2018, insbes. S. 27–32 (Kapitel „Der Mythos
von der Großfamilie“). 3
Charlotte Hedenstierna-Jonson u. a., „A Female Viking Warrior Confirmed by Geno
mics“, in: American Journal of Physical Anthropology 164 / 4 (2017), S. 853–860, https://doi.
org/10.1002/ajpa.23308 (abgerufen am 25.03.2020).i 6
U. a. Rebekka Habermas, Frauen und Männer des Bürgertums. Eine Familiengeschichte
(1750–1850), Göttingen 2000; Stephanie Coontz, „Historical Perspectives on Family Studies“,
in: Journal of Marriage and Family 62 / 2 (2000), S. 283–297; Andreas Gestrich, Jens-Uwe Krau
se und Michael Mitterauer, Geschichte der Familie, Stuttgart 2003; François-Joseph Ruggiu,
L’Individu et la famille dans les sociétés urbaines anglaise et française (1720–1780), Paris
2007; Albrecht Koschorke u. a., Vor der Familie. Grenzbedingungen einer modernen Institu
tion, Konstanz und München 2010; Inken Schmidt-Voges (Hrsg.), Ehe – Haus – Familie. Soziale
Institutionen im Wandel 1750–1850, Köln u. a. 2010 und Christina von Braun, Blutsbande. Ver
wandtschaft als Kulturgeschichte, Berlin 2018.
7
Vgl. dazu auch Michaela Hohkamp, „Wer ist mit wem, warum und wie verheiratet? Über
legungen zu Ehe, Haus und Familie als gesellschaftliche Schlüsselbeziehungen am Beginn
des 19. Jahrhunderts – samt einem Beispiel aus der Feder eines Mörders“, in: Schmidt-Voges
(Hrsg.) 2010, Ehe – Haus – Familie, S. 31–47; Inken Schmidt-Voges, „Strategien und Inszenierun
gen häuslichen Lebens zwischen 1750 und 1820. Eine Einführung“, in: ebd., S. 9–28. 6
U. a. Rebekka Habermas, Frauen und Männer des Bürgertums. Eine Familiengeschichte
(1750–1850), Göttingen 2000; Stephanie Coontz, „Historical Perspectives on Family Studies“,
in: Journal of Marriage and Family 62 / 2 (2000), S. 283–297; Andreas Gestrich, Jens-Uwe Krau
se und Michael Mitterauer, Geschichte der Familie, Stuttgart 2003; François-Joseph Ruggiu,
L’Individu et la famille dans les sociétés urbaines anglaise et française (1720–1780), Paris
2007; Albrecht Koschorke u. a., Vor der Familie. Grenzbedingungen einer modernen Institu
tion, Konstanz und München 2010; Inken Schmidt-Voges (Hrsg.), Ehe – Haus – Familie. Soziale
Institutionen im Wandel 1750–1850, Köln u. a. 2010 und Christina von Braun, Blutsbande. Ver
wandtschaft als Kulturgeschichte Berlin 2018 wandtschaft als Kulturgeschichte, Berlin 2018.
7
Vgl. dazu auch Michaela Hohkamp, „Wer ist mit wem, warum und wie verheiratet? Über
legungen zu Ehe, Haus und Familie als gesellschaftliche Schlüsselbeziehungen am Beginn
des 19. Jahrhunderts – samt einem Beispiel aus der Feder eines Mörders“, in: Schmidt-Voges
(Hrsg.) 2010, Ehe – Haus – Familie, S. 31–47; Inken Schmidt-Voges, „Strategien und Inszenierun
gen häuslichen Lebens zwischen 1750 und 1820. Eine Einführung“, in: ebd., S. 9–28. I. Über ‚Familie‘ nachdenken und schreiben: Kritik am impliziten Modell
der Kleinfamilie Besonders wichtig bei der Analyse von Familienstrukturen (auch
und gerade vor dem bürgerlichen 19. Jahrhundert) scheint es daher, genauer
nachzufragen, was ‚Familie‘ in der jeweiligen Zeit heißt, ohne dabei freilich
ins Gegenteil jenes bürgerlichen Modells zu verfallen, d. h. ein anderes all
gemeingültiges Modell zu propagieren, etwa dem einem ‚Mythos von der
Großfamilie‘ das Wort zu reden 5 This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 36 Melanie Unseld Die neuere Familienforschung geht von einem historisch-kritischen Fami
lienbegriff aus, der das Soziotop Familie (a) zeitlich differenziert und (b) wei
tere Differenzkriterien wie Stand, Religion, Profession, Region, ökonomische
Verhältnisse etc. berücksichtigt.6 Diese Differenzierungen verbieten denn
auch, allzu grobmaschig von persistenten Modellen auszugehen: Weder
war das Modell der (nachträglich idealisierten) Großfamilie grundsätzlich
etabliert, noch das Modell der Kern- oder Kleinfamilie. Den Historikerin
nen Inken Schmidt-Voges, Michaela Hohkamp und anderen ist daher zuzu
stimmen, dass die Bandbreite möglicher Lebensverhältnisse (eheähnliche
Lebensgemeinschaften, gewollte und ungewollte Ehelosigkeit, Lebensge
meinschaften mit und ohne Kinder, gleich- und nicht-gleichgeschlechtliche,
generationelle Diversität, leibliche / angenommene Kindschaftsverhältnisse
etc.) im 18. und frühen 19. Jahrhundert groß und – jenseits der diskursbe
stimmenden Schriften und den in den Quellen sichtbaren „Inszenierungen
häuslichen Lebens“ – eine enorme Vielfalt der „Ehe- und Paarbeziehungen,
der Ausgestaltung von familiärer Ökonomie und Haushaltsstrukturen“ zu
beobachten ist.7 Dass aber Familien einer ökonomisch stabilen Grundlage
bedürfen, dass deshalb ökonomisch sinnvolle Haushaltsstrukturen etabliert
wurden und dabei Ausbildung und Professionalisierungsprozesse substan
tieller Bestandteil von Familienstrukturen waren, ist bei all dieser Vielfalt
unbenommen. Auf diesen Ausschnitt des Familialen sei daher im Folgen
den der Fokus gelegt, und insbesondere dorthin, wo implizite Vorannahmen
des Familialen Phänomene unsichtbar zu machen drohen. Dabei gehe ich
davon aus, dass (1) Ausbildung und Profession näher beieinanderliegen als
in modernen Vorstellung von Professionalisierungsprozessen angenommen
wird und dass (2) Formen des professionellen Handelns in Musikerfami This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 Musikerfamilien als Ort der Ausbildung und Professionalisierung 37 lien existierten, die dann unsichtbar bleiben, wenn man „family affair“ und
„profession“ als getrennte und trennbare Sphären des Familialen versteht. Ausbildung und Professionalisierungsprozesse werden im Folgenden mithin
als Bestandteil des Konzepts des Familialen gedacht. So ergibt sich nicht zu
letzt die Möglichkeit, auch jene Tätigkeiten von weiblichen Familienmitglie
dern als Teil eines professionellen Selbstverständnisses wahrnehmbar zu
machen, die ansonsten allzu rasch als Assistenz, Zuarbeit oder Vergnügen
– mithin als unprofessionell im doppelten Wortsinne – abgewertet wurden. 8
Erkennbar wird dies u. a. an der Situation der Mägde in der Frühen Neuzeit. Vgl. dazu
auch Renate Dürr, Mägde in der Stadt. Das Beispiel Schwäbisch Hall in der Frühen Neuzeit,
Frankfurt a. M. 1995. I. Über ‚Familie‘ nachdenken und schreiben: Kritik am impliziten Modell
der Kleinfamilie Auf diese Weise geht es den folgenden Überlegungen um einen neuen Blick
auf bekannte Familien(konstellationen) der Musikkultur des 18. Jahrhun
derts. Die Überlegungen gehen davon aus, dass wir auf bekannte Quellen
anders schauen, wenn wir einen anderen Blickwinkel einnehmen. Einen sol
chen möchte der folgende Text aufzeigen; die Erschließung weiterer Quellen
zu der hier vorgestellten Neukonzeption des Familialen muss weiterer (Mu
sikerfamilien-)Forschung vorbehalten bleiben. lien existierten, die dann unsichtbar bleiben, wenn man „family affair“ und
„profession“ als getrennte und trennbare Sphären des Familialen versteht. Ausbildung und Professionalisierungsprozesse werden im Folgenden mithin
als Bestandteil des Konzepts des Familialen gedacht. So ergibt sich nicht zu
letzt die Möglichkeit, auch jene Tätigkeiten von weiblichen Familienmitglie
dern als Teil eines professionellen Selbstverständnisses wahrnehmbar zu
machen, die ansonsten allzu rasch als Assistenz, Zuarbeit oder Vergnügen
– mithin als unprofessionell im doppelten Wortsinne – abgewertet wurden 9
Mit Erfolg ist hier explizit eine möglichst lang andauernde ökonomische Stabilität der
Familie gemeint, womit ein markanter Unterschied zu einem Erfolgsbegriff deutlich wird, wie
er für Musik als Kunst veranschlagt wird und der insbesondere mit der Neukonturierung des
Genie-Begriffs im ausgehenden 18. Jahrhundert in Verbindung steht. Dort meint Erfolg ins
besondere eine individuelle Leistung, die nicht notwendigerweise auf finanziellen Erfolg zu
Lebzeiten, viel mehr auf Persistenz von Namen und Werk in der Zukunft (Geschichtsschrei
bung, Kanon etc.) abzielt.
10 Ephraim Chambers, Cyclopædia. Or, an Universal Dictionary of Arts and Sciences, Bd. 2,
London 1728, S. 10.
11 Heide Wunder, „Er ist die Sonn’, sie ist der Mond“. Frauen in der Frühen Neuzeit, München
1992.
12 Funcke und Hildenbrand 2018, Ursprünge und Kontinuität der Kernfamilie, S. 108. 13 Professionalisierungsprozesse werden darüber hinaus auch als Abbild gesellschaftlicher
Machtstrukturen, Verteilungskämpfe und Aushandlungsprozesse verstanden, wobei Formen
der gesellschaftlichen Arbeitsteilung und der Professionalisierung nicht geschlechtsneutral
waren und sind. Vgl. dazu Angelika Wetterer, Arbeitsteilung und Geschlechterkonstruktion.
„Gender at work“ in theoretischer und historischer Perspektive, Konstanz 2002.
14 Rudolf Stichweh, „Professionen in einer funktional differenzierten Gesellschaft“, in: Pä
dagogische Professionalität. Untersuchungen zum Typus pädagogischen Handelns, hrsg. von
Arno Combe und Werner Helsper, Frankfurt a. M. 1996, S. 49–69. Vgl. auch Gudrun Ehlert, Art.
„Professionalisierung“, in: socialnet Lexikon, 2019, https://www.socialnet.de/lexikon/Profes
sionalisierung (abgerufen am 09.05.2020).
15 U. a. Niklas Luhmann, Das Erziehungssystem der Gesellschaft, hrsg. von Dieter Lenzen,
Frankfurt a. M. 2002. 16 Der deutsche Begriff „Beruf“ etwa setzte sich – wie der Begriff „Familie“ – erst im 19. Jahr
hundert allmählich durch. Zuvor war von „Gewerben“, „Handwerken“ und „Hantierungen“,
auch von „Professionen“ die Rede. Im Französischen überwogen, etwa in der Diderotschen
Encyclopédie (1751–1780) die Begriffe „sciences“, „arts libéraux“ und „arts méchaniques“, was
an mittelalterliche Vorstellungen erinnert. Vgl. Thomas Sokoll, Art. „Beruf“, in: Enzyklopädie
der Neuzeit, Bd. 2, hrsg. von Friedrich Jaeger, Stuttgart 2005, Sp. 43–50, hier Sp. 45. 13 Professionalisierungsprozesse werden darüber hinaus auch als Abbild gesellschaftlicher
Machtstrukturen, Verteilungskämpfe und Aushandlungsprozesse verstanden, wobei Formen
der gesellschaftlichen Arbeitsteilung und der Professionalisierung nicht geschlechtsneutral
waren und sind. Vgl. dazu Angelika Wetterer, Arbeitsteilung und Geschlechterkonstruktion.
„Gender at work“ in theoretischer und historischer Perspektive, Konstanz 2002.
14 Rudolf Stichweh, „Professionen in einer funktional differenzierten Gesellschaft“, in: Pä
dagogische Professionalität. Untersuchungen zum Typus pädagogischen Handelns, hrsg. von
Arno Combe und Werner Helsper, Frankfurt a. M. 1996, S. 49–69. Vgl. auch Gudrun Ehlert, Art.
„Professionalisierung“, in: socialnet Lexikon, 2019, https://www.socialnet.de/lexikon/Profes
sionalisierung (abgerufen am 09.05.2020).
15 U. a. Niklas Luhmann, Das Erziehungssystem der Gesellschaft, hrsg. von Dieter Lenzen,
Frankfurt a. M. 2002.
16 Der deutsche Begriff „Beruf“ etwa setzte sich – wie der Begriff „Familie“ – erst im 19. Jahr
hundert allmählich durch. Zuvor war von „Gewerben“, „Handwerken“ und „Hantierungen“,
auch von „Professionen“ die Rede. Im Französischen überwogen, etwa in der Diderotschen
Encyclopédie (1751–1780) die Begriffe „sciences“, „arts libéraux“ und „arts méchaniques“, was
an mittelalterliche Vorstellungen erinnert. Vgl. Thomas Sokoll, Art. „Beruf“, in: Enzyklopädie
der Neuzeit, Bd. 2, hrsg. von Friedrich Jaeger, Stuttgart 2005, Sp. 43–50, hier Sp. 45.
17 Bernd Dewe und Hans-Uwe Otto, Art. „Profession“, in: Handbuch Soziale Arbeit. Grundla
gen der Sozialarbeit und Sozialpädagogik, hrsg. von dems. und Hans Thiersch, München und
Basel 42011, S. 1131–1142, hier S. 1131. Hierzu gehört freilich auch, was Andreas Reckwitz als pro
fessionelles Selbstverständnis des bürgerlichen Arbeitssubjekts des ausgehenden 18. und
frühen 19. Jahrhunderts beschreibt. Vgl. Andreas Reckwitz, Das hybride Subjekt. Eine Theorie
der Subjektkulturen von der bürgerlichen Moderne zur Postmoderne, Weilerswist 2006. 13 Professionalisierungsprozesse werden darüber hinaus auch als Abbild gesellschaftlicher
Machtstrukturen, Verteilungskämpfe und Aushandlungsprozesse verstanden, wobei Formen
der gesellschaftlichen Arbeitsteilung und der Professionalisierung nicht geschlechtsneutral
waren und sind. Vgl. dazu Angelika Wetterer, Arbeitsteilung und Geschlechterkonstruktion.
„Gender at work“ in theoretischer und historischer Perspektive, Konstanz 2002. II. ‚Familie‘ im 18. Jahrhundert Will man das Gefüge, aus dem viele Musiker_innen (auch) des 18. Jahr
hunderts stammen und in dem sie wesentliche Züge ihrer musikalischen
Ausbildung erhalten haben, betrachten, bieten sich neuzeitliche Vorstel
lungen des Familialen an, insbesondere die Berücksichtigung einer über
generationellen Familie, die als ökonomisch-moralische Einheit verstanden
wird: ökonomisch, da sich die so verstandene soziale Einheit einer Ordnung
unterwirft, die sich gemeinschaftlich-arbeitsteilig um das Einkommen und
finanzielle Auskommen der gesamten, nicht bloß blutsverwandtschaftlich
verbundenen Gemeinschaft kümmert; moralisch, da die Einheit allen ihren
Mitgliedern einen zwar hierarchisch strukturierten, aber unter religiös-mo
ralischen Gesichtspunkten gesicherten Platz zuweist. Dies war insbesonde
re für Mädchen und unverheiratete Frauen ein bestimmender, wenngleich
auch besonderer Kontrolle unterworfener Faktor.8 This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 38 Melanie Unseld Für diese familiale Ökonomie wesentlich war der wirtschaftliche Erfolg.9
Darum waren einerseits die (arbeitsteiligen) Strukturen so geschaffen, dass
alle an ebendiesem Erfolg mitarbeiten konnten / mussten – woraus sich die
Selbstverständlichkeit ableitete, dass Lehrlinge, Kostzöglinge, Dienerschaft
etc. zur ‚Familie‘ hinzugerechnet wurden. In Ephraim Chambers’ Cyclopædia
(1728) etwa heißt es zum Lemma „Familia, Family“: „commonly implies all
the Servants, belonging to a particular Master.“10 Andererseits war dies die
Grundlage dafür, dass innerhalb einer Familie nicht notwendigerweise eine
Profession ausgeübt wurde, sondern dass Professionen grundsätzlich so
auszuwählen, zu kombinieren und zu realisieren waren, dass die Familie
ökonomisch erfolgreich agieren konnte. Zur Struktur der Familie gehörte eine gattenzentrierte Lebensweise, die
vom Hausvater- / Hausmutter-Paar11 ausgehend neben blutsverwandtschaft
lichen Familienmitgliedern Gesinde und Lehrlinge umfasste (einschließlich
der Eingriffsrechte in die Familienverhältnisse ebendieser), dazu auch Pa
ten- und Vormundschaften. Wiederverehelichung galt ebenso als ökono
misch stabilisierend wie eine Heiratspraxis, die – anders als in Adelsfamilien – darauf abzielte, „rationale Formen der Arbeitsorganisation zu sichern“.12
Für die familialen Ausbildungszusammenhänge in Musiker-, aber auch In
strumentenbauer-, Theater- oder anderen Künstlerfamilien (zumindest
des 18. Jahrhunderts) ist die Situation nicht anders: neben blutsverwandt
schaftlichen und angeheirateten Familienmitgliedern gehören auch solche
Personen hinzu, die zur Ausbildung in eine Familie geschickt wurden, ohne
dass irgendeine Art von Verwandtschaft vorlag. Daraus ergaben sich höchst
unterschiedliche (temporäre oder dauerhafte) Familienkonstellationen, die
nicht selten enge Schnittstellen zu beruflichen Netzwerken aufweisen. This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 16 Der deutsche Begriff „Beruf“ etwa setzte sich – wie der Begriff „Familie“ – erst im 19. Jahr
hundert allmählich durch. Zuvor war von „Gewerben“, „Handwerken“ und „Hantierungen“,
auch von „Professionen“ die Rede. Im Französischen überwogen, etwa in der Diderotschen
Encyclopédie (1751–1780) die Begriffe „sciences“, „arts libéraux“ und „arts méchaniques“, was
an mittelalterliche Vorstellungen erinnert. Vgl. Thomas Sokoll, Art. „Beruf“, in: Enzyklopädie
der Neuzeit, Bd. 2, hrsg. von Friedrich Jaeger, Stuttgart 2005, Sp. 43–50, hier Sp. 45.
17 Bernd Dewe und Hans-Uwe Otto, Art. „Profession“, in: Handbuch Soziale Arbeit. Grundla
gen der Sozialarbeit und Sozialpädagogik, hrsg. von dems. und Hans Thiersch, München und
Basel 42011, S. 1131–1142, hier S. 1131. Hierzu gehört freilich auch, was Andreas Reckwitz als pro
fessionelles Selbstverständnis des bürgerlichen Arbeitssubjekts des ausgehenden 18. und
frühen 19. Jahrhunderts beschreibt. Vgl. Andreas Reckwitz, Das hybride Subjekt. Eine Theorie
der Subjektkulturen von der bürgerlichen Moderne zur Postmoderne, Weilerswist 2006. 17 Bernd Dewe und Hans-Uwe Otto, Art. „Profession“, in: Handbuch Soziale Arbeit. Grundla
gen der Sozialarbeit und Sozialpädagogik, hrsg. von dems. und Hans Thiersch, München und
Basel 42011, S. 1131–1142, hier S. 1131. Hierzu gehört freilich auch, was Andreas Reckwitz als pro
fessionelles Selbstverständnis des bürgerlichen Arbeitssubjekts des ausgehenden 18. und
frühen 19. Jahrhunderts beschreibt. Vgl. Andreas Reckwitz, Das hybride Subjekt. Eine Theorie
der Subjektkulturen von der bürgerlichen Moderne zur Postmoderne, Weilerswist 2006. 21 Vgl. dazu u. a. der Professorenhaushalt: Ulrike Gleixner, „Der Professorenhaushalt“, in:
Das Athen der Welfen. Die Reformuniversität Helmstedt 1576–1810, hrsg. von Jens Bruning,
Wolfenbüttel 2010, S. 129–143; Elizabeth Harding, Der Gelehrte im Haus – Ehe, Familie und
Haushalt in der Standeskultur der frühneuzeitlichen Universität Helmstedt, Wiesbaden 2014;
Melanie Unseld, „Medea auf der Récamière. Transformation zwischen Text, Musik und Perfor
manz im Melodram um 1800“, in: SprachMedienWelten. Wissen und Geschlecht in Musik*The
ater*Film* (mdw Gender Wissen 8), hrsg. von Andrea Ellmeier, Doris Ingrisch und Claudia
Walkensteiner-Preschl, Wien u. a. 2020, S. 83–111. 18 Elisabeth Joris, „Profession und Geschlecht. Das Haus als Ort der Ausbildung und Be
rufstätigkeit im 19. Jahrhundert“, in: Das Haus in der Geschichte Europas. Ein Handbuch, hrsg.
von Joachim Eibach, Inken Schmidt-Voges und Simone Derix, Berlin und Boston 2015, S. 355–
372, hier S. 356. 372, hier S. 356.
19 Zur Auseinandersetzung mit dem Begriff des ‚ganzen Hauses‘ (Otto Brunner) in der
deutschsprachigen Forschung (‚Brunner-Debatte‘) vgl. Philip Hahn, „Trends der deutschspra
chigen historischen Forschung nach 1945: Vom ‚ganzen Haus‘ zum ‚offenen Haus‘“, in: Eibach,
Schmidt-Voges und Derix (Hrsg.) 2015, Das Haus in der Geschichte Europas, S. 47–63. Siehe
dort auch die gegenüber dem Brunner’schen Haus-Begriff formulierte Kritik seitens der Gen
derforschung, die für die Musikgeschichte von besonderer Relevanz ist.
20
Ebd S 54 Musikerfamilien als Ort der Ausbildung und Professionalisierung 39 Musikerfamilien als Ort der Ausbildung und Professionalisierung Wenn Familie, auf diese Weise verstanden, auch ein Ort von Professiona
lisierung13 war, lässt sich hier auch das Erlernen von professionstypischen
Handlungsprozessen und von mit der Profession verbundenen habituellen
Anforderungen beobachten, wobei nicht zu vergessen ist, dass der Pro
fessionen-Begriff selbst historisch zu verstehen ist, zumal an der Schwel
le von ständischer zur funktional differenzierten Gesellschaft.14 Damit ist
festzuhalten, nimmt man arbeitssoziologische Ansätze15 in die historische
Familienforschung mit auf, dass die Vorstellung von Profession selbst im
18. Jahrhundert im Wandel begriffen war und sich deutlich von der heutigen
Vorstellung von Beruf unterschied,16 Professionalisierung aber auch in der
Familienstruktur des 18. Jahrhunderts wichtiger Bestandteil der Berufsbio
graphie war, sowie auch das Erlernen von Professionalität, mithin das „ha
bitualisierte[], szenisch-situativ zum Ausdruck kommende[] Agieren[] unter
typischerweise sowohl hochkomplexen wie auch paradoxen Handlungsan
forderungen“.17 Festzuhalten ist außerdem, dass sich ab der zweiten Hälfte
des 18. Jahrhunderts durch die (allmähliche) Auslagerung von Berufsquali
fikationen an Universitäten die Bedeutung der Familie für Ausbildung und
Professionalisierungsprozesse insofern veränderte, als diese Veränderun
gen auf Verwissenschaftlichung und den Fokus auf eine exklusiv männli
che Individualisierung qua Beruf abzielten. Zugleich aber blieb der soziale This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 40 Melanie Unseld Raum des ‚Hauses‘ ein Ort, an dem Frauen auch professionell agieren konn
ten. „Das berufliche Tun […] von Frauen“ sei zwar, so Elisabeth Joris, „im Haus
verankert [ge]blieben, das jedoch im Sinne eines ‚offenen Hauses‘ nicht als
ein dem Öffentlichen Entgegengesetztes zu verstehen ist.“18 Eingedenk dieser Beobachtungen zum Familialen scheint es sinnvoll zu
sein, im Kontext der Familienforschung mit jenem differenzierten ‚Haus‘-Be
griff zu arbeiten, den die Geschichtswissenschaft – nicht zuletzt eingedenk
der Problematik des Begriffs und in kritischer Auseinandersetzung damit
– in jüngerer Zeit vorgeschlagen hat.19 Für die hier skizzierten Überlegungen
sind dabei vor allem die Perspektiven auf ‚Haus‘ als Ordnungskonzept und
als sozialer Raum von Bedeutung: Ersteres meint sowohl die rechtlich-in
stitutionelle Ebene dieser Lebensform, die auf soziale Ordnung abzielt, als
auch die Bedeutung, die das ‚Haus‘ als „Bezugspunkt kollektiver und indi
vidueller Identitätsbildungsprozesse“ hatte.20 Mit ‚Haus‘ als sozialem Raum
fällt der Blick nicht nur auf Fragen der Bedeutung von Verwandtschaft (kin
ship) und die variablen Grenzen des ‚Hauses‘ etwa durch temporär öffentli
che Nutzung konkreter Räume, sondern damit auch auf verschiedene Haus
typen, die an Spezifika der Professionen gebunden sind.21 ,
21 Vgl. dazu u. a. 18 Elisabeth Joris, „Profession und Geschlecht. Das Haus als Ort der Ausbildung und Be
rufstätigkeit im 19. Jahrhundert“, in: Das Haus in der Geschichte Europas. Ein Handbuch, hrsg.
von Joachim Eibach, Inken Schmidt-Voges und Simone Derix, Berlin und Boston 2015, S. 355–
372, hier S. 356.
19 Zur Auseinandersetzung mit dem Begriff des ‚ganzen Hauses‘ (Otto Brunner) in der
deutschsprachigen Forschung (‚Brunner-Debatte‘) vgl. Philip Hahn, „Trends der deutschspra
chigen historischen Forschung nach 1945: Vom ‚ganzen Haus‘ zum ‚offenen Haus‘“, in: Eibach,
Schmidt-Voges und Derix (Hrsg.) 2015, Das Haus in der Geschichte Europas, S. 47–63. Siehe
dort auch die gegenüber dem Brunner’schen Haus-Begriff formulierte Kritik seitens der Gen
derforschung, die für die Musikgeschichte von besonderer Relevanz ist.
20 Ebd., S. 54.
21 Vgl. dazu u. a. der Professorenhaushalt: Ulrike Gleixner, „Der Professorenhaushalt“, in:
Das Athen der Welfen. Die Reformuniversität Helmstedt 1576–1810, hrsg. von Jens Bruning,
Wolfenbüttel 2010, S. 129–143; Elizabeth Harding, Der Gelehrte im Haus – Ehe, Familie und
Haushalt in der Standeskultur der frühneuzeitlichen Universität Helmstedt, Wiesbaden 2014;
Melanie Unseld, „Medea auf der Récamière. Transformation zwischen Text, Musik und Perfor
manz im Melodram um 1800“, in: SprachMedienWelten. Wissen und Geschlecht in Musik*The
ater*Film* (mdw Gender Wissen 8), hrsg. von Andrea Ellmeier, Doris Ingrisch und Claudia
Walkensteiner-Preschl, Wien u. a. 2020, S. 83–111. 18 Elisabeth Joris, „Profession und Geschlecht. Das Haus als Ort der Ausbildung und Be
rufstätigkeit im 19. Jahrhundert“, in: Das Haus in der Geschichte Europas. Ein Handbuch, hrsg.
von Joachim Eibach, Inken Schmidt-Voges und Simone Derix, Berlin und Boston 2015, S. 355–
372, hier S. 356.
19 Zur Auseinandersetzung mit dem Begriff des ‚ganzen Hauses‘ (Otto Brunner) in der
deutschsprachigen Forschung (‚Brunner-Debatte‘) vgl. Philip Hahn, „Trends der deutschspra
chigen historischen Forschung nach 1945: Vom ‚ganzen Haus‘ zum ‚offenen Haus‘“, in: Eibach,
Schmidt-Voges und Derix (Hrsg.) 2015, Das Haus in der Geschichte Europas, S. 47–63. Siehe
dort auch die gegenüber dem Brunner’schen Haus-Begriff formulierte Kritik seitens der Gen
derforschung, die für die Musikgeschichte von besonderer Relevanz ist.
20 Ebd., S. 54.
21
Vgl dazu u a der Professorenhaushalt Ulrike Gleixner
Der Professorenhaushalt“ in Musikerfamilien als Ort der Ausbildung und Professionalisierung der Professorenhaushalt: Ulrike Gleixner, „Der Professorenhaushalt“, in:
Das Athen der Welfen. Die Reformuniversität Helmstedt 1576–1810, hrsg. von Jens Bruning,
Wolfenbüttel 2010, S. 129–143; Elizabeth Harding, Der Gelehrte im Haus – Ehe, Familie und
Haushalt in der Standeskultur der frühneuzeitlichen Universität Helmstedt, Wiesbaden 2014;
Melanie Unseld, „Medea auf der Récamière. Transformation zwischen Text, Musik und Perfor
manz im Melodram um 1800“, in: SprachMedienWelten. Wissen und Geschlecht in Musik*The
ater*Film* (mdw Gender Wissen 8), hrsg. von Andrea Ellmeier, Doris Ingrisch und Claudia
Walkensteiner-Preschl, Wien u. a. 2020, S. 83–111. This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 Musikerfamilien als Ort der Ausbildung und Professionalisierung 41 22 Southey 2006, Music-Making in North-East England, S. 15. 25 Dazu vgl. Carola Bebermeier, Celeste Coltellini (1760–1828). Lebensbilder einer Sängerin
und Malerin (Biographik. Theorie – Kritik – Praxis 4), Köln u. a. 2015. 24 Melanie Unseld, „Musikerfamilien. Kontinuitäten und Veränderungen im Mikrokosmos
der Musikkultur um 1800“, in: ebd., S. 25–54. y
g
g
23 Vgl. z. B. Süddeutsche Hofkapellen im 18. Jahrhundert, hrsg. von Silke Leopold und Bär
bel Pelker, Heidelberg 2014; siehe ebenda insbesondere die Musikerlisten zu den hohenlo
hischen Residenzen und zur württembergischen Hofmusik sowie die Anmerkungen zu den
dichten familiären Querverbindungen von Bärbel Pelker. S. a. Birgit Lodes, Elisabeth Reisinger
und John D. Wilson (Hrsg.), Beethoven und andere Hofmusiker seiner Generation. Bericht über
den internationalen musikwissenschaftlichen Kongress Bonn, 3.–6. Dezember 2015 (Musik am
Bonner kurfürstlichen Hof 1) Bonn 2018 22 Southey 2006, Music Making in North East England, S. 15.
23 Vgl. z. B. Süddeutsche Hofkapellen im 18. Jahrhundert, hrsg. von Silke Leopold und Bär
bel Pelker, Heidelberg 2014; siehe ebenda insbesondere die Musikerlisten zu den hohenlo
hischen Residenzen und zur württembergischen Hofmusik sowie die Anmerkungen zu den
dichten familiären Querverbindungen von Bärbel Pelker. S. a. Birgit Lodes, Elisabeth Reisinger
und John D. Wilson (Hrsg.), Beethoven und andere Hofmusiker seiner Generation. Bericht über
den internationalen musikwissenschaftlichen Kongress Bonn, 3.–6. Dezember 2015 (Musik am
Bonner kurfürstlichen Hof 1), Bonn 2018.
24 Melanie Unseld, „Musikerfamilien. Kontinuitäten und Veränderungen im Mikrokosmos
der Musikkultur um 1800“, in: ebd., S. 25–54.
25 Dazu vgl. Carola Bebermeier, Celeste Coltellini (1760–1828). Lebensbilder einer Sängerin
und Malerin (Biographik. Theorie – Kritik – Praxis 4), Köln u. a. 2015. III. Im Haus einer Musikerfamilie: Ökonomie, Arbeitserziehung
und Berufsvielfalt Wer im 18. Jahrhundert professionell (im oben genannten Sinne) Musik
machte, kam nicht selten aus einer Musikerfamilie. Wenngleich quantita
tive Erhebungen dazu ausstehen, dürfte es eine Viel-, wenn nicht gar die
Mehrzahl der professionellen Musiker und Musikerinnen gewesen sein,
und zwar von so unterschiedlichen Bereichen (und strukturell-institutio
nellen Zusammenhängen) wie höfische Kapelle, städtische Musikdarbie
tungen, Instrumentalisten- und Virtuosentum, Kantorat und Kapellmeister
bis hin zu Stadtpfeiferei, Tanz- und Militärmusik, Instrumentenbau, Musik
verlagswesen und -handel. Dies betrifft, um nur wenige Beispiele aus dem
16. / 17. Jahrhundert zu nennen, die italienisch-englische Musikerfamilie
Bassano ebenso wie die englischen Musikerfamilien Gibbons, Purcell, Tom
kins ebenso wie die Familie Avison in Newcastle, in der der junge Charles
Avison zu Beginn des 18. Jahrhunderts sein „early training […] at his father’s
hands and at those of the musical community that surrounded these fami
lies“22 erhielt. Nicht zu vergessen sind hierbei auch die Hofmusikerfamilien23
Mozart, Cramer, Cannabich, Stamitz, Beethoven und Ries an den Höfen in
Salzburg, Mannheim oder Bonn, die in verschiedenen Musikprofessionen
tätigen Familien wie die Familien Arne, Storace und Dussek / Corri,24 die in
Musik, Literatur und Bildender Kunst professionalisierte Familie Burney, die
juristisch-bildnerisch-musikalisch-literarisch tätige Familie Coltellini,25 die
vor allem in den Tasteninstrumenten reüssierende französische Musikerfa
milie Couperin, die italienische Instrumentenbauerfamilien Guadagnini oder
die aus Augsburg stammende, in Wien erfolgreiche Familie Stein-Streicher,
die unzähligen Theaterfamilien, darunter die irische Familie Hamilton, die This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 Melanie Unseld 42 (Tänzer-), Schauspieler-, Manager-Familien Hallam oder Kemble / Siddons26,
zahllose Opern- und Wandertruppen u. v. m. Wie explizit sich jede einzelne
der Familien im beruflichen Zusammenhang als solche auswies, auf welche
Medien sie dabei zurückgriff (visuelle Medien, Notendruck27, Firmen-Name28,
Biographik / Lexikographik29, Stammbaum u. a. m.), und welche Traditionsli
nien dabei ausgestellt wurden (patrilinear / uxorilinear, Geschwister, Schüler
u. a.), ist ebenso unterschiedlich wie die Funktion und Wirksamkeit dieser
öffentlichen Sichtbarmachung. Auch die Sichtbarkeit des Familialen in der
Musikgeschichtsschreibung ist verschieden. Wenngleich zu einer Reihe von
Musikerfamilien Einzelstudien vorliegen – die angeführte Literatur mag hier
punktuelle Hinweise geben – steht eine Studie, die vergleichend Musiker
familien als sozialen Raum in den Blick nimmt, noch aus. Musikausbildung fand in ganz Europa – abgesehen von frühen Ausbil
dungsstätten wie den italienischen conservatori oder den Singschulen im
kirchlichen Kontext („Sängerknaben“) – in Familien statt.30 Somit war die
Ausbildung der folgenden Generation(en) grundlegender Teil der Arbeitsge
meinschaft im ‚Haus‘. 26 Wobei die Schauspielerin Sarah Siddons auch als Bildhauerin angesehen war.
27 Vgl. u. a. der Hinweis auf die geschwisterliche Verbindung auf Notendrucken von Ve
ronika Cianchettini, die auf Titelblättern ihrer gedruckten Kompositionen als „Sister to Mr.
Dussek“ ausgewiesen wird. Siehe dazu auch Unseld 2018, „Musikerfamilien“.
28 Vgl. dazu die Firmenbezeichnungen der Klavierbauerfamilie Broadwood (John Broadwood
& Son[s], ab 1728 bzw. ab 1808) oder die wechselnden Firmennamen der Steinschen Klavier
bauer-Familie, u. a. „Frère et Sœur Stein d’Augsbourg à Vienne“, ab 1802: „Nannette Streicher
neé Stein“. Zu letzterer s. Michael Latcham, „The development of the Streicher firm of piano
builders under the leadership of Nannette Streicher, 1792 to 1823“, in: Das Wiener Klavier
bis 1850. Bericht des Symposiums „Das Wiener Klavier bis 1850“ veranstaltet von der Samm
lung alter Musikinstrumente des Kunsthistorischen Museums Wien vom 16. bis 18.10.2003,
hrsg. von Beatrix Darmstädter, Alfons Huber und Rudolf Hopfner, Tutzing 2007, S. 43–71; Klaus
Martin Kopitz, Art. „Nannette Streicher“, in: MUGI. Musikvermittlung und Genderforschung:
Lexikon und multimediale Präsentationen, hrsg. von Beatrix Borchard und Nina Noeske, 2018,
http://mugi.hfmt-hamburg.de/artikel/Nannette_Streicher (abgerufen am 06.11.2020). g
30 Dies galt sowohl für die Kinder von Musikerfamilien, als auch für Kinder, die sich für
Musik talentiert zeigten, aber aus Nicht-Musikerfamilien stammten: „For talented children
from non-musical families, an apprenticeship was the usual way into the profession.“ Lowell
Lindgren und Colin Timms, „The Correspondence of Agostino Steffani and Giuseppe Riva,
1720–1728, and Related Correspondence with J. P. F. von Schönborn and S. B. Pallavicini“, in:
Royal Musical Association Research Chronicle 36 (2003), S. 1–174, hier S. 165. Ein anderes Ziel
verfolgte das Unterrichten von Schülerinnen oder Schülern, das zwar durchaus auch privatim
erteilt wurde, aber nicht auf eine professionelle Tätigkeit abzielte. 26 Wobei die Schauspielerin Sarah Siddons auch als Bildhauerin angesehen war.
27 Vgl. u. a. der Hinweis auf die geschwisterliche Verbindung auf Notendrucken von Ve
ronika Cianchettini, die auf Titelblättern ihrer gedruckten Kompositionen als „Sister to Mr.
Dussek“ ausgewiesen wird. Siehe dazu auch Unseld 2018, „Musikerfamilien“.
28 Vgl. dazu die Firmenbezeichnungen der Klavierbauerfamilie Broadwood (John Broadwood
& Son[s], ab 1728 bzw. ab 1808) oder die wechselnden Firmennamen der Steinschen Klavier
bauer-Familie, u. a. „Frère et Sœur Stein d’Augsbourg à Vienne“, ab 1802: „Nannette Streicher
neé Stein“. Zu letzterer s. Michael Latcham, „The development of the Streicher firm of piano
builders under the leadership of Nannette Streicher, 1792 to 1823“, in: Das Wiener Klavier
bis 1850. Bericht des Symposiums „Das Wiener Klavier bis 1850“ veranstaltet von der Samm
lung alter Musikinstrumente des Kunsthistorischen Museums Wien vom 16. bis 18.10.2003,
hrsg. von Beatrix Darmstädter, Alfons Huber und Rudolf Hopfner, Tutzing 2007, S. 43–71; Klaus
Martin Kopitz, Art. „Nannette Streicher“, in: MUGI. Musikvermittlung und Genderforschung:
Lexikon und multimediale Präsentationen, hrsg. von Beatrix Borchard und Nina Noeske, 2018,
http://mugi.hfmt-hamburg.de/artikel/Nannette_Streicher (abgerufen am 06.11.2020).
29 Für die Familie Dussek vgl. Unseld 2018, „Musikerfamilien“.
30 Dies galt sowohl für die Kinder von Musikerfamilien, als auch für Kinder, die sich für
Musik talentiert zeigten, aber aus Nicht-Musikerfamilien stammten: „For talented children
from non-musical families, an apprenticeship was the usual way into the profession.“ Lowell
Lindgren und Colin Timms, „The Correspondence of Agostino Steffani and Giuseppe Riva,
1720–1728, and Related Correspondence with J. P. F. von Schönborn and S. B. Pallavicini“, in:
Royal Musical Association Research Chronicle 36 (2003), S. 1–174, hier S. 165. Ein anderes Ziel
verfolgte das Unterrichten von Schülerinnen oder Schülern, das zwar durchaus auch privatim
erteilt wurde, aber nicht auf eine professionelle Tätigkeit abzielte. p
g
g
g
29 Für die Familie Dussek vgl. Unseld 2018, „Musikerfamilien“. Wobei die Schauspielerin Sarah Siddons auch als Bildhauerin angesehen war. 31 Andreas Gestrich, Art. „Familie“, in: Enzyklopädie der Neuzeit, Bd. 3, hrsg. von Friedrich Jae
ger, Stuttgart 2006, Sp. 790–812, hier Sp. 803. Vgl. dazu auch Reinhard Sieder, Vom Patriarchat
zur Partnerschaft. Zum Strukturwandel der Familie, München 41991 (engl. Übersetzung: The
European Family. Patriarchy to Partnership from the Middle Ages to the Present, Oxford 41989).
32 Edward Palmer Thompson, The Making of the English Working Class, London 1963, S. 332.
In dieser groß angelegten Studie zur englischen Arbeiterklasse wird „musician“ gleichauf mit
zahlreichen anderen Berufen genannt, die die „society of artisans“ (S. 156) bildeten.
33 Wobei die kollektive Professionalisierung qua Akademisierung Musikberufe nicht zuletzt
auch – wie andere Berufsgruppen – zu höherer gesellschaftlicher Anerkennung in der beruf
lichen Hierarchie verhelfen sollte.
34 S. Melanie Unseld, „‚Verehrter LandsMann!‘. Die Netzwerke der Musikerfamilien Salomon,
Ries und Beethoven zwischen Bonn und London“, Vortrag auf dem 3. Hauptsymposium („Der
Bonner Beethoven“) im Rahmen des Internationalen wissenschaftlichen Kongresses Beetho
ven-Perspektiven, Beethoven-Haus Bonn, 10.–14. Februar 2020 (Druck in Vorb.). III. Im Haus einer Musikerfamilie: Ökonomie, Arbeitserziehung
und Berufsvielfalt Da Musikberufe insgesamt noch stark mit der Idee des
Handwerks verbunden waren, lässt sich für die musikalische Ausbildung im
‚Haus‘ durchaus der Begriff der ‚Arbeitserziehung‘ veranschlagen. Der Histo
riker Andreas Gestrich erläutert den Begriff in Anlehnung an Reinhard Sie This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 Musikerfamilien als Ort der Ausbildung und Professionalisierung 43 der als eine Ausbildungsweise, die gleichsam „naturwüchsig“ zu denken sei,
„die von den Kindern zu übernehmenden Einstellungen, Verhaltensweisen
und Fertigkeiten durch unmittelbares Erleben im Rahmen der Hausgemein
schaft vermittelt wurden.“31 Auch Edward Palmer Thompson unterstrich in
seiner bereits 1963 erschienenen Studie The Making of the English Working
Class die Bedeutung der Kinderarbeit, wobei „[t]he most prevalent form of
child labour was in the home or within the family economy.“32 Musikerfamilien waren der Nucleus der musikalischen Professionalisie
rung vor der Gründung entsprechender Ausbildungsinstitutionen, die eine
Akademisierung33 der Musikberufe seit dem späten 18., vor allem aber im
19. Jahrhundert vorantrieb. In der Familie wurde musikalisches Wissen tra
diert, über Generationen hinweg stabilisiert und weitergegeben. Dabei wa
ren Musikerfamilien Kern- und Knotenpunkte für weiter reichende, zum Teil
sich überschneidende professionelle, künstlerische, ökonomische, religiöse
u. a. Netzwerke,34 die als wichtige Basis (auch) der Musikausbildung zu ver
stehen sind. Familien, die im weiten Feld der Professionen der performa
tiven Künste tätig waren, sponnen untereinander ein enges Netz, in dem
Kinder, Kindeskinder, aber auch Kostzöglinge oder andere im Haushalt Le
bende, in der eigenen oder anderen ortsansässigen, regional bis internatio
nal vernetzten Familie unterrichtet und früh in die Praxis eingeführt wurden. Diese Form der Ausbildung qua Lehre bedeutete auch, in der ausbilden
den Familie zu leben. Damit wird erkennbar, dass es sich in diesem Sin
ne beim Familialen nicht notwendigerweise um blutsverwandtschaftliche
Zusammenhänge handelte. Gerade weil Musik, Theater, Schauspiel, Tanz
– mithin die performativen Künste – mit all ihren notwendigen Tätigkeits
bereichen viele Hände benötigte, waren diese Familien, insbesondere in This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 Melanie Unseld 44 ihren ökonomisch erfolgreichen Phasen, kontinuierlich auf der Suche nach
Lehrlingen. Diese kamen für die eigene Professionalisierung in die Familie
temporär hinein und arbeiteten hier bereits professionell mit, z. B. indem
sie Kopistenarbeit übernahmen, Begleitstimmen aussetzten oder Partien
für Nebenrollen komponierten, bei Aufführungen mitwirkten etc. Auf der
Bühne zu stehen, zunächst als Kinderdarstellerin oder Tänzerin, später als
Schauspielerin, Sängerin, dann auch für die Bühne zu schreiben, ggf. zu
komponieren, ggf. 35 Vgl. dazu etwa die weiblichen Mitglieder der Schauspieler-Musikerfamilie Brandes.
36 Vgl. Southey 2006, Music-Making in North-East England, S. 165: „Apprentices frequently
played in concert and dancing assembly bands but were not necessarily always trouble-free;
Avison listed the maintaining of such apprentices as a necessary part of the expenses of a
subscription series, and a burdensome one, although he did not state precisely why; he may
have been thinking, as he wrote, of the difficulties he had had with one of his apprentices,
George William, who had run away in 1739, prompting Avison to advertise for him in local
papers.“ III. Im Haus einer Musikerfamilie: Ökonomie, Arbeitserziehung
und Berufsvielfalt – etwa als Witwe – eine Theatertruppe zu leiten – solche
Berufsbiographien sind für das 18. Jahrhundert auch für Frauen, etwa aus
Schauspielerfamilien, nicht unüblich.35 Solche Ausbildungswege waren ebenso wenig akademisiert wie stan
dardisiert, damit hochgradig kontingent, folgten aber der Grundidee, dass
durch die frühe Einbindung in den Alltag der performativen Künste Routi
nen erlernt, Talente erkannt, entwickelt und erprobt werden konnten. Auf
diese Weise im ‚Haus‘ ausgebildet zu werden, bereitete die Heranwachsen
den auf eine professionelle Laufbahn in mehreren künstlerischen Feldern,
nicht notwendigerweise in einem einzelnen Fach („profession“) vor. Die Zeit
als Lehrling war dabei keineswegs immer konfliktfrei: Quellen sprechen von
Ausbeutung und Misshandlung, aber auch von unzuverlässigen Lehrlingen,
die ihren Pflichten nicht nachkamen.36 Dass Kinder und Heranwachsende zum Zweck der Ausbildung migrierten,
war eher erwartbar als die Ausnahme. Und nicht unüblich war, dass Lehr
linge nach der Ausbildungszeit in einer Musikerfamilie auf Reisen gingen. Finanziert werden konnten diese Reisen, die als weiterer Teil der Ausbildung
verstanden wurden, durchaus häufig durch die ausbildende Musikerfamilie
selbst. Das ist immerhin erstaunlich, denn auf diese Weise übernahm die
Musikerfamilie Kosten, die alternativ durch andere, etwa die Aristokratie bzw. der Hof oder andere Patronatspersonen finanziert wurden. Hier wird deut
lich, wie eng die Lehrlinge zum ‚Haus‘ als ökonomischer Einheit verbunden This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 Musikerfamilien als Ort der Ausbildung und Professionalisierung 45 blieben und welche Bedeutung sie auch dann noch für das ‚Haus‘ hatten,
wenn sie nicht mehr buchstäblich unter dem gemeinsamen Dach wohnten. Die Reisen dienten zwei wesentlichen Zwecken: der weiteren Ausbildung
des Lehrlings und der Vernetzung. Auf diese Weise konnte einerseits das Re
nommee der Musikerfamilie (der ‚Name‘) etabliert, andererseits die für die
Musikkultur so wichtige Vernetzung initiiert und gestärkt werden (Mobili
tät). In diesem Sinne war eine (Ausbildungs)Reise nach London und seinem
im 18. Jahrhundert bereits stark entwickeltem Musikmarkt mit dem Versuch
verbunden, hier eine freie musikalische Karriere zu starten und entspre
chend für zahllose Musikerinnen und Musiker hoch attraktiv, was an zahl
reichen Musiker_innen-Biographien erkennbar ist.37 Wie lange dann jede / r Einzelne und in welcher Form des „apprentice
ships“ in Musikerfamilien (vor Ort und auf Reisen) verblieb, differiert von we
nigen Wochen bis zu Jahren, von engem Studium bis hin zur losen Nutzung
der Familie als ‚Kontaktbörse‘. Musikerfamilien des 18. Jahrhunderts sind, so
wäre daher zu verallgemeinern, gerade nicht (bzw. 38 Thomas Sokoll geht sogar davon aus, dass die Geschichte der Berufe „vornehmlich
männlich bestimmt gewesen“ sei. Es habe zwar einige wenige „Frauenberufe“ (und auch
Frauenzünfte) gegeben, im Grunde aber gehörte die arbeitende Frau zum Teil der männli
chen Berufswelt: „[…] die Frau des Meisters [arbeitete] im Betrieb (v. a. in Verkauf und Buch
führung) mit[…] oder Töchter [wurden] ‚angelernt‘ […]. Aber als solche blieb sie im berufl.
Schatten des Mannes, aus dem sie erst heraustreten konnte, wenn sie als Meisterwitwe sei
nen Betrieb weiterführte.“ Sokoll 2006, Art. „Beruf“, Sp. 49. Da Sokoll in seiner Übersicht die 37 Vgl. dazu auch Lindgren und Timms 2003, „The Correspondence of Agostino Steffani and
Giuseppe Riva“. Zur (ökonomisch erzwungenen) Mobilität von Musikerfamilien im ausgehen
den 18. und 19. Jahrhundert vgl. auch Ann V. Beedell, The Decline of the English Musician
1788–1888. A Family of English Musicians in Ireland, England, Mauritius, and Australia, Oxford
1992. Künste als Beruf nicht inkludiert, dürfte hier Anlass zur Differenzierung geben sein. Zur Frage
der Frauenberufe vgl. auch Wetterer 2002, Arbeitsteilung und Geschlechterkonstruktion.
39 Vgl. u. a. Suzanne Aspden, The Rival Sirens. Performance and Identity on Handel’s Ope
ratic Stage, Cambridge 2013. Die moralische Vulnerabilität von Künstlerinnen, insbesondere
Bühnenkünstlerinnen, existierte noch weit über das 18. Jahrhundert hinaus. Ein familialer
Kontext (Heirat, Familie etc.) konnte diese Vulnerabilität abschwächen, wenngleich Heirat
nicht selten den Verzicht auf öffentliches Auftreten nach sich zog.
40 Claudia Schweitzer, „…ist übrigens als Lehrerinn höchst empfehlungswürdig“. Kulturge
schichte der Clavierlehrerin, Oldenburg 2008. III. Im Haus einer Musikerfamilie: Ökonomie, Arbeitserziehung
und Berufsvielfalt nicht ausschließlich) als
räumlich stabile Einheit einer zusammenhängenden blutsverwandtschaft
lichen Familie zu denken, sondern auch als Nucleus von ästhetischen und
ökonomischen Einheiten, zu deren Interesse es gehörte, gut vernetzt und
hoch mobil zu sein. Versteht man Familie schließlich auch als moralische Einheit, lag ein gro
ßes Interesse auch auf der Stabilisierung der moralischen Integrität. Bei
des war auch für Musikerfamilien essentiell. Die moralische Integrität wird
insbesondere dann plausibel, berücksichtigt man zum einen die schmale
Trennlinie der Profession zu unteren sozialen Schichten (Spielleute), zum
anderen den hohen Anteil der Mädchen und Frauen, die als professionelle
Akteurinnen in der Öffentlichkeit (Bühne) standen.38 In Theater- und Mu
sikerfamilien des 18. Jahrhunderts waren – wie in der ökonomischen Ein This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 46 Melanie Unseld heit Familie vorgesehen – Mädchen und Frauen selbstverständlich Teil der
Arbeitsgemeinschaft und damit auch moralisch abgesichert, was aufgrund
der hohen moralischen Vulnerabilität von Bühnenkünstlerinnen nicht un
wesentlich war.39 Aus der Couperin-Familie beispielsweise waren mehrere
weibliche Familienmitglieder, etwa als Töchter des renommierten François
Couperin (le Grand) moralisch abgesichert, was sich daran erkennen lässt,
dass sie etablierter wie angesehener Teil der Versailler Hofmusik waren, da
runter etwa Marguerite-Antoinette Couperin, jüngste Tochter von Couperin
und Marie-Anne Ansault. Auch sie war im Familienkontext ausgebildet wor
den und reüssierte als Cembalistin am Versailler Hof. Dass sie zunächst den
Titel als „Maîtresse de Clavecin de Mesdames de France“, mithin als Lehrerin
der Töchter Louis’ XV., erhielt, später (1736) auch den Titel der königlichen Ti
tularcembalistin, verband künstlerische mit institutioneller Anerkennung.40
Aus der böhmisch-englischen Musikerfamilie Dussek wiederum stammten
u. a. die Cembalistin, Komponistin und Pädagogin Veronika Dussek, verhei
ratet mit dem Londoner Musikverleger Francesco Cianchettini, oder auch die
Harfenistin, Sängerin, Pädagogin und Komponistin Sophia Dussek, Schwä
gerin von Veronika Dussek und aus der Edinburgher Musikerfamilie Corri
stammend. Die Schwägerinnen, beide aus Musikerfamilien kommend und in
solche wiederum einheiratend, waren mit ihren verschiedenen Tätigkeiten
über Jahrzehnte in das Londoner Musikleben integriert. Die Vielfachbezeichnungen bei den Schwägerinnen Cianchettini / Dussek
– ein in der modernen Lexikographik eher nivelliertes, denn thematisiertes
Phänomen – verweist schließlich auch auf das Phänomen der ‚Berufsviel
falt‘. Als ökonomisch stabilisierend galt das Arbeitsteilige innerhalb eines
‚Hauses‘ bzw. das Selbstverständnis, dass zum wirtschaftlichen Erfolg viele
Teilbereiche notwendig waren: Alle Beteiligten (unabhängig von Alter, Ge
schlecht etc.) trugen ebenso grundsätzlich wie arbeitsteilig zum Einkommen
bei. 41 In Theaterfamilien und solchen, die generell performative Künste als Profession aus
übten, war dies kaum anders. Hinzu kommen Professionen wie Literatur (Libretti, Dramen,
Reise- und Memoirenliteratur, pädagogische Literatur), Journalismus, Verlags- und Publika
tionswesen, aber auch bildende Künste u. a. m., die weitere professionelle Schnittstellen und
Synergien boten. III. Im Haus einer Musikerfamilie: Ökonomie, Arbeitserziehung
und Berufsvielfalt Die Vielzahl an notwendigen Tätigkeiten etwa, die Musik zu dem macht,
was sie ist – ein klingendes Ereignis –, bedurfte einer Vielzahl an Menschen, This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 Musikerfamilien als Ort der Ausbildung und Professionalisierung 47 die in der Lage waren, diese Vielzahl an Tätigkeiten gemeinsam und / oder
arbeitsteilig auszuführen.41 Je nach Profession konnte dies sein: Kopisten-
und andere Schreibarbeiten, Instrumentenstimmung und -reparaturen,
unterrichten, die Organisation von Auftritten, das Leiten von Proben, arran
gieren, Generalbass spielen, üben, komponieren, mit dem Publikum kom
munizieren, Noten drucken, Geschäftsmodelle entwickeln, Kontakte pflegen
u. a. m. Für kaum eine dieser (Teil)Tätigkeiten gab es eine spezialisierte Aus
bildung, vieles wurde im täglichen Betrieb bzw. nach täglichem Bedarf er
lernt und jedes Mitglied der Familie tat gut daran, ein Bündel dieser (Teil-)
Tätigkeiten zu beherrschen. Die ökonomische Stabilität einer Musikerfamilie
wurde durch das möglichst reibungslose Ineinandergreifen dieser vielfälti
gen Tätigkeiten jedes / r einzelnen unterstützt. Vice versa waren vom Schei
tern einer solchen ökonomischen Einheit alle Beteiligten betroffen. Es ist vor diesem Hintergrund nicht erstaunlich, dass in zahlreichen Mu
sikerfamilien zahlreiche Tätigkeiten gebündelt zu finden waren, die heute
jede einzelne für sich als „Beruf“ bezeichnet werden, im Verständnis des
18. Jahrhunderts aber zur Existenz des Musikerhauses zusammengehörig
gedacht waren. Denn zum (v. a. ökonomischen) Erfolg einer Musikerfamilie
zählte insbesondere dort, wo keine Anbindung an einen Hof für eine gewis
se ökonomische Stabilität sorgte, ein Portfolio an Tätigkeiten, die sich am
Bedarf orientierten, sich bestenfalls ergänzten und auf diese Weise nicht
nur individuellen Fähigkeiten Raum gaben, sondern vor allem auch öko
nomische Multiplikationseffekte hatten. Das Beispiel der in London tätigen,
mehrgenerationellen Musikerfamilien Dussek / Corri / Cianchettini mit Be
rufsfeldern wie Virtuosentum (Tasteninstrumente, Harfe, Gesang), kompo
nieren, unterrichten, Musik verlegen und Musikalienhandel bestätigt das
ebenso wie der aus der Mannheimer Musikerfamilie stammende Johann
Baptist Cramer, der in London als Pädagoge, Komponist, Klaviervirtuose, Mu
sikverleger, Musikalienhändler und Instrumentenbauer erfolgreich war. Musikprofessionen waren, darauf mögen die knappen Beispiele hinge
deutet haben, im 18. Jahrhundert notwendigerweise ein weites Feld, durch
aus kontingent und volatil, am Markt orientiert und noch nicht selbstver This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 48 Melanie Unseld ständlich und in umfassendem Maße von Spezialisierung gekennzeichnet,
die sich dann im Verlauf des 19. Jahrhunderts zu etablieren begann und
hierbei mit der akademischen Institutionalisierung der Musikerausbildung
Hand in Hand ging. III. Im Haus einer Musikerfamilie: Ökonomie, Arbeitserziehung
und Berufsvielfalt Jene kontingent-volatilen Professionen aber ließen sich
durch einen sozialen Zusammenhang, wie es das Haus als ökonomische
Einheit bot, stabilisieren. 42 Vgl. grundlegend zu einzelnen Familienmitgliedern https://www.mcgill.ca/burneycentre/
(abgerufen am 01.04.2020). IV. Familie Burney. Ein Beispiel für künsteübergreifende Berufsvielfalt,
Arbeitserziehung und Professionalisierungs(un)möglichkeiten Wie aber lassen sich diese allgemeinen Beobachtungen zu Musikerfamilien
weiter konkretisieren? Wie lassen sie sich vor allem bei der Darstellung der
einzelnen Akteur_innen und ihrer Handlungsfelder nutzbringend einsetzen? Wie werden in den Quellen Hinweise auf künsteübergreifendes Handeln,
arbeitsteiliges Handeln und innerfamiliäre Professionalisierungsprozesse
erkennbar? Dies sei im Folgenden kurz am Beispiel der Familie Burney42
in den Blick genommen. Der 1726 geborene Charles Burney war Sohn von
James Macburney, der zeittypisch in mehreren Professionen tätig war. Die
Quellen lassen ihn als Tänzer, Geiger und als Porträtmaler auftreten. Die Mu
sik war u. a. in der Familie durch Charles’ Halbbruder James verankert, James
Burney war Organist in Shrewsbury und unterrichtete den heranwachsen
den Charles, bevor dieser für sieben Jahre nach London in das Haus des
Komponisten Thomas Augustine Arne und seiner Frau, der bei Francesco
Geminiani ausgebildeten Sängerin Cecilia Arne, geb. Young, gegeben wurde. Damit durchlief Burney bereits in jungen Jahren geradezu prototypisch die
Ausbildung zum Musiker: innerhalb der Musikerfamilien Burney und Arne,
wobei das Haus Arnes mit seinem professionellen Netzwerk in London zu
künftige berufliche Vorteile versprach. Immerhin wurde Burney so in die
Lage versetzt, unter der Leitung von Georg Friedrich Händel als Orchester
musiker spielen zu können. Die Zeit als Lehrling war für Burney gleichwohl
nicht konfliktfrei, so dass er 1748 durch seinen Patron Fulke Greville aus dem
Lehrverhältnis zu Arne „ausgelöst“ wurde. An dieser Stelle wird noch ein
mal deutlich, dass die Ausbildung in einer Musikerfamilie eng mit ökonomi This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 Musikerfamilien als Ort der Ausbildung und Professionalisierung 49 schen Bedingungen verknüpft war. Wollte ein Lehrling das Haus verlassen,
musste ggf. sogar eine Form der Ablöse gezahlt werden. Auch die Aspekte von Mobilität und professioneller wie personeller Vo
latilität von Musikerfamilien lassen sich in der Burney-Familie zeigen.43 Auf
fällig ist hier, dass sich sowohl die Grenzen zwischen den Professionen, als
auch die zwischen den Künsten immer wieder auflösten bzw. durchlässig
blieben: Betrachtet man die Familie nur im Ausschnitt dreier Generationen,
ist auffällig, dass sich die einzelnen Familienmitglieder in (zum Teil meh
reren) Bereichen der Bildenden Kunst (Malerei, Karikatur), der praktischen
Musik und der Literatur (Drama, Reise- und Erinnerungsliteratur, Musikkritik,
Musikgeschichte u. a.) bewegten. Vom Vater Macburney war schon die Rede,
dessen Beruf als Porträtmaler nach zwei Generationen durch Edward Fran
cisco Burney wieder aufgegriffen wurde.44 Letzterer arbeitete vorwiegend als
Porträtmaler und Illustrator bzw. 43 Die nachfolgenden biographischen Angaben sind den von Philip Olleson herausgege
benen Tagebüchern und Briefen von Susanna Elizabeth (Susan) Burney entnommen, ebenso
der „Biographical Introduction“ von Olleson, ebd. Olleson vertritt dabei nicht explizit die
Perspektive einer Musikerfamilie als ökonomisch-moralische Einheit, ist sich der Problematik
in der Darstellung, insbesondere der Aktivitäten der Töchter von Burney aber wohl bewusst.
Insofern lassen sich Darstellung und Kommentierung der Arbeitssituation als Musikerfamilie
auch jene Friktionen entnehmen, die dann entstehen, wenn eine stärker am bürgerlichen
Kernfamilienmodell orientierte Lesart zum Vorschein kommt. Siehe zu Ollesons weiterer Aus
einandersetzung mit Musikerfamilien auch ders., „Father and Sons. Charles Wesley, Samuel
Wesley, and Charles Wesley ‚the Younger‘“, in: Music and the Wesleys, hrsg. von Stephen
Banfield und Nicholas Temperley, Urbana 2010, S. 175–182 und ders., „Charles Wesley and his
Children“, in: Charles Wesley. Life, Literature and Legacy, hrsg. von Kenneth Newport und Ted
Campbell, Peterborough 2007, S. 124–140.
44 Vgl. https://www.mcgill.ca/burneycentre/resources/edward-francisco-burney-1760-1848
(abgerufen am 01 04 2020) 43 Die nachfolgenden biographischen Angaben sind den von Philip Olleson herausgege
benen Tagebüchern und Briefen von Susanna Elizabeth (Susan) Burney entnommen, ebenso
der „Biographical Introduction“ von Olleson, ebd. Olleson vertritt dabei nicht explizit die
Perspektive einer Musikerfamilie als ökonomisch-moralische Einheit, ist sich der Problematik
in der Darstellung, insbesondere der Aktivitäten der Töchter von Burney aber wohl bewusst.
Insofern lassen sich Darstellung und Kommentierung der Arbeitssituation als Musikerfamilie
auch jene Friktionen entnehmen, die dann entstehen, wenn eine stärker am bürgerlichen
Kernfamilienmodell orientierte Lesart zum Vorschein kommt. Siehe zu Ollesons weiterer Aus
einandersetzung mit Musikerfamilien auch ders., „Father and Sons. Charles Wesley, Samuel
Wesley, and Charles Wesley ‚the Younger‘“, in: Music and the Wesleys, hrsg. von Stephen
Banfield und Nicholas Temperley, Urbana 2010, S. 175–182 und ders., „Charles Wesley and his
Children“, in: Charles Wesley. Life, Literature and Legacy, hrsg. von Kenneth Newport und Ted
Campbell, Peterborough 2007, S. 124–140.
44 Vgl. https://www.mcgill.ca/burneycentre/resources/edward-francisco-burney-1760-1848
(abgerufen am 01.04.2020).
45 S. https://www.tate.org.uk/art/artworks/burney-amateurs-of-tye-wig-music-musicians-
of-the-old-school-t07278 (abgerufen am 01.04.2020). Vgl. dazu auch Thomas Tolley, „‚Stamp of
Genius‘. Beethoven as Satire in Regency Britain“, auf dem 1. Hauptsymposium („Der politische
Beethoven“) im Rahmen des Internationalen wissenschaftlichen Kongresses Beethoven-Per
spektiven, Beethoven-Haus Bonn, 10.–14. Februar 2020 (Druck in Vorb.).
46 Burney selbst beschrieb seine Tätigkeit zu Beginn seiner Karriere folgendermaßen: „[I]
began to be in fashion in the City, as a Master, and had my hands full of professional business
of all kinds with scholars at both ends of the town, Composition, & public playing.“ Zit. aus
den Memoirs in: Olleson 2012, The Journals and Letters of Susan Burney, S. [5]. 45 S. https://www.tate.org.uk/art/artworks/burney-amateurs-of-tye-wig-music-musicians-
of-the-old-school-t07278 (abgerufen am 01.04.2020). Vgl. dazu auch Thomas Tolley, „‚Stamp of
Genius‘. Beethoven as Satire in Regency Britain“, auf dem 1. Hauptsymposium („Der politische
Beethoven“) im Rahmen des Internationalen wissenschaftlichen Kongresses Beethoven-Per
spektiven, Beethoven-Haus Bonn, 10.–14. Februar 2020 (Druck in Vorb.). 45 S. https://www.tate.org.uk/art/artworks/burney-amateurs-of-tye-wig-music-musicians-
of-the-old-school-t07278 (abgerufen am 01.04.2020). Vgl. dazu auch Thomas Tolley, „‚Stamp of
Genius‘. Beethoven as Satire in Regency Britain“, auf dem 1. Hauptsymposium („Der politische
Beethoven“) im Rahmen des Internationalen wissenschaftlichen Kongresses Beethoven-Per
spektiven, Beethoven-Haus Bonn, 10.–14. Februar 2020 (Druck in Vorb.).
46 Burney selbst beschrieb seine Tätigkeit zu Beginn seiner Karriere folgendermaßen: „[I]
began to be in fashion in the City, as a Master, and had my hands full of professional business
of all kinds with scholars at both ends of the town, Composition, & public playing.“ Zit. aus
den Memoirs in: Olleson 2012, The Journals and Letters of Susan Burney, S. [5]. Genius. Beethoven as Satire in Regency Britain , auf dem 1. Hauptsymposium („Der politische
Beethoven“) im Rahmen des Internationalen wissenschaftlichen Kongresses Beethoven-Per
spektiven, Beethoven-Haus Bonn, 10.–14. Februar 2020 (Druck in Vorb.).
46 Burney selbst beschrieb seine Tätigkeit zu Beginn seiner Karriere folgendermaßen: „[I]
began to be in fashion in the City, as a Master, and had my hands full of professional business
of all kinds with scholars at both ends of the town, Composition, & public playing.“ Zit. aus
den Memoirs in: Olleson 2012, The Journals and Letters of Susan Burney, S. [5]. 44 Vgl. https://www.mcgill.ca/burneycentre/resources/edward-francisco-burney-1760-1848
(abgerufen am 01.04.2020). 47 Hetty Burney spielte Harfe und Cembalo und taucht in den Familiendokumenten als
herausragende Interpretin auf. Sie bekam sieben (vgl. Olleson 2012, The Journals and Letters
of Susan Burney, Family Tree) bzw. zehn Kinder (vgl. Philip H. Highfill, Jr., Kalman A. Burnim
und Edward A. Langhans, A Biographical Dictionary of Actors, Actresses, Musicians, Dancers,
Managers, and Other Stage Personnel in London, 1660–1800, Bd. 1, Carbondale 1973, S. 428).
48 Vgl. dazu u. a. Barbara Darby, Frances Burney Dramatist. Gender, Performance, and the
Late-Eighteenth-Century Stage, Lexington / Kentucky 1997, sowie https://www.mcgill.ca/bur
neycentre/resources/frances-fanny-burney-darblay-1752-1840 (abgerufen am 01.04.2020).
49 Vgl. Olleson 2012, The Journals and Letters of Susan Burney.
50 James Burney begleitete Captain Cook auf zwei seiner Expeditionen (1772–1773, Antarktis
und Pazifik) und veröffentlichte zahlreiche Bücher darüber, u. a. die fünfbändige Chronologi
cal History of the Discoveries in the South Sea or Pacific Ocean, London 1803–1817. Für weitere
Informationen s. https://www.mcgill.ca/burneycentre/resources/james-burney-1750-1821 (ab
gerufen am 01.04.2020).
51 S. https://www.mcgill.ca/burneycentre/resources/charles-burney-jr-1757-1817 (abgerufen
am 01.04.2020). IV. Familie Burney. Ein Beispiel für künsteübergreifende Berufsvielfalt,
Arbeitserziehung und Professionalisierungs(un)möglichkeiten Karikaturist (in der Tradition von William
Hogarth, James Gillray u. a.), und dass seine musikbezogenen Karikaturen
(zumindest) von profundem musikhistorischem Wissen zeugen, ist etwa
dem Gemälde Amateurs of Tye-Wig Music (‚Musicians of the Old School‘)
(ca. 1820) zu entnehmen.45 Charles Burney selbst verstand sich46 als Musik
historiker, Komponist, Organist und Lehrer. Schließlich gilt der Fokus auch
der nachfolgenden Generation, die Kinder von Burney aus seiner ersten Ehe 46 Burney selbst beschrieb seine Tätigkeit zu Beginn seiner Karriere folgendermaßen: „[I]
began to be in fashion in the City, as a Master, and had my hands full of professional business
of all kinds with scholars at both ends of the town, Composition, & public playing.“ Zit. aus
den Memoirs in: Olleson 2012, The Journals and Letters of Susan Burney, S. [5]. This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 50 Melanie Unseld mit Esther, geb. Sleep: Die älteste Tochter, die Cembalistin Esther (genannt:
Hetty) Burney47 heiratete ihren Cousin, den Geiger und Cembalisten Charles
Rousseau Burney, beide waren professionell musikalisch tätig; die zweite
Tochter, Frances (genannt Fanny), war höchst erfolgreiche Schriftstellerin
und Dramatikerin (zunächst pseudonym veröffentlichend).48 Auch die dritte
Burney-Tochter, Susanna Elisabeth, war schreibend tätig, allerdings ohne zu
veröffentlichen.49 Und auch die Söhne waren literarisch tätig: James Burney
war Reiseschriftsteller (und Admiral50), Charles Burney d. J. war Lehrer, Intel
lektueller und bibliophiler Sammler: „His collection included about 13,500
printed books and manuscript volumes, nearly 400 volumes of notes, cut
tings, playbills and other material related to the history of the English thea
tre, and about 700 volumes of newspapers spanning the 17th, 18th, and 19th
centuries.“51 – Betrachtet man das professionelle Spektrum der Burneys im
Ausschnitt dieser Generationen, wäre es dann, statt von einer Musiker-Fa
milie zu sprechen, ebenso plausibel, von einer Literaten-Familie oder auch
einer Maler-Familie zu schreiben? In der Tat – was freilich nicht gegen den
Begriff der Musikerfamilie spricht, sondern ausschließlich gegen die Veren
gung der Vorstellung solcher als Musikerfamilien bezeichneten familiären
Konstellationen auf eine (Kunst)Sparte oder Profession. Zugleich sind die drei Burney-Töchter gute Beispiele für Wesentliches in
Bezug auf Ausbildung, Partizipation von Frauen und die künstlerischen Pro
fessionen im Rahmen einer Musikerfamilie im England des 18. Jahrhunderts:
Burney, der Vater, kannte, wie erwähnt, selbst das Prinzip, die nachfolgende
Generation in einer Musikerfamilie auszubilden. Wie gestaltet er dies nun
für seine Töchter? 52 1767 heiratete Burney in zweiter Ehe die Witwe Elizabeth Allen, die selbst drei Kinder mit
in die Ehe brachte. Zwei weitere gemeinsame Kinder folgten 1768 und 1772.
53 Brief Charles Burney an Denis Diderot, 10.10.1771, zit. nach Olleson 2012, The Journals and
Letters of Susan Burney, S. 13.
54 Todd Gilman, „The Evolution of Charles Burney’s Musical Taste Between 1770 and 1811“,
Forschungsbericht, https://www.mcgill.ca/burneycentre/files/burneycentre/2014_fellowship_
report_todd_gilman_0.pdf (abgerufen am 01.04.2020). Brief Fanny Burney an Samuel Crisp,
02.03.1774, in: The Early Journals and Letters of Fanny Burney, Bd. 2, 1774–1777, hrsg. von Lars E.
Troide, Oxford 1990, S. 77–78. 52 1767 heiratete Burney in zweiter Ehe die Witwe Elizabeth Allen, die selbst drei Kinder mit
in die Ehe brachte. Zwei weitere gemeinsame Kinder folgten 1768 und 1772. IV. Familie Burney. Ein Beispiel für künsteübergreifende Berufsvielfalt,
Arbeitserziehung und Professionalisierungs(un)möglichkeiten Denn obwohl sie als das musi
kalisch begabteste Kind galt, obwohl auch ihr jene „Arbeitserziehung“ zuteil
wurde und sie als Musikerin wie als Diskussionspartnerin zu einem festen
Bestandteil des Londoner Salons ihres Vaters gehörte, erwuchs ihr daraus
nicht die Möglichkeit einer professionellen Entfaltung. Das berufliche Um
feld ihres Ehemannes Molesworth Philips, einem Offizier der Royal Navy,
ließen keine professionellen Ambitionen der gebildeten und hochmusika
lischen Susan Burney zu. Ihre musikalischen Aktivitäten nach ihrer Heirat
lassen gleichwohl erkennen, dass die Basis ihrer musikalischen Ausbildung
profund war: Sowohl während ihrer Besuche in London, als auch in ihrem
Haus in Mickleham (Surrey), wohin die junge Familie nach der Hochzeit ge
zogen war, spielte sie als Cembalistin mit professionellen Musikern (etwa
dem aus der Schweiz stammenden Geiger Scheener55 oder Johann Peter
Salomon), die sie regelmäßig besuchten. Sie führte damit nach der Heirat
fort, was sie aus dem ‚Haus‘ ihres Vaters gewohnt war, war aber zugleich
abgeschnitten von der Idee der musiko-literarischen Professionalität, die
bei Burney (Vater) und ihren Schwestern Hetty und Frances weiter existier
te. Obwohl weiterhin Teil der familialen Künste-Netzwerke (die wiederum
weitere Musikerfamilien mit einschloss und auf diese Weise ebenso breit
wie tief in die Londoner Musik- und Theaterkultur verwoben war), verlor
Susan Burney durch ihre Rolle als Offiziersgattin zwar nicht ihre musiko-lite
raten Kompetenzen, aber doch den Status des arbeitsteilig-professionellen
Handelns innerhalb des ‚Hauses‘ Burney. Denn dass sie weiterhin Konzerte
und musikalische Salons mit deren gängigen konzertähnlichen Musikdar
bietungen mit großer Kenntnis rezipieren und kritisieren konnte, zeigen ihre
Tagebuch-Eintragungen immer wieder. Über das Konzert der französischen
Geigerin Louise Gautherot in den Hanover Square Rooms etwa schrieb sie: Der Fall von Susan Burney wiederum zeigt, dass es für Frauen aus Musiker
familien für die Frage weiterer professioneller Handlungsspielräume exis
tentiell war, welchen Partner sie heirateten. Denn obwohl sie als das musi
kalisch begabteste Kind galt, obwohl auch ihr jene „Arbeitserziehung“ zuteil
wurde und sie als Musikerin wie als Diskussionspartnerin zu einem festen
Bestandteil des Londoner Salons ihres Vaters gehörte, erwuchs ihr daraus
nicht die Möglichkeit einer professionellen Entfaltung. Das berufliche Um
feld ihres Ehemannes Molesworth Philips, einem Offizier der Royal Navy,
ließen keine professionellen Ambitionen der gebildeten und hochmusika
lischen Susan Burney zu. 55 Auch „Shanere“ oder „Sheneer“. Weitere Informationen s. Olleson 2012, The Journals and
Letters of Susan Burney, S. 200, FN 4. IV. Familie Burney. Ein Beispiel für künsteübergreifende Berufsvielfalt,
Arbeitserziehung und Professionalisierungs(un)möglichkeiten In Anbetracht seiner vielfältigen Tätigkeiten und zudem This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 Musikerfamilien als Ort der Ausbildung und Professionalisierung 51 zwischen 1762 und 1767 Witwer von sechs Kindern,52 konnte er, wie er 1771
an Denis Diderot schrieb, nicht viel Zeit für die Ausbildung erübrigen. Über
seine Tochter Susan berichtete Burney: „She […] is very fond of Music, has
a good Ear, & talents Which I have not had leisure to cultivate.“53 Dennoch
zeigen die Tagebücher und Briefe der Schwestern, dass alle eine profunde
Musikausbildung erhalten hatten und zwar nicht primär durch den Vater,
sondern – eben im Musikerfamilien-Prinzip – durch Personen, die im ‚Haus‘
Burney lebten. Dazu gehörten Kollegen des Vaters, durchreisende Musike
rinnen und Musiker, aber auch Aktivitäten wie gemeinsame Konzert- und
Opernbesuche, Austausch über Musik, musikalische Aufführungen und Un
terricht im Haus: Diese Form der literat-musikalischen „Arbeitserziehung“
legte – um nur die Kinder aus erster Ehe zu berücksichtigen – nicht zuletzt
die Grundlage für die Musikerin Hetty Burney, die Dramatikerin Fanny Bur
ney, die ‚Musikexpertin‘ Susan Burney, den Reiseschriftsteller James Burney
und den ebenso enzyklopädisch wie historisch sammelnden Wissensenthu
siasten Charles Burney d. J. Darüber hinaus ist in den Familiendokumenten
erkennbar, dass die Phase der „Arbeitserziehung“ gleichsam nahtlos in die
professionellen Praktiken überging. So wurde beispielsweise um 1775 im
Haus Burney über die Musik von Carl Philipp Emanuel Bach diskutiert – zu
diesem Zeitpunkt war Hetty Burney bereits mit Charles Rousseau Burney
verheiratet und Mutter dreier Kinder: In the mid-1770s C. P. E. Bach’s music was also a staple of the Burney household,
for in a letter of 2 March 1775 to family friend Samuel Crisp (1707–1783), Fanny Bur
ney (1752–1840) writes of entertaining the Italian soprano Lucrezia Aguiari or Agu
jari (1743–1783): „After Tea, we went into the Library, & Hetty [i.e., Esther, Charles
Burney’s daughter] was prevailed upon to play a Lesson of Bach of Berlin’s, upon
our Merlin Harpsichord. It was very sweet, & she [Aguiari] appeared to be really
much pleased with it, & spoke highly of the Taste & feeling with which she [Hetty]
played. Mr Burney sat down next. They all stared, as usual, at his performance.54 This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 52 Melanie Unseld Der Fall von Susan Burney wiederum zeigt, dass es für Frauen aus Musiker
familien für die Frage weiterer professioneller Handlungsspielräume exis
tentiell war, welchen Partner sie heirateten. IV. Familie Burney. Ein Beispiel für künsteübergreifende Berufsvielfalt,
Arbeitserziehung und Professionalisierungs(un)möglichkeiten – It is true I heard Sirmen before I had heard any great Violin Players – & now
perhaps she wd not seem so charming to me as she did in those early days […].56 Susan Burney zeigt sich hier als kenntnisreiche, kritisch abwägende und
durchaus unabhängige Kritikerin. Denn die Londoner Presse sah die Leis
tung von Gautherot weitaus positiver: Der Morning Chronicle and London
Advertiser schrieb (2. März 1789): „Madame Gautherot’s concerto was so
delicious and charming a performance, that the audience could not con
tain their raptures till the end of the respective movements“. In der London
Evening Post (26. Februar 1789) hieß es: „Madame Gautherot played on the
violin a Concerto of Viotti’s, a Parisian Composer, with amazing taste and
brilliancy. There are few professional men who can surpass her exertion“. Und die Whitehall Evening Post vom gleichen Tag hielt fest: „Madame Gau
therot’s concerto on the violin was equal to any performance on the same
instrument by the first musical master of the present times“.57 Die Konzert
berichte, die Susan Burney in ihr Tagebuch notierte, zeigen mithin nicht nur
eine sehr eigenständig kritische Haltung und eine profunde Kenntnis der
(Violin)Spielpraxis, sondern die weite Kenntnis verschiedener Interpretin
nen und Interpreten (Gautherot, Cramer, Sirmen). Susan Burney zeigt sich hier als kenntnisreiche, kritisch abwägende und
durchaus unabhängige Kritikerin. Denn die Londoner Presse sah die Leis
tung von Gautherot weitaus positiver: Der Morning Chronicle and London
Advertiser schrieb (2. März 1789): „Madame Gautherot’s concerto was so
delicious and charming a performance, that the audience could not con
tain their raptures till the end of the respective movements“. In der London
Evening Post (26. Februar 1789) hieß es: „Madame Gautherot played on the
violin a Concerto of Viotti’s, a Parisian Composer, with amazing taste and
brilliancy. There are few professional men who can surpass her exertion“. Und die Whitehall Evening Post vom gleichen Tag hielt fest: „Madame Gau
therot’s concerto on the violin was equal to any performance on the same
instrument by the first musical master of the present times“.57 Die Konzert
berichte, die Susan Burney in ihr Tagebuch notierte, zeigen mithin nicht nur
eine sehr eigenständig kritische Haltung und eine profunde Kenntnis der
(Violin)Spielpraxis, sondern die weite Kenntnis verschiedener Interpretin
nen und Interpreten (Gautherot, Cramer, Sirmen). IV. Familie Burney. Ein Beispiel für künsteübergreifende Berufsvielfalt,
Arbeitserziehung und Professionalisierungs(un)möglichkeiten Ihre musikalischen Aktivitäten nach ihrer Heirat
lassen gleichwohl erkennen, dass die Basis ihrer musikalischen Ausbildung
profund war: Sowohl während ihrer Besuche in London, als auch in ihrem
Haus in Mickleham (Surrey), wohin die junge Familie nach der Hochzeit ge
zogen war, spielte sie als Cembalistin mit professionellen Musikern (etwa
dem aus der Schweiz stammenden Geiger Scheener55 oder Johann Peter
Salomon), die sie regelmäßig besuchten. Sie führte damit nach der Heirat
fort, was sie aus dem ‚Haus‘ ihres Vaters gewohnt war, war aber zugleich
abgeschnitten von der Idee der musiko-literarischen Professionalität, die
bei Burney (Vater) und ihren Schwestern Hetty und Frances weiter existier
te. Obwohl weiterhin Teil der familialen Künste-Netzwerke (die wiederum
weitere Musikerfamilien mit einschloss und auf diese Weise ebenso breit
wie tief in die Londoner Musik- und Theaterkultur verwoben war), verlor
Susan Burney durch ihre Rolle als Offiziersgattin zwar nicht ihre musiko-lite
raten Kompetenzen, aber doch den Status des arbeitsteilig-professionellen
Handelns innerhalb des ‚Hauses‘ Burney. Denn dass sie weiterhin Konzerte
und musikalische Salons mit deren gängigen konzertähnlichen Musikdar
bietungen mit großer Kenntnis rezipieren und kritisieren konnte, zeigen ihre
Tagebuch-Eintragungen immer wieder. Über das Konzert der französischen
Geigerin Louise Gautherot in den Hanover Square Rooms etwa schrieb sie: Made Gautherot played a Violin Duet with Cramer – I was glad to see she was
supposed to do herself credit in it – but for my own part it seemed a great disad
vantage to her – she executed all the passages – but it was wth evident Labour –
nothing was distinct – nothing clear – the powerful tone, the freedom – decision,
& most of all the perfect facility wth wch Cramer repeated every passage after her, This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 Musikerfamilien als Ort der Ausbildung und Professionalisierung 53 disgraced all that she attempted, & betrayed the weakness & inferiority, wch tho’
certainly it was no wonder to perceive wd have appeared far less glaring hat she
not subjected herself to so close & immediate a comparison. She has labored
infinitely there can be no doubt to attain such rapid execution, & so much preci
sion – but in the most valuable points is I believe very inferiour to Sirmen indeed. 56 Tagebucheintrag Susan Burney, 25.04.–01.05.1789, zit. nach Olleson 2012, The Journals and
Letters of Susan Burney, S. 226–227 (Hervorheb. orig.).
57 Zit. nach Volker Timmermann, Art. „Gautherot, Louise, geb. Deschamps“, in: Europäische
Instrumentalistinnen des 18. und 19. Jahrhunderts, 2008, https://www.sophie-drinker-institut.
de/gautherot-louise (abgerufen am 01.04.2020); s. dort auch weitere Informationen zu Gau
therot sowie das Konzertprogramm des von Susan Burney kommentierten Konzerts. 59 Ollsen 2012, The Journals and Letters of Susan Burney, S. 14 (Hervorheb. M. U.). Zu beden
ken ist bei dem Begriff ‚Sekretär‘, dass der Beruf des Sekretärs / der Sekretärin im 19. Jahr
hundert eine starke Feminisierung des Berufsbildes durchlief, insbesondere aufgrund der
geringen Aufstiegschancen. In der heutigen Begriffsverwendung spielt diese Feminisierung
eine starke Rolle, was bei der Verwendung des Begriffs für die vorindustrielle Berufssituation
(vgl. hierfür etwa den Eintrag „Sekretär“ im Grimmschen Wörterbuch: http://woerterbuchnetz.
de/cgi-bin/WBNetz/wbgui_py?sigle=DWB&mode=Vernetzung&lemid=GS25724#XGS25724, abge
rufen am 01.04.2020) mitberücksichtigt werden muss. 58 Dewe und Otto 42011, Art. „Profession“, S. 1131. 60 Es wäre weiterer Überlegungen wert, warum sich das Konzept der Arbeitserziehung ins
besondere in den Musikerfamilien erfolgreich behaupten konnte, trotz einer allgemeinen
Tendenz zur Institutionalisierung der Erziehung, die für das so genannte „pädagogische Jahr
hundert“ zu verzeichnen ist. IV. Familie Burney. Ein Beispiel für künsteübergreifende Berufsvielfalt,
Arbeitserziehung und Professionalisierungs(un)möglichkeiten Ist mithin die schiere Wahrnehmung der künstlerischen Tätigkeiten und
ihrer professionell-familialen Settings der Burney-Schwestern bereits eine
Herausforderung, ist die historiographische Darstellung eine zweite Hürde,
insbesondere dann, wenn die Idee von Familie als Lebenszusammenhang
außer Acht bleibt: die arbeitsteilig, künsteüberschreitend, innerfamiliäre
Ausbildung hin zur Professionalität, letztere im Sinne eines „habitualisierten,
szenisch-situativ zum Ausdruck kommenden Agierens unter typischerweise This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 Melanie Unseld 54 sowohl hochkomplexen wie auch paradoxen Handlungsanforderungen“.58
Im Fall der Burney-Familie betrifft das insbesondere diejenigen Familien
mitglieder, die in den großen Publikationsprojekten von Charles Burney mit
ihrer Expertise eingebunden waren, insbesondere Fanny und Susan Burney,
beide zu diesem Zeitpunkt noch unverheiratete, junge Frauen in ihren frü
hen zwanziger Jahren: sowohl hochkomplexen wie auch paradoxen Handlungsanforderungen“.58
Im Fall der Burney-Familie betrifft das insbesondere diejenigen Familien
mitglieder, die in den großen Publikationsprojekten von Charles Burney mit
ihrer Expertise eingebunden waren, insbesondere Fanny und Susan Burney,
beide zu diesem Zeitpunkt noch unverheiratete, junge Frauen in ihren frü
hen zwanziger Jahren: By the mid-1770s, Charles Burney’s reputation was at its height. Following the
success of the two volumes of Tours and an intensive period of writing in which
both Susan and Fanny were employed as secretaries and assistants, he publis
hed the first volume of the General History of Music in January 1776 […]; the se
cond volume would follow in 1782 and the third and fourth in 1789.59 Das Dilemma, wie ein solcher Arbeitszusammenhang adäquat bezeichnet
werden könnte, ist offensichtlich. Bezeichnet man Fanny und Susan Bur
ney allerdings als Sekretärinnen ihres Vaters, wird das Missverständnis über
Musikerfamilien deutlich: Im Sinne des ‚Hauses‘ war es selbstverständlich,
dass ein Großprojekt wie das Schreiben einer Musikgeschichte ein ökono
misches Unterfangen war und dabei alle zur Verfügung stehenden Kräfte
des Hauses Burney mitarbeiteten. Doch ‚Sekretärin‘ ist eine moderne Be
rufsbezeichnung, noch dazu eine, die seit dem 19. Jahrhundert zutiefst in
die Wirrungen abwertender Gender-Hierarchien verstrickt wurde. Wie aber
soll man insbesondere Susan Burney, verh. Philips, alternativ bezeichnen? Der oben verwendete Begriff ‚Musikexpertin‘ bringt immerhin ihre Expertise
und Professionalität zum Ausdruck, ohne dass sich daraus der (moderne)
Anspruch ableitet, die Bezeichnung ‚Musikexpertin‘ als Profession oder gar
als Beruf zu verstehen. This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 Musikerfamilien als Ort der Ausbildung und Professionalisierung 55 V. Verlorene Gewissheiten und neue Einblicke. Ein Ausblick Nimmt man Musikerfamilien als ökonomisch-moralische Einheit wahr, nicht
als Abbild einer (früh- / vor-)bürgerlichen Idealform von Kernfamilie, erwei
tert sich das Wissen über Produktionsbedingungen von Musik erheblich. Verloren gehen dabei Gewissheiten über die genaue Grenzziehung zwischen
Berufsfeldern und auch über jene implizit angenommene Grenze zwischen
„family affair“ und „profession“. Erkennbar wird, dass zwischen Profession
und Handeln als Experte bzw. Expertin dort ebensowenig eine Grenze gezo
gen werden kann wie zwischen ‚öffentlichem‘ und ‚privatem‘ Handeln, dass
gerade das gemeinschaftliche Handeln und Arbeiten im ‚Haus‘ gegen eine
solche Distinktion spricht. Deutlicher zum Vorschein treten allerdings die
Bedingungen musikbezogenen Handelns im 18. Jahrhundert mitsamt ihren
gelebten und immer wieder neu ausgestalteten Übergängen zu anderen
Künsten, anderen Professionen. Es werden dabei Spezifika der Professionalisierung wie die der ‚Arbeits
erziehung‘ sichtbar, die wesentliche Bedeutung vor der Gründung von Mu
sikausbildungsstätten und der damit einhergehenden Akademisierung hat
ten.60 Prägnanter tritt auch hervor, was im 18. Jahrhundert unter ‚(Musik‑)
Beruf‘, besser: Musik-Professionen, verstanden wurde. Das ‚Haus‘ als ökono
mische Einheit ist Basis für eine Vielfalt an Tätigkeiten, die – je nach ökono
mischem Wert und des mit dem Haus (bzw. Namen) verbundenen kulturel
len Kapitals – zur Stabilisierung des Hauses dienen kann. Dass hierbei auch
jene Grenzen überschritten wurden, die die akademischen Disziplinen der
Gegenwart ausschließlich mit dem Impetus der Interdisziplinarität über
schreiten, etwa Bildende Kunst, Literatur und Musik, war in diesem Modell
gang und gäbe. Das Beispiel der ‚Musikerfamilie‘ Burney ließe sich entspre
chend auch als ‚Malerfamilie‘ Burney oder auch als ‚Literatenfamilie‘ Burney
beschreiben. Auf diese Weise wird auch besser erklärlich, woher ein Karika
turist wie Edward Burney seine profunden musikalisch-musikhistorischen
Kenntnisse hatte, und wie das Schreiben über Reisen, über Geschichte, über
Musik u. a. m. miteinander zusammenhängen, warum Reiseliteratur, Thea
terdichtung, Musikgeschichtsschreibung und -kritik in der Burney-Familie This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 Melanie Unseld 56 nicht unverbunden nebeneinander stehen. Schließlich kann mit einem
Modell von Musikerfamilie als ökonomisch-moralischer Einheit die Berück
sichtigung von Frauen adäquater gelingen. Das Beispiel der Burney-Töch
ter konnte zeigen, dass sie in die Produktions- und Rezeptionsbedingungen
von Musik ebenso einbezogen waren wie ihre männlichen Altersgenossen,
auch wenn ihnen – etwa durch Heirat – Handlungsspielräume beschränkt
wurden. Um all jenes historiographisch wahrnehmbar zu machen, was jen
seits der Beschränkungen für Musikerinnen (und andere Künstlerinnen) des
18. Jahrhunderts möglich war zu rezipieren und zu produzieren, kann das
Modell der Familie als ökonomisch-moralische Einheit hilfreich sein. V. Verlorene Gewissheiten und neue Einblicke. Ein Ausblick So ist
das Modell nicht zuletzt dazu geeignet, angemessenere Begrifflichkeiten für
die hier vorhandene, hohe Expertise zu finden, die sich – etwa im Haus Bur
ney – (auch) in den Töchtern zeigte. nicht unverbunden nebeneinander stehen. Schließlich kann mit einem
Modell von Musikerfamilie als ökonomisch-moralischer Einheit die Berück
sichtigung von Frauen adäquater gelingen. Das Beispiel der Burney-Töch
ter konnte zeigen, dass sie in die Produktions- und Rezeptionsbedingungen
von Musik ebenso einbezogen waren wie ihre männlichen Altersgenossen,
auch wenn ihnen – etwa durch Heirat – Handlungsspielräume beschränkt
wurden. Um all jenes historiographisch wahrnehmbar zu machen, was jen
seits der Beschränkungen für Musikerinnen (und andere Künstlerinnen) des
18. Jahrhunderts möglich war zu rezipieren und zu produzieren, kann das
Modell der Familie als ökonomisch-moralische Einheit hilfreich sein. So ist
das Modell nicht zuletzt dazu geeignet, angemessenere Begrifflichkeiten für
die hier vorhandene, hohe Expertise zu finden, die sich – etwa im Haus Bur
ney – (auch) in den Töchtern zeigte. This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115 This work is available under CC BY-NC-SA 4.0; DOI: https://doi.org/10.25366/2020.115
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https://openalex.org/W2794473010
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https://journal.ugm.ac.id/bkm/article/download/23904/20534
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Indonesian
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Hubungan kualitas udara hasil pembakaran avtur dengan gangguan fungsi paru pekerja di bandara Supadio Pontianak
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Berita kedokteran masyarakat/Berita Kedokteran Masyarakat
| 2,017
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cc-by-sa
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Dikirim: 11 April 2017
Diterbitkan: 1 Juni 2017 1 Departemen Perilaku Kesehatan, Lingkungan, dan Kedokteran Sosial, Fakultas Kedokteran, Universitas Gadjah Mada
(Email:zuzun.nazila@gmail.com)
2 Departemen Kedokteran Keluarga, Komunitas dan Bioetika, Fakultas Kedokteran, Universitas Gadjah Mada Zuzun Nazila & Adi Heru Sutomo
1
2 Zuzun Nazila & Adi Heru Sutomo
1
2 Zuzun Nazila & Adi Heru Sutomo
1
2 g
2 Departemen Kedokteran Keluarga, Komunitas dan Bioetika, Fakultas Kedokteran, Universitas Gadjah Mada Berita Kedokteran Masyarakat
(BKM Journal of Community Medicine and Public Health) Berita Kedokteran Masyarakat
(BKM Journal of Community Medicine and Public Health) Volume 33 Nomor 6
Halaman 277-284 Abstract Purpose: This study aimed to determine the relationship of air quality
aviation fuel combustion products with impaired lung function in workers in
Pontianak Supadio International Airport. Methods: This research was an
observational study using a cross-sectional approach involving 60 people. Sampling technique used total sampling. The statistical analysis used Fisher
exact and logistic regression tests, with significance level of p < 0.05 and 95%
confidence interval and odds ratios. Results: Based on bivariate analysis from
36 samples the results were: NO2 variable (p = 0.04), SO2 (p = 0.15), dust (p =
0.25), age (p = 0.20), working period (p = 0.08), nutritional status (p = 0.09),
and smoking habits (p = 0.03). Conclusion: There is significant correlation
between the variables of NO2
and smoking habits with impaired lung
function, while the variables: SO2, dust, age, working period and nutritional
status had no significant correlation with lung function disorders. Keywords: air quality; the burning aircraft fuel; impaired lung function 1 Departemen Perilaku Kesehatan, Lingkungan, dan Kedokteran Sosial, Fakultas Kedokteran, Universitas Gadjah Mada
(Email:zuzun.nazila@gmail.com)
2 Departemen Kedokteran Keluarga, Komunitas dan Bioetika, Fakultas Kedokteran, Universitas Gadjah Mada 277 Berita Kedokteran Masyarakat, Volume 33 No. 6 Tahun 2017 Berita Kedokteran Masyarakat, Volume 33 No. 6 Tahun 2017 masalah
polusi
udara
lokal
yang
berpengaruh
terhadap orang yang tinggal di sekitar bandara. Risiko
terbesar kemungkinan dialami pekerja yang mendapat
paparan secara terus menerus. Emisi pesawat adalah
salah satu faktor yang diduga menyebabkan gangguan
kesehatan terutama pada saluran pernapasan (1). PENDAHULUAN Frekuensi penerbangan di dalam negeri saat ini
meningkat
seiring
pertambahan
jumlah
operasi
maskapai penerbangan yang beroperasi baik domestik
dan
internasional. Kepadatan
lalu
lintas
udara
meningkat, hal tersebut tentu semakin meningkatkan
penggunaan
bahan
bakar
pesawat
dan
semakin
meningkat pula polutan udara yang dilepaskan hasil
dari
pembakaran
tersebut
yang
dikhawatirkan
memiliki dampak merugikan terhadap kesehatan (1). Selain faktor pekerjaan, fungsi paru dapat dipe-
ngaruhi oleh usia, jenis kelamin, ukuran paru, kelom-
pok etnik, kebiasaan merokok, kebiasaan latihan (9). Selain itu gangguan fungsi paru dipengaruhi oleh
beberapa faktor, antara lain umur, jenis kelamin,
status gizi, riwayat penyakit, kebiasaan merokok, masa
kerja dan penggunaan alat pelindung diri (10). Konsentrasi
polutan
ambien
di
bandara
ber-
hubungan dengan aktivitas landing take off (LTO)
pesawat (2). Penelitian ini fokus pada aktivitas pesawat
udara pada fase LTO karena terkait dengan kualitas
udara lokal di bandar udara mengingat ada jeda waktu
yang cukup lama antara pesawat yang akan take off
dari tempat parkir menuju landasan pacu dan pesawat
yang landing dari landasan pacu menuju tempat parkir
(3). Saat jeda waktu tersebut mesin pesawat dalam
keadaan hidup dan terjadi pembakaran avtur pesawat
sedangkan posisi pesawat masih berada di tempat
parkir atau sekitar landasan pacu, dimana banyak
pekerja ground handling yang berada di sekitar tempat
tersebut
yang
setiap
hari
bekerja
dengan
tidak
menggunakan alat pelindung diri (APD). Pekerja di
bandara
yang
berisiko
terpapar
adalah
baggage
handlers,
catering
drivers,
cleaning
staff,
airside
security, landside security (4). Penelitian mengenai polusi udara yang bersumber
dari
transportasi
udara
belum
banyak
diketahui,
apalagi jika dikaitkan dengan kesehatan pekerja di
sekitar tempat terpaparnya polusi. Oleh karena itu,
penelitian ini penting untuk dilakukan. Penelitian ini
dilakukan dengan tujuan untuk mengetahui hubungan
kualitas
udara
hasil
pembakaran
avtur
dengan
gangguan
fungsi
paru
pada
pekerja
di
bandara
internasional Supadio Pontianak. METODE Penelitian ini merupakan penelitian observasional
analitik dengan desain cross sectional. Studi cross
sectional merupakan studi yang dilakukan dengan
menelaah sebab akibat pada waktu yang bersamaan
(11). Besar sampel penelitian sebanyak 36 orang yang
bekerja sebagai ground handling di bandara interna-
sional Supadio Pontianak. Kriteria eksklusi meliputi
mempunyai riwayat penyakit paru, sedang sakit infeksi
saluran pernapasan akut (ISPA), merokok berat, dan
tidak berada di lokasi penelitian ketika penelitian
dilaksanakan. Penelitian ini dilaksanakan pada bulan
Februari 2017. Dokumen aviation and emission a prime (2005) yang
dikeluarkan
Federal
Aviation
Administration
(FAA)
menyatakan
mesin
pesawat
terbang menghasilkan
emisi gas buang
yang dapat berpotensi mencemari
udara, seperti nitrogen oksida (NOx), sulfur oksida
(SOx), karbon dioksida (CO2), uap air (H2O), karbon
monoksida
(CO)
(5). Mesin pesawat terbang akan
menghasilkan emisi yang dilepaskan ke udara meliputi
CO, CO2, H2O, SO2, NO2, HC (6). Variabel bebas adalah kualitas udara yang diukur
dari
paparan SO2 dan NO2, variabel terikat yaitu
gangguan
fungsi
paru,
variabel
pengganggu
yaitu
umur, masa kerja, status gizi, kebiasaan merokok dan
debu. Instrumen dalam penelitian ini menggunakan
air sampler impinger merk inscien pro US-1012 untuk
mengukur
kualitas
udara
(paparan
SO2,
NO2),
sensidyne 80570 nephelometer untuk mengukur kadar
debu, spirometer dengan merk spiro analyzer ST-75
untuk
mengukur
kapasitas paru, timbangan berat
badan merk omron HN 383 untuk mengetahui berat
badan, meteran tinggi badan merk ZT 120 untuk
mengetahui tinggi badan, dan lembar isian data untuk
memperoleh informasi tambahan yang meliputi umur, Organ
pernapasan
merupakan
bagian
pertama
yang berhubungan dengan berbagai bahan pencemar
udara yang dapat berpengaruh terhadap sistem organ
(7). Beberapa
polutan
di
udara
yang
dapat
menyebabkan gangguan pernapasan adalah SO2, NO2,
dan
partikel
debu. Polutan
ini
dapat
mengiritasi
saluran pernapasan, menyebabkan gangguan fungsi
paru dan masalah pernapasan (8). Bandar
udara
internasional
Supadio
Pontianak
yang dikelola oleh PT. Angkasa Pura II merupakan
bandar
udara yang mempunyai tingkat kesibukan
cukup tinggi, dengan rata-rata jumlah pesawat terbang
yang
beroperasi
sekitar
40
pesawat
per
hari. Peningkatan
frekuensi
penerbangan
menimbulkan 278 Berita Kedokteran Masyarakat, Volume 33 No. 6 Tahun 2017 masa kerja, kebiasaan merokok, riwayat penyakit, dan
riwayat pekerjaan. Sedangkan OR antara variabel debu dengan gangguan
fungsi paru menunjukkan nilai 1,3. Artinya secara
klinis debu tidak berpeluang menimbulkan gangguan
fungsi paru karena nilai OR < 1,5 yaitu 1,3. Analisis data dilakukan dengan analisis univariat,
analisis bivariat dengan uji statistik menggunakan chi
square dan fisher, serta analisis multivariat meng-
gunakan uji regresi logistik. HASIL Tabel
1
menunjukkan
bahwa
hasil
pengukuran
kualitas udara yang dilakukan di sekitar landasan
apron
yaitu
pada
titik
A
dengan
titik
koordinat
00o08’4921‘ ‘S-109o24 ‘1403 ‘ ‘E, titik B dengan titik
koordinat 00o08’5103’’S 109o24 1489 ‘ ‘E dan titik C
dengan titik koordinat 00o08’4321 ‘ ‘S 109o24 ‘1403 ‘ ‘E
tidak terdapat hasil yang melebihi ambang batas baku
mutu udara ambien menurut PP Nomor 41 Tahun
1999. Tabel 2. Odds ratio variabel penelitian
Variabel
Gangguan Fungsi Paru
OR
CI 95%
Ya
Tidak
%
%
SO2
NO2
Debu
Umur
30 tahun
≤30 tahun
Masa Kerja
10 tahun
≤10 tahun
Status Gizi
Tidak normal
Normal
Merokok
Merokok sedang
Merokok ringan
Tidak merokok
19,4
19,4
19,4
28,6
6,7
35,7
9,1
31,6
5,9
41,7
18,2
0
80,6
80,6
80,6
71,4
93,3
64,3
90,9
68,4
94,1
58,3
81,8
100
1,1
1,2
1,3
5,6
5,6
7,4
3
1,8
0,97-1,17
1,00-1,39*
0,84-1,89
0,54-275,59
0,70-65,68
0,71-360,38
0,69-13,89
0,39-8,78
Keterangan: *signifikan (p<0,05) Tabel 2. Odds ratio variabel penelitian Tabel 1. Hasil pengukuran kualitas udara
Parameter Satuan Baku
Mutu
Hasil Pengukuran
Kualitas Udara
Rerata
Titik A Titik B Titik C
SO2
µg/Nm3 900*
301,90 310,41 281,07 281,07
NO2
µg/Nm3 400*
170,34 181,11 169,14 169,14
Debu
µg/Nm3 90*
33,96
34,55
28,37
28,37 Tabel 1. Hasil pengukuran kualitas udara Paparan SO2, NO2 dan debu paling banyak terdapat
pada
titik
B. Hal
ini
disebabkan
karena
titik
B
merupakan
tempat yang dekat dengan lalu lintas
pesawat yang masuk dan keluar dari landasan pacu. Tabel 2 menjelaskan nilai p 0,08 (p > 0,05) yang
berarti secara statistik tidak terdapat hubungan yang
bermakna antara masa kerja dengan gangguan fungsi
paru. Sedangkan secara klinis berdasarkan perhitu-
ngan OR menunjukkan bahwa pekerja dengan masa
kerja > 10 tahun memiliki peluang 5,6 kali lebih besar
mengalami
gangguan
fungsi
paru
dibandingkan
dengan pekerja dengan masa kerja ≤ 10 tahun. Tabel 2 menunjukkan bahwa paparan SO2 dengan
gangguan fungsi paru menunjukkan hubungan yang
tidak bermakna dengan nilai p 0,15 (p > 0,05). Artinya
secara statistik tidak terdapat hubungan antara SO2
dengan gangguan fungsi paru pekerja. Hasil perhitu-
ngan OR antara variabel SO2 dengan gangguan fungsi
paru didapatkan 1.1. Artinya secara klinis SO2 tidak
berpeluang
menimbulkan
gangguan
fungsi
paru
karena nilai OR < 1,5 yaitu 1,1. METODE Keseluruhan tes meng-
gunakan odd ratio (OR), dengan confidence interval (CI)
95% dan tingkat kemaknaan p 0,05 (12,13). Penelitian
ini telah mendapat persetujuan dari komite etik FK
UGM. Tabel 2 menunjukkan secara statistik tidak terdapat
hubungan
yang
bermakna
antara
variabel
umur
dengan gangguan fungsi paru dengan nilai p 0,20. Secara
klinis
berdasarkan
perhitungan
OR
me-
nunjukkan bahwa pekerja yang berusia > 30 tahun
memiliki
peluang
5,6
kali
lebih besar mengalami
gangguan fungsi paru dibandingkan dengan pekerja
yang berusia ≤ 30 tahun. BAHASAN Polutan udara yang dihasilkan pesawat terbang dan
kendaraan bermotor adalah pada kadar polutan yang
dihasilkan (14). Mesin pesawat terbang akan meng-
hasilkan emisi yang dilepaskan ke udara meliputi CO,
CO2, H2O, SO2, NO2, HC (6). Pencemaran udara menim-
bulkan dampak buruk bagi kesehatan manusia, pe-
nyakit yang timbul antara lain infeksi saluran perna-
fasan atas, gangguan fungsi paru, hipertensi, jantung,
kanker dan lain sebagainya (15). Polutan di udara yang
dapat mengiritasi saluran pernapasan dan gangguan
fungsi paru adalah SO2, NO2 dan debu (8). Secara klinis pekerja yang berumur lebih dari 30
tahun berisiko 5,6 kali memiliki gangguan fungsi paru
dibanding pekerja yang berusia kurang dari 30 tahun,
namun uji statistik menunjukkan umur tidak memiliki
hubungan yang bermakna dengan gangguan fungsi
paru (p 0,20). Hasil ini sejalan dengan penelitian
Wulandari, dkk, Zarandi, dkk dan Anes, dkk yang
menyatakan tidak ada hubungan signifikan antara
umur dengan gangguan fungsi paru (24-26). Uji statistik menunjukkan tidak terdapat hubungan
yang bermakna antara paparan SO2 dengan gangguan
fungsi paru (p 0,15). Hasil ini sejalan dengan penelitian
Husaini bahwa tidak ada hubungan yang bermakna
antara pajanan SO2 dengan gangguan fungsi paru
restriktif
(16). Hal
tersebut diperkuat oleh Damri
bahwa
paparan
SO2
tidak
ada
hubungan
yang
signifikan terhadap kejadian penyakit pekerja parkir
mall dengan nilai p 0,122 (17). Perubahan usia berpengaruh terhadap perubahan
organ tubuh. Fungsi faal paru meningkat pada usia
22-30 tahun, dan menurun secara perlahan sesuai pe-
rubahan
usia
(22,27). Umur
merupakan
variabel
penting dalam gangguan fungsi paru. Semakin ber-
tambah umur disertai kondisi lingkungan buruk, maka
kemungkinan
terkena
suatu
penyakit
penurunan
fungsi paru menjadi lebih besar. Secara statistik umur
tidak memengaruhi kapasitas paru pekerja. Hal ini bisa
disebabkan karena pola hidup yang sehat dan status
gizi yang baik, bisa juga dipengaruhi faktor lain seperti
jenis kelamin, berat badan, tinggi badan dan kebiasaan
olah raga (28). Hasil penelitian menyatakan tidak ada hubungan
yang bermakna antara paparan SO2 dengan gangguan
fungsi paru, karena paparan SO2 yang diperoleh tidak
melebihi nilai ambang batas (NAB) dan masa kerja
pegawai yang kurang dari 10 tahun sehingga efek dari
paparan SO2 yang terhirup saat bekerja belum tampak. Hasil uji statistik menunjukkan tidak ada hubungan
yang bermakna antara masa kerja dengan gangguan
fungsi paru. Hasil ini sejalan dengan penelitian Draid,
dkk dan Rahmansyah yang menyatakan bahwa masa
kerja dengan gangguan fungsi paru tidak memiliki
hubungan bermakna (29,30). HASIL Tabel 3 Model regresi logistik
Variabel
OR
SE
Z
p
95% CI
NO2
SO2
Umur
Masa
Kerja
Status Gizi
1,529
0,901
103,3
0,088
14,64
0,381
0,092
254,3
0,186
21,39
1,70
-1,02
1,88
-1.15
1,84
0,089
0,308
0,059
0,249
0,066
0,937924-2,495697
0,737333-1,101104
0,831526-12851,87
0,001448-5,443808
0,836566-256,4489
N
Prob chi2
Pseudo R2
36
0,0185
0,3827 Tabel
2
juga
menjelaskan
terdapat
hubungan
bermakna
antara
paparan
NO2
dengan
gangguan
fungsi paru dengan nilai p 0,04 (p < 0,05). Hasil
perhitungan OR antara variabel NO2 dengan gangguan
fungsi paru menunjukkan nilai 1,2. Artinya secara
klinis NO2 tidak berpeluang menimbulkan gangguan
fungsi paru karena nilai OR < 1,5 yaitu 1,2. Tabel
3
menunjukkan
analisis
regresi
logistik. Penelitian ini menemukan hubungan signifikan secara
bersama-sama antara variabel NO2, SO2, umur, masa
kerja dan status gizi dengan gangguan fungsi paru Tabel 2 menunjukkan secara statistik tidak terdapat
hubungan
yang
bermakna
antara
paparan
debu
dengan gangguan fungsi paru dengan nilai p 0,25. 279 Berita Kedokteran Masyarakat, Volume 33 No. 6 Tahun 2017 dengan p 0,0185. Namun jika dilihat dari masing
masing
variabel
tidak
terdapat
hubungan
yang
signifikan dengan gangguan fungsi paru (NO2 p 0,089,
SO2 p 0,308, Umur p 0,059, masa kerja p 0,249 dan
status gizi p 0,066). Ketika keberadaan faktor itu secara
bersama-sama maka variabel NO2, SO2, umur, masa
kerja dan status gizi berkontribusi terhadap gangguan
fungsi paru sebesar 38% dan sebesar 62% dipengaruhi
oleh variabel lain yang tidak diteliti. menerus maka akan mengakibatkan gangguan fungsi
paru. Hal ini diperkuat oleh penelitian Ionel, dkk. bahwa jumlah paparan NO2 yang berbanding lurus
dengan
kepadatan lalu lintas pesawat di bandara
Romania (20). Paparan polusi di bandara berhubungan
dengan
peningkatan
risiko
terhadap
penyakit
pernapasan dan kardiovaskuler (21). Selain paparan di lingkungan kerja kapasitas paru
dipengaruhi
oleh
anatomi
tubuh,
jenis
kelamin,
merokok, riwayat penyakit, pencemaran udara dan
aktivitas
fisik
(22). Gangguan
fungsi
paru
juga
dipengaruhi oleh beberapa faktor, antara lain umur,
jenis kelamin, riwayat penyakit, status gizi, kebiasaan
merokok, masa kerja dan penggunaan alat pelindung
diri (23). BAHASAN Variabel yang berpotensi
menyebabkan gangguan fungsi paru adalah lamanya
seseorang terpapar polutan, artinya semakin lama
masa kerja seseorang, maka semakin lama pula waktu
paparan terhadap polutan tersebut (27). Secara statistik terdapat hubungan yang bermakna
antara paparan NO2 dengan gangguan fungsi paru
dengan
p
0,04
(< 0,05). Hasil ini sejalan dengan
penelitian Wulansari bahwa terdapat hubungan yang
bermakna
antara
paparan
NO2
dengan
gangguan
fungsi paru (18). Hal ini diperkuat dengan penelitian
Tunnicliffe bahwa ada hubungan antara paparan emisi
bahan
bakar
pesawat
dengan
gejala
gangguan
pernapasan pada pekerja di bandara (19). Paparan emisi bahan bakar pesawat berhubungan
dengan
gejala
gangguan
pernapasan
pekerja
di
bandara (1). Hal ini disebabkan karena paparan NO2 di
lingkungan kerja akan masuk ke dalam tubuh saat kita
bernapas,
jika
hal
tersebut
terjadi
secara
terus Status
gizi
tidak menunjukkan hubungan yang
bermakna dengan gangguan fungsi paru (p 0,09). Hasil
ini sejalan dengan penelitian Widjanarti dan
Pratiwi
bahwa status gizi dengan gangguan fungsi paru tidak
memiliki hubungan bermakna (31,32). 280 Berita Kedokteran Masyarakat, Volume 33 No. 6 Tahun 2017 Pekerja dengan status gizi baik, produktivitas kerja
akan baik pula, karena status gizi memengaruhi daya
tahan
dan
kemampuan
kerja. Kekurangan
gizi
menyebabkan kemampuan detoksifikasi tubuh terha-
dap benda asing menurun karena penurunan sistem
imunitas dan antibodi sehingga orang menjadi lebih
mudah sakit (33). Kekurangan status gizi menyebabkan
organ tubuh tidak berkembang dan berfungsi dengan
baik, sedangkan kelebihan status gizi menyebabkan
penumpukan lemak berlebih yang dapat mengganggu
proses kerja organ dalam tubuh (34). Selain status gizi
faktor
lain
yang
memengaruhi
kondisi
kesehatan
adalah kebiasaan merokok, tekanan darah, aktivitas
fisik dan riwayat genetik (30). Seorang perokok mem-
butuhkan
asupan
nutrisi
yang
mengandung
anti-
oksidan
tinggi,
karena
rokok
dapat
menimbulkan
kerusakan organ pernapasan (35). Kebiasaan merokok
menyebabkan gangguan ventilasi paru, karena me-
nyebabkan iritasi dan produksi sekresi berlebih pada
bronkus (36). SIMPULAN Penelitian menyimpulkan bahwa secara statistik
terdapat hubungan yang signifikan antara paparan
NO2 dengan gangguan fungsi paru. Sedangkan faktor
lain yang tidak berhubungan dengan gangguan fungsi
paru adalah SO2, debu, umur, masa kerja, status gizi,
kebiasaan merokok, karena responden rata-rata masih
berusia 30 tahun dengan status gizi yang baik serta
masa kerja yang kurang dari 10 tahun. Adapun
saran
yang
dapat
diberikan
bagi
PT. Angkasa Pura II Pontianak adalah agar memberikan
pembinaan tentang Keselamatan dan Kesehatan Kerja
(K3) kepada para pekerja, melakukan pemeriksaan
kesehatan
secara
berkala
termasuk
pemeriksaan
fungsi paru, menyediakan alat pelindung diri (APD)
bagi
pekerja. Bagi
peneliti
lain
agar
melakukan
penellitian lebih lanjut dengan dengan penambahan
jumlah
sampel
dan
variabel
yang berbeda untuk
mengetahui faktor lain yang berhubungan dengan
terjadinya gangguan fungsi paru. Penelitian ini tidak menemukan hubungan bermak-
na antara kebiasaan merokok dengan gangguan fungsi
paru. Hasil ini sejalan dengan penelitian Indira dan
Kumendong
bahwa
tidak
ada
hubungan
antara
merokok dengan gangguan fungsi paru (37,38). Hasil
penelitian tidak sesuai dengan teori yang ada karena
asupan nutrisi yang baik dan aktivitas fisik yang tinggi
menunjang
daya
tahan
tubuh
dan
meningkatkan
kapasitas paru. PUSTAKA pp
18. Wulansari, A. ‘Faktor-Faktor Yang Berhubungan
Dengan
Fungsi
Paru
Anak
Sekolah
Dasar
di
Lingkungan Lalu Lintas Padat dan Tidak Padat
Yogyakarta’,
Tesis,
Universitas
Gadjah
Mada,
Yogyakarta; 2015. 1. Touri, L., Marchetti, H., Sari-Minodier, I., Molinari,
N. and
Chanez,
P.,
The
airport
atmospheric
environment:
respiratory
health
at
work. European
respiratory
review
2013;
22(128),
pp.124-130. gy
19. Tunnicliffe,
W.S., O'Hickey, S.P., Fletcher, T.J.,
Miles, J.F., Burge, P.S. and Ayres, J.G. Pulmonary
function
and
respiratory
symptoms
in
a
population of airport workers. Occupational and
environmental medicine 1999;56(2), pp.118-123. pp
2. Hsu,
H.H.,
Adamkiewicz,
G.,
Houseman,
E.A.,
Vallarino, J., Melly, S.J., Wayson, R.L., Spengler,
J.D. and
Levy,
J.I. The
relationship
between
aviation activities and ultrafine particulate matter
concentrations
near
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dan
Kepatuhan
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Jayanti. S.,
&
Ekawati. Faktor-Faktor
yang
Berhubungan
dengan
Gangguan
Fungsi
Paru
pada
Pekerja
yang
Terpapar
Potassium
Permanganate
dan
Phosphoric Acid di Industri Garmen. Universitas
Diponegoro, Semarang; 2014. 27. Widjanarti, M.P. ‘Hubungan Antara Kadar Debu
Respirabel dengan Parameter Uji Fungsi Paru
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Kerja, Fungsi Paru dan Kelelahan Kerja Pada
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g
11. Swarjana, I.K. Metodologi Penelitian Kesehatan
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13 Penyakit Statistik.CV Sagung Seto. Jakarta; 2010. 29. Sunuh, H. S. ‘Hubungan Kualitas Udara Ruang
Perawatan
Dengan
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Fungsi
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g
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M.S. Analisis
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Pekerja dan Pemakaian Alat Pelindung Pernafasan
(Masker)
dengan
Kapasitas
Fungsi Paru pada
Pekerja Wanita Bagian Pengampelasan di Industri
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Besi dan Debu Besi Dengan Gangguan Fungsi Paru
Dan Kadar Imunoglobulin Serum Perajin Logam’,
Disertasi, Universitas Gadjah Mada, Yogyakarta;
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L. Gizi
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Dengan
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Paru
Pekerja
Paving
Block
CV
Sumber
Galian,
Artikel,
Universitas Hasanuddin, Makasar; 2014. 17. Damri., Ilza, M., Afandi, D. Abstrak Tujuan: Penelitian bertujuan mengetahui hubungan
produk bahan bakar avtur dengan kualitas udara
dengan
gangguan
fungsi
paru
pada
pekerja
di
bandara
Supadio
Pontianak. Metode:
Penelitian
observasional
menggunakan
pendekatan
cross
sectional. Teknik
sampling
menggunakan
total
sampling dengan populasi 60 orang. Uji statistik
menggunakan regresi fisher exact dan logistik. Uji
keseluruhan menggunakan tingkat signifikansi p
0,05 dengan interval kepercayaan 95% dan rasio
odds. Hasil: Berdasarkan analisis bivariat dari 36
sampel yang dilakukan, variabel NO2 (p 0,04), SO2 (p
0,15), debu (p 0,25), umur (p 0,20), periode kerja (p
0,08), status gizi (p 0,09), kebiasaan merokok (p 0,03). Simpulan: Secara statistik terdapat hubungan yang
signifikan
antara
variabel
NO2
dan
kebiasaan
merokok dengan gangguan fungsi paru, sedangkan
variabel SO2, debu, umur, masa kerja dan status gizi
tidak memiliki hubungan yang signifikan dengan
kelainan fungsi paru. Hasil uji statistik menunjukkan tidak ada hubungan
yang bermakna antara paparan debu dengan gangguan
fungsi paru. Hasil ini sejalan dengan penelitian Sunuh
yang menyatakan tidak ada hubungan yang bermakna
antara paparan debu dengan gangguan fungsi paru
(33). Hal ini diperkuat penelitian Widjanarti bahwa
tidak
terdapat
hubungan
yang
bermakna
antara
paparan debu dengan kapasitas paru pekerja (31). Paparan debu yang terhirup oleh pekerja berkaitan
dengan
efek
kesehatan
baik akut maupun kronis
terutama pada sistem pernapasan dan kinerja fungsi
paru (39,40). Hal ini sejalan dengan
yang dilakukan
Masngut,
dkk
yang
menjelaskan
hubungan
efek
paparan debu terhadap kesehatan termasuk gejala
pernapasan dan perubahan fungsi paru (41). Hasil
penelitian
menyatakan
bahwa
tidak
ada
hubungan
yang
bermakna
antara
paparan
debu
dengan gangguan fungsi paru, karena paparan debu
yang diperoleh tidak melebihi NAB dan masa kerja
pegawai yang kurang dari 10 tahun sehingga efek dari
paparan
debu
yang
terhirup
saat
bekerja
belum
tampak. Kata kunci: kualitas udara; bahan bakar pesawat
terbakar; gangguan fungsi paru Kata kunci: kualitas udara; bahan bakar pesawat
terbakar; gangguan fungsi paru 281 Berita Kedokteran Masyarakat, Volume 33 No. 6 Tahun 2017 di
Kota
Pekanbaru. Dinamika
Lingkungan
2016;3(1), pp.42-47. Berita Kedokteran Masyarakat, Volume 33 No. 6 Tahun 2017 PUSTAKA Analisis Paparan Co
Dan So2 Pada Petugas Parkir di Basement Mall Ska 282 Berita Kedokteran Masyarakat, Volume 33 No. 6 Tahun 2017 33. Indira, R.A.L. ‘Paparan Debu Terhirup Terhadap
Hasil Tes Fungsi Paru Pada Pekerja Sentra Perajin
Pisau “Rukun Karya Lestasi” di Kabupaten Bantul’,
Tesis, Universitas Gadjah Mada, Yogyakarta; 2015. 34. Kumendong, D.J., Rattu, J.A. and Kawatu, P.A. Hubungan
Antara
Lama
Paparan
dengan
Kapasitas Paru Tenaga Kerja Industri Mebel di CV. Sinar Mandiri Kota Bitung. KESMAS 2012;1(1),
pp.5-10. 39. pp
35. Attarchi, M., Dehghan, F., Afrasyabi, M., Sadeghi,
Z., & Mohammadi, S. Combined Effect of Cigarette
Smoking and Occupational Exposures on Lung
Function : A Cross-sectional Study; 2013. y
36. Yanagi,
N.,
Kitamura,
H.,
Mizuno,
M.,
Hata,
K.,Uchiyama, T., Kuga, H., & Higashi, T. A 4-Year
Follow-up Cohort Study of Respiratory Function in
Toner-handling Workers. Safety and Health at
Work; 2014 5(4), pp. 222-226. pp
37. Masngut, M.I., Baharudin, M.R., & Rahman, A.A. A
Systematic Review On Risk Factor For Reduce
Lung Function Due To Occupational Respirable
Dust Exposure 2005-2015. International Journal of
Public Health and Clinical Sciences 2015;2(4), pp. 44-62. 283 284
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English
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Supplementary Data from Chromatin Remodeling Induced by ARID1A Loss in Lung Cancer Promotes Glycolysis and Confers JQ1 Vulnerability
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Supplementary Figures Figure S1. ARID1A is frequently mutated across human cancers and is downregulated in
Kras-driven lung tumors in mice. Figure S2. Identification of TTF-1, P63 expression and cell apoptosis in KP, KPAfl/+ and KPAfl/fl
lung tumors. Figure S2. Identification of TTF-1, P63 expression and cell apoptosis in KP, KPAfl/+ and KPAfl/fl
lung tumors. Figure S3. ARID1A loss alters the transcriptome of lung cancer in murine models. Figure S4. Validation of the EMT features predicted by bioinformatic analysis in KPAfl/fl tumors. Figure S5. Analysis of the mRNA level of glycolysis-related genes in TCGA-LUAD patients with
higher or lower ARID1A expression. Figure S6. The mRNA levels of PGAM1, PKM, and PGK1 are negatively correlated with
ARID1A expression across various cancer cell lines and human cancers. Figure S7. The expression of ARID1A, SMARCC1 and SAMRCA4 in KP and KPA tumor cells. Figure S8. Knockdown of BRD4 decreases the expression of Pgk1, Pgam1 and Pkm in KPA cells
and attenuates the tumorigenic ability of KPA cells in vivo. Figure S9. Effects of 6-Aminonicotinamide, Oxamate, Shikonin and 2-DG on the tumorigenicity
of KP and KPA cells. Figure S10. Expression of PGAM1 and PKM2 in the lung tumors treated with pSECC lentivirus.. Figure S11. Expression of Pgam1, Pkm, and Pgk1 in JQ1-treated cell lines. Figure S12. Expression of PGAM1, PKM2, and PGK1 in JQ1-treated mice. Figure S13. Knockdown of SMARCA4 did not affect the binding of BRD4 to the promoters of
Pgk1, Pkm and Pgam1 in KP and KPA cells. 1 Figure S14. Knockdown of SMARCA4 up-regulated the mRNA levels of OXPHOS-related genes
(ATP5L and PGC-1α) but not that of glycolysis-related enzymes (PGAM1, PGK1 and PKM). Supplementary Tables Table S1. Primers used in this study. Table S2. Details of mutations identified in ARID1A in clinical cohorts. Table S3. Differentially expressed genes between KPAfl/fl and KP tumors identified using
RNA-seq. Table S4. The NES (normalized enrichment score) and FDR value of pre-rank GSEA analysis of
differently expressed genes. Table S5. ARID1A was negatively associated with mRNA levels of PGAM1, PKM, and PGK1 in
Cancer Cell Line Encyclopedia dataset and in various human cancers in TCGA database. Table S6. The significant differentially enriched genomic locus of KPAfl/fl and KP tumors
identified by Diffbind (p<0.01, DEseq2 method) using ATAC-seq data. Table S6. The significant differentially enriched genomic locus of KPAfl/fl and KP tumors
identified by Diffbind (p<0.01, DEseq2 method) using ATAC-seq data. 2 2 Figure S1. ARID1A is frequently mutated across human cancers and is downregulated in
Kras-driven lung tumors in mice. (A) The landscape of ARID1A expression and its alternations
across various human cancers. (B-C) Western blot was performed to examine the expression of
ARID1A, SMARCA4, SMARCC1 in KP tumors and adjacent tissues. The ImageJ software was used
for quantification. Data were analyzed by Welch’s t-test and presented with mean ± s.e.m. in barplot. (D-E) Western blot was performed to examine the expression of ARID1A, SMARCA4 and HIF2 in
KP tumors and KPAfl/fl tumors. The ImageJ software was used to for quantification. Data were
analyzed by Welch’s t-test and presented with mean ± s.e.m. in barplot. 3 Figure S2. Identification of TTF-1, P63 expression and cell apoptosis in KP, KPAfl/+ and KPAfl/fl
lung tumors. (A) Immunohistochemistry (IHC) analysis of TTF-1 and P63 expression in tumors
derived from KP, KPAfl/-, and KPA fl/fl mice. (B) TUNEL staining of tumors derived from KP, KPAfl/-,
and KPA fl/fl mice. The nuclei were labeled using DAPI, and the TUNEL-positive cells were labeled
using green fluorescent marker. (C)The apoptotic cells in KP, KPAfl/+ and KPAfl/fl lung tumors were
quantified according to the TUNEL staining. Data were analyzed by one-way ANOVA and presented
with mean ± s.e.m. in barplot. Figure S2. Identification of TTF-1, P63 expression and cell apoptosis in KP, KPAfl/+ and KPAfl/fl
lung tumors. (A) Immunohistochemistry (IHC) analysis of TTF-1 and P63 expression in tumors
derived from KP, KPAfl/-, and KPA fl/fl mice. (B) TUNEL staining of tumors derived from KP, KPAfl/-,
and KPA fl/fl mice. The nuclei were labeled using DAPI, and the TUNEL-positive cells were labeled
using green fluorescent marker. (C)The apoptotic cells in KP, KPAfl/+ and KPAfl/fl lung tumors were
quantified according to the TUNEL staining. Data were analyzed by one-way ANOVA and presented
with mean ± s.e.m. in barplot. Figure S2. Identification of TTF-1, P63 expression and cell apoptosis in KP, KPAfl/+ and KPAfl/fl
lung tumors. (A) Immunohistochemistry (IHC) analysis of TTF-1 and P63 expression in tumors
derived from KP, KPAfl/-, and KPA fl/fl mice. (B) TUNEL staining of tumors derived from KP, KPAfl/-,
and KPA fl/fl mice. The nuclei were labeled using DAPI, and the TUNEL-positive cells were labeled
using green fluorescent marker. (C)The apoptotic cells in KP, KPAfl/+ and KPAfl/fl lung tumors were
quantified according to the TUNEL staining. Data were analyzed by one-way ANOVA and presented
with mean ± s.e.m. in barplot. Figure S3. ARID1A loss alters the transcriptome of lung cancer in murine models. (A) Boxplot
of the mRNA level (normalized reads count) of Arid1a, which was significantly reduced in tumors
derived from KPA fl/fl mice (p < 0.001). Data were analyzed by Welch’s t-test. (B) Heatmap of the top
200 different differentially expressed genes between KPAfl/fl and KP tumors. (C) Volcano plot of the
landscape of differentially expressed mRNA between KPAfl/fl and KP tumors, with fold change ≥2
and BH-adjusted p-value < 0.05. Data were analyzed using the R software (package ‘DESeq2’
version 1.32.0). *, p < 0.05; **, p < 0.01; ***, p < 0.001. Figure S3. ARID1A loss alters the transcriptome of lung cancer in murine models. (A) Boxplot
of the mRNA level (normalized reads count) of Arid1a, which was significantly reduced in tumors
derived from KPA fl/fl mice (p < 0.001). Data were analyzed by Welch’s t-test. (B) Heatmap of the top
200 different differentially expressed genes between KPAfl/fl and KP tumors. (C) Volcano plot of the
landscape of differentially expressed mRNA between KPAfl/fl and KP tumors, with fold change ≥2
and BH-adjusted p-value < 0.05. Data were analyzed using the R software (package ‘DESeq2’
version 1.32.0). *, p < 0.05; **, p < 0.01; ***, p < 0.001. 5 Figure S4. Validation of the EMT features in KPAfl/fl tumors. (A) Representative images of
immunofluorescent (IF) stained E-cadherin and Vimentin in KP and KPAfl/fl lung tumors. (B)
Quantification of the expression level of E-cadherin and Vimentin protein for (A). The ImageJ
software was used for quantification, and the IF intensity of Vimentin and E-cadherin was
normalized to that of DAPI. Data were analyzed by Welch’s t-test and presented with mean ± s.e.m. in barplot. (C-D) Western blot was performed to examine the expression of E-cadherin and Vimentin
in KP tumors and KPAfl/fl tumors. The ImageJ software was used for quantification. Data were
analyzed by Welch’s t-test and presented with mean ± s.e.m. in barplot. 6 Figure S5. Analysis of the mRNA level of glycolysis-related genes in TCGA-LUAD patients
with higher or lower ARID1A expression. (A) Unsupervised clustering of transcriptome data from
patients with the top 5% (n=26) and bottom 5% (n=26) of ARID1A expression in TCGA-LUAD (n =
533) dataset. The scale indicated normalized z-score of the sample-sample distance calculated using
Euclidean method. (B) The mRNA level of glycolysis-related genes, including PGAM1, PGK1,
PKM, ENO1, and LDHA in patients with top and bottom 5% of ARID1A expression in the
TCGA-LUAD dataset. Data were analyzed by Student’s t-test. n = 26 per group. Figure S5. Analysis of the mRNA level of glycolysis-related genes in TCGA-LUAD patients
with higher or lower ARID1A expression. (A) Unsupervised clustering of transcriptome data from
patients with the top 5% (n=26) and bottom 5% (n=26) of ARID1A expression in TCGA-LUAD (n =
533) dataset. The scale indicated normalized z-score of the sample-sample distance calculated using
Euclidean method. (B) The mRNA level of glycolysis-related genes, including PGAM1, PGK1,
PKM, ENO1, and LDHA in patients with top and bottom 5% of ARID1A expression in the
TCGA-LUAD dataset. Data were analyzed by Student’s t-test. n = 26 per group. 7 Figure S6. The mRNA levels of PGAM1, PKM, and PGK1 are negatively correlated with
ARID1A expression across various cancer cell lines and human cancers. mRNA levels of PGK1,
PGAM1, and PKM are negatively correlated with ARID1A level across different cancer cell lines (A)
and human cancers, including glioblastoma multiforme (B), stomach adenocarcinoma (C), and
pancreatic adenocarcinoma (D), according to TCGA datasets. The Pearson Correlation Coefficient
and p value were calculated for each group. Statistical analysis was performed using cbioportal
(https://www.cbioportal.org/) webtool. 8 Figure S7. The expression of ARID1A, SMARCC1 and SAMRCA4 in KP and KPA tumor cells.
The KP cells were derived from KP lung tumors. Next, we knocked down the expression of ARID1A
in KP cells using shRNA to generate the KPA cells. Expression of ARID1A, SMARCC1 and
SAMRCA4 in KP and KPA tumor cells was validated with Western blot. Figure S7. The expression of ARID1A, SMARCC1 and SAMRCA4 in KP and KPA tumor cells. The KP cells were derived from KP lung tumors. Next, we knocked down the expression of ARID1A
in KP cells using shRNA to generate the KPA cells. Expression of ARID1A, SMARCC1 and
SAMRCA4 in KP and KPA tumor cells was validated with Western blot. 9 Figure S8. Knockdown of BRD4 decreases the expression of Pgk1, Pgam1 and Pkm in KPA
cells and attenuates the tumorigenic ability of KPA cells in vivo. (A) Validation of the effect of
siRNAs on BRD2, BRD3 and BRD4 expression in KPA cells using Quantitative Real-time PCR
(qPCR). Data were analyzed by Welch’s t-test and presented with mean ± s.e.m. in barplot. (B)
Validation of the effect of BRD2, BRD3 and BRD4 knockdown on expression of Pgk1, Pgam1 and
Pkm in KPA cells using qPCR. Data were analyzed by Welch’s t-test and presented with mean ±
s.e.m. in barplot. (C) Representative images of subcutaneous KPA tumors with knockdown of BRD2,
BRD3 and BRD4 expression, respectively. (D) Quantification of tumor weight across different
groups in (C). Data were analyzed by Welch’s t-test and presented with mean ± s.e.m. in barplot. 10 10 Figure S9. Inhibitory effects of 6-Aminonicotinamide, Oxamate, Shikonin and 2-DG on the
tumorigenicity of KP and KPA cells. (A) Representative images of subcutaneous KP and KPA
tumors with treatment of saline, 6-Amino (6-Aminonicotinamide) (30 mg/kg body weight, i.p),
oxamate (600 mg/kg body weight, i.p), shikonin (30 mg/kg body weight, i.p) and 2-DG (30 mg/kg
body weight, i.p) daily for three weeks, respectively. (B) Quantification of tumor weight across
different groups in (A). Data were analyzed by Welch’s t-test and presented with mean ± s.e.m. in
barplot. Figure S9. Inhibitory effects of 6-Aminonicotinamide, Oxamate, Shikonin and 2-DG on the
tumorigenicity of KP and KPA cells. (A) Representative images of subcutaneous KP and KPA
tumors with treatment of saline, 6-Amino (6-Aminonicotinamide) (30 mg/kg body weight, i.p),
oxamate (600 mg/kg body weight, i.p), shikonin (30 mg/kg body weight, i.p) and 2-DG (30 mg/kg
body weight, i.p) daily for three weeks, respectively. (B) Quantification of tumor weight across
different groups in (A). Data were analyzed by Welch’s t-test and presented with mean ± s.e.m. in
barplot. 11 11 Figure S10. Expression of PGAM1 and PKM2 in the lung tumors treated with pSECC
lentivirus. (A-D) Representative images of IF staining for PGAM1 and PKM2 expression in tumors
treated with pSECC-sgTom, pSECC-sgPgam1, or pSECC-sgPgam1 lentivirus in KP and KPAfl/fl
mice. (E-F) The IF intensity of PGAM1 and PKM2 was determined using ImageJ software and was
normalized to the IF intensity of DAPI. Data were analyzed by Welch’s t-test and presented with
mean ± s.e.m. in barplot. Figure S10. Expression of PGAM1 and PKM2 in the lung tumors treated with pSECC
lentivirus. (A-D) Representative images of IF staining for PGAM1 and PKM2 expression in tumors
treated with pSECC-sgTom, pSECC-sgPgam1, or pSECC-sgPgam1 lentivirus in KP and KPAfl/fl
mice. (E-F) The IF intensity of PGAM1 and PKM2 was determined using ImageJ software and was
normalized to the IF intensity of DAPI. Data were analyzed by Welch’s t-test and presented with
mean ± s.e.m. in barplot. Figure S10. Expression of PGAM1 and PKM2 in the lung tumors treated with pSECC
lentivirus. (A-D) Representative images of IF staining for PGAM1 and PKM2 expression in tumors
treated with pSECC-sgTom, pSECC-sgPgam1, or pSECC-sgPgam1 lentivirus in KP and KPAfl/fl
mice. (E-F) The IF intensity of PGAM1 and PKM2 was determined using ImageJ software and was
normalized to the IF intensity of DAPI. Data were analyzed by Welch’s t-test and presented with
mean ± s.e.m. in barplot. 12 12 Figure S11. Expression of Pgam1, Pkm, and Pgk1 in JQ1-treated cell lines. (A) Expression of
Pgam1, Pkm, and Pgk1 in NRAS mutant melanoma cells with or without JQ1 treatment (GSE95153). Data were analyzed by Welch’s t-test and presented with mean ± s.e.m. in barplot. (B) Expression of
Pgam1, Pkm, and Pgk1 in rheumatoid synovial fibroblast cells with or without JQ1 treatment
(GSE9148395). Data were analyzed by Welch’s t-test and presented with mean ± s.e.m. in barplot. (C)
Expression of Pgam1 and Pgk1 in colon cancer cell lines (including HCT116, HCT15, COLO320,
COLO205, SW480, and HT29) with or without JQ1 treatment (GSE73318). Data were analyzed by
Welch’s t-test and presented with mean ± s.e.m. in barplot. *, p < 0.05; **, p < 0.01. 13 13 Figure S12. Expression of PGAM1, PKM2, and PGK1 in JQ1-treated mice. (A) Representative
images of IF staining for PGAM1, PGK1 and PKM2 expression in the tumors of KP and KPAfl/fl
genetically engineered mice treated with saline or JQ1(30 mg/kg body weight, i.p.). (B) Quantifying
the expression of PGAM1, PGK1 and PKM2 in in the tumors of KP (n=6) and KPAfl/fl (n=6)
genetically engineered mice according to IF staining. The IF intensity of the target protein was
determined using ImageJ software and was normalized to the IF intensity of DAPI. Data were
analyzed by Welch’s t-test and presented with mean ± s.e.m. in barplot. 14 14 Figure S13. Knockdown of SMARCA4 did not affect the binding of BRD4 to the promoters of
Pgk1, Pkm and Pgam1 in KP and KPA cells. (A) ChIP assay was performed to examine the
binding of SMARCA4 to the promoters of Pgam1, Pkam and Pgk1 in KP and KPA cells. (B-D) ChIP
assay was performed to examine the binding of BRD4 to the promoters of Pgam1, Pkam and Pgk1 in
KP and KPA cells after knockdown of SMARCA4. N.s, not significant. Figure S13. Knockdown of SMARCA4 did not affect the binding of BRD4 to the promoters of
Pgk1, Pkm and Pgam1 in KP and KPA cells. (A) ChIP assay was performed to examine the
binding of SMARCA4 to the promoters of Pgam1, Pkam and Pgk1 in KP and KPA cells. (B-D) ChIP
assay was performed to examine the binding of BRD4 to the promoters of Pgam1, Pkam and Pgk1 in
KP and KPA cells after knockdown of SMARCA4. N.s, not significant. Figure S13. Knockdown of SMARCA4 did not affect the binding of BRD4 to the promoters of
Pgk1, Pkm and Pgam1 in KP and KPA cells. (A) ChIP assay was performed to examine the
binding of SMARCA4 to the promoters of Pgam1, Pkam and Pgk1 in KP and KPA cells. (B-D) ChIP
assay was performed to examine the binding of BRD4 to the promoters of Pgam1, Pkam and Pgk1 in
KP and KPA cells after knockdown of SMARCA4. N.s, not significant. 15 15 Figure S14. Knockdown of SMARCA4 up-regulated the mRNA levels of OXPHOS-related
genes (ATP5L and PGC-1α) but not that of glycolysis-related enzymes (PGAM1, PGK1 and
PKM). (A) qPCR was performed to examine the mRNA levels of SMARCA4. (B-C) The mRNA
levels of ATP5L and PGC-1α were examined in KP and KPA cells by qPCR. (D-F) The mRNA levels
of PGAM1, PGK1 and PKM were examined in KP and KPA cells by qPCR. N.s, not significant. Figure S14. Knockdown of SMARCA4 up-regulated the mRNA levels of OXPHOS-related
genes (ATP5L and PGC-1α) but not that of glycolysis-related enzymes (PGAM1, PGK1 and
PKM). (A) qPCR was performed to examine the mRNA levels of SMARCA4. (B-C) The mRNA
levels of ATP5L and PGC-1α were examined in KP and KPA cells by qPCR. (D-F) The mRNA levels
of PGAM1, PGK1 and PKM were examined in KP and KPA cells by qPCR. N.s, not significant. Figure S14. Knockdown of SMARCA4 up-regulated the mRNA levels of OXPHOS-related
genes (ATP5L and PGC-1α) but not that of glycolysis-related enzymes (PGAM1, PGK1 and
PKM). (A) qPCR was performed to examine the mRNA levels of SMARCA4. (B-C) The mRNA
levels of ATP5L and PGC-1α were examined in KP and KPA cells by qPCR. (D-F) The mRNA levels
of PGAM1, PGK1 and PKM were examined in KP and KPA cells by qPCR. N.s, not significant. 16
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https://openalex.org/W2884171652
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https://journalofinequalitiesandapplications.springeropen.com/track/pdf/10.1186/s13660-018-1773-0
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English
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Approximate weakly efficient solutions of set-valued vector equilibrium problems
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Journal of inequalities and applications
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cc-by
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© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, pro-
vided 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. R ES EA RCH Open Access Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181
https://doi.org/10.1186/s13660-018-1773-0 Abstract In this paper, we introduce a new kind of approximate weakly efficient solutions to
the set-valued vector equilibrium problems with constraints in locally convex
Hausdorfftopological vector spaces; then we discuss a relationship between the
weakly efficient solutions and approximate weakly efficient solutions. Under the
assumption of near cone-subconvexlikeness, by using the separation theorem for
convex sets we establish Kuhn–Tucker-type and Lagrange-type optimality conditions
for set-valued vector equilibrium problems, respectively. MSC: 90C33; 90C46; 90C59 Keywords: Set-valued vector equilibrium problem; Approximate weakly efficient
solution; Near cone-subconvexlikeness; Optimality condition Jian Chen1, Yihong Xu1* and Ke Zhang1 Jian Chen1, Yihong Xu1* and Ke Zhang1 *Correspondence:
xuyihong@ncu.edu.cn
1Department of Mathematics,
Nanchang University, Nanchang,
China 1 Introduction Vector optimization problems, vector variational inequality problems, vector complemen-
tarity problems, and vector saddle point problems are particular cases of vector equilib-
rium problems. As an extensive mathematical model, the vector equilibrium problem is
a hot topic in the fields of operations research and nonlinear analysis (see [1–8]). Gong
[2–4] obtained optimality conditions for vector equilibrium problems with constraints
under the assumption of cone-convexity, and by using a nonlinear scalarization function
and Ioffe subdifferentiability he derived optimality conditions for weakly efficient solu-
tions, Henig solutions, super efficient solutions, and globally efficient solutions to non-
convex vector equilibrium problems. Long et al. [5] obtained optimality conditions for
Henig efficient solutions to vector equilibrium problems with functional constrains under
the assumption of near cone-subconvexlikeness. Luu et al. [7, 8] established sufficient and
necessary conditions for efficient solutions to vector equilibrium problems with equality
and inequality constraints and obtained the Fritz John and Karush–Kuhn–Tucker neces-
sary optimality conditions for locally efficient solutions to vector equilibrium problems
with constraints and sufficient conditions under assumptions of appropriate convexities. It is well known that models describe only simplified versions of real problems and nu-
merical algorithms generate only approximate solutions. Hence it is interesting and mean-
ingful to have a theoretical analysis of the notion of an approximate solution. For example,
Loridan [9, 10] introduced the concept of ϵ-solutions in general vector optimization prob-
lems. Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181 Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181 Page 2 of 17 As far as we know, there are few papers dealing with approximate weakly efficient solu-
tions to the set-valued vector equilibrium problems. Li et al. [11] introduced a new kind
of approximate solution set of a vector approximate equilibrium problem; it is uncertain
if ϵ tends to zero, whether or not the approximate solution set equals to the original so-
lution set? It is a natural question how to define approximate weakly efficient solutions
to the set-valued vector equilibrium problems and under what condition the set of ap-
proximate weakly efficient solutions equals to the set of weakly efficient solutions? This
has great theoretical significance and applicable value in the research of optimality condi-
tions for approximate weakly efficient solutions to the set-valued vector equilibrium prob-
lems. On the other hand, convexity plays an important role in the study of vector equilib-
rium problems. In 2001, Yang et al. [12] introduced a new convexity, named near cone-
subconvexlikeness, and proved that it is a generalization of cone-convexness and cone-
subconvexlikeness. In 2005, Sach (see [13]) introduced another new convexity called ic-
cone-convexness, Xu et al. [14] proved that near cone-subconvexlikeness is also a gener-
alization of ic-coneconvexness. Up to now, near cone-subconvexlikeness is considered to
be the most generalized convexity. Motivated by works in [3, 12, 15], in this paper, we introduce a new kind of approximate
weakly efficient solutions to the set-valued vector equilibrium problems and reveal the re-
lationship between weakly efficient solutions and approximate weakly efficient solutions. We establish Kuhn-Tucker type and Lagrange-type optimality conditions for set-valued
vector equilibrium problems under the assumption of the near cone-subconvexlikeness. The organization of the paper is as follows. Some preliminary facts are given in Sect. 2
for our later use. Section 3 is devoted to the relationship between weakly efficient solutions
and approximate weakly efficient solutions. In Sect. 4, we establish Kuhn–Tucker-type suf-
ficient and necessary optimality conditions for approximate weakly efficient solutions to
the set-valued vector equilibrium problems. In Sect. 5, we establish Lagrange-type suf-
ficient and necessary optimality conditions for approximate weakly efficient solutions to
the set-valued vector equilibrium problems. At the end of the paper, we draw some con-
clusions. Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181 2 Preliminaries Let X be a real topological vector space, and let Y and Z be real locally convex Hausdorff
topological vector spaces with topological dual spaces Y ∗and Z∗, respectively. Let C ⊂Y
and D ⊂Z be pointed closed convex cones with intC ̸= ∅and intD ̸= ∅. The dual cones C∗
of C and D∗of D are defined as C∗= {φ ∈Y ∗: φ(c) ≥0,∀c ∈C} and D∗= {ψ ∈Z∗: ψ(d) ≥
0,∀d ∈D}, respectively. Let X0 be a nonempty convex subset in X, and let G : X0 →2Z and
: X0 × X0 →2Y be mappings. We denote by L(Z,Y) the set of all continuous linear operators from Z to Y. A subset
L+(Z,Y) of L(Z,Y) is defined as L+(Z,Y) = {T ∈L(Z,Y) : T(D) ⊂C}. We denote by L(Z,Y) the set of all continuous linear operators from Z to Y. A subset
L+(Z,Y) of L(Z,Y) is defined as L+(Z,Y) = {T ∈L(Z,Y) : T(D) ⊂C}. We denote the feasible set by A =
x ∈X0 : G(x) ∩(–D) ̸= ∅
. Page 3 of 17 Page 3 of 17 Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181 Consider the set-valued vector equilibrium problem with constraints (for short, -
SVEPC): find x ∈A such that (x,y) ∩(–P) = ∅,
∀y ∈A, where P ∪{0} is a convex cone in Y. Definition 2.1 A vector ¯x ∈A satisfying Definition 2.1 A vector ¯x ∈A satisfying (¯x,y) ∩(–intC) = ∅,
∀y ∈A, is called a weakly efficient solution to the -SVEPC. The set of all weakly efficient solutions
to the -SVEPC is denoted by XW min(,A). Let F : X0 →2Y be a set-valued map. We consider the following set-valued optimization
problem: (SOP)
minF(x), s.t. x ∈A =
x ∈X0 : G(x) ∩(–D) ̸= ∅
. We assume that the feasible set A ⊂X0 of (SOP) is nonempty. Journal of Inequalities and Applications ( 2018) 2018:181 Page 4 of 17 Definition 2.6 The map F : X0 →2Y is said to be C-convex on X0 if, for all x1,x2 ∈X0 and
λ ∈[0,1], we have λF(x1) + (1 – λ)F(x2) ⊂F
λx1 + (1 – λ)x2
+ C. Definition 2.7 ([16]) The map F : X0 →2Y is said to be C-subconvexlike on X0 iffthere
exists θ ∈intC such that, for all x1,x2 ∈X0, λ ∈[0,1], yi ∈F(xi), i = 1,2, and α > 0, there
exists x3 ∈X0 such that αθ + λy1 + (1 – λ)y2 ∈F(x3) + C. Definition 2.8 ([17]) The map F : X0 →2Y is said to be generalized C-subconvexlike on
X0 iffthere exists θ ∈intC such that, for all x1,x2 ∈X0, λ ∈[0,1], and α > 0, there exist
x3 ∈X0 and ρ > 0 such that αθ + λF(x1) + (1 – λ)F(x2) ⊂ρF(x3) + C. Definition 2.9 ([12]) The map F : X0 →2Y is called nearly C-subconvexlike on X0 iff
clcone(F(X0) + C) is convex. If ∅̸= S1 ⊂Y, ∅̸= S2 ⊂Y, ¯y ∈Y, and ψ ∈Y ∗, then
ψ(S1) ≥ψ(S2) stands for ψ(s1) ≥ψ(s2),
∀s1 ∈S1,s2 ∈S2,
and
ψ(S1) ≥ψ(¯y) stands for ψ(s1) ≥ψ(¯y),
∀s1 ∈S1. Definition 2.9 ([12]) The map F : X0 →2Y is called nearly C-subconvexlike on X0 iff
clcone(F(X0) + C) is convex. Definition 2.9 ([12]) The map F : X0 →2Y is called nearly C-subconvexlike on X0 iff
clcone(F(X0) + C) is convex. If ∅̸= S1 ⊂Y, ∅̸= S2 ⊂Y, ¯y ∈Y, and ψ ∈Y ∗, then We assume that the feasible set A ⊂X0 of (SOP) is nonempty. We assume that the feasible set A ⊂X0 of (SOP) is nonempty. Definition 2.2 A feasible solution ¯x of (SOP) is said to be a weakly efficient solution of
(SOP) if there exists ¯y ∈F(¯x) such that (F(A) – ¯y) ∩(–intC) = ∅. In this case, (¯x, ¯y) is said
to be a weakly efficient pair to (SOP). Definition 2.3 Let ϵ ∈C. A feasible solution ¯x of (SOP) is said to be an ϵ-weakly efficient
solution of (SOP) if there exists ¯y ∈F(¯x) such that (F(A)– ¯y+ϵ)∩(–intC) = ∅. In this case,
(¯x, ¯y) is said to be an ϵ-weakly efficient pair to (SOP). Let ¯T ∈L+(Z,Y). Consider the following unconstrained set-valued optimization prob-
lem induced by (SOP): (USOP) ¯T
min
x∈X0 L(x, ¯T), where L(x, ¯T) = F(x) + ¯T(G(x)), (x, ¯T) ∈X0 × L+(Z,Y). where L(x, ¯T) = F(x) + ¯T(G(x)), (x, ¯T) ∈X0 × L+(Z,Y). Definition 2.4 A vector ¯x ∈X0 is said to be a weakly efficient solution of (USOP) ¯T if there
exists ¯y ∈F(¯x) such that (L(X0, ¯T)– ¯y)∩(–intC) = ∅, where L(X0, ¯T) =
x∈X0 L(x, ¯T). In this
case, (¯x, ¯y) is said to be a weakly efficient pair to (USOP) ¯T. Definition 2.4 A vector ¯x ∈X0 is said to be a weakly efficient solution of (USOP) ¯T if there
exists ¯y ∈F(¯x) such that (L(X0, ¯T)– ¯y)∩(–intC) = ∅, where L(X0, ¯T) =
x∈X0 L(x, ¯T). In this
case, (¯x, ¯y) is said to be a weakly efficient pair to (USOP) ¯T. Definition 2.5 Let ϵ ∈C. A vector ¯x ∈X0 is said to be an ϵ-weakly efficient solution
of (USOP) ¯T if ∃¯y ∈F(¯x) such that (L(X0, ¯T) – ¯y + ϵ) ∩(–intC) = ∅, where L(X0, ¯T) =
x∈X0 L(x, ¯T). In this case, (¯x, ¯y) is said to be an ϵ-weakly efficient pair to (USOP) ¯T. Definition 2.5 Let ϵ ∈C. A vector ¯x ∈X0 is said to be an ϵ-weakly efficient solution
of (USOP) ¯T if ∃¯y ∈F(¯x) such that (L(X0, ¯T) – ¯y + ϵ) ∩(–intC) = ∅, where L(X0, ¯T) =
x∈X0 L(x, ¯T). In this case, (¯x, ¯y) is said to be an ϵ-weakly efficient pair to (USOP) ¯T. Several definitions of generalized convexities have been introduced in the literature. Chen et al. 3 Approximate weakly efficient solutions Firstly, we introduce approximate weakly efficient solutions to the set-valued vector equi-
librium problems with constraints. Definition 3.1 Let ϵ ∈C. A vector ¯x ∈A satisfying Definition 3.1 Let ϵ ∈C. A vector ¯x ∈A satisfying
(¯x,y) + ϵ
∩(–intC) = ∅,
∀y ∈A, is called an ϵ-weakly efficient solution to the -SVEPC. The set of all ϵ-weakly efficient
solutions to the -SVEPC is denoted by ϵ-XW min(,A). Let ϒ : X0 × X0 →2Y be a mapping. Consider the following unconstrained set-valued
vector equilibrium problem (for short, ϒ-USVEP): find x ∈X0 such that ϒ(x,y) ∩(–P) = ∅,
∀y ∈X0, where P ∪{0} is a convex cone in Y. Page 5 of 17 Page 5 of 17 Page 5 of 17 Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181 Definition 3.2 Let ϵ ∈C. A vector ¯x ∈X0 satisfying
ϒ(¯x,y) + ϵ
∩(–intC) = ∅,
∀y ∈X0, is called an ϵ-weakly efficient solution to the ϒ-USVEP. The set of all ϵ-weakly efficient
solutions to the ϒ-USVEP is denoted by ϵ-XW min(ϒ,X0). Proposition 3.1 For any ϵ ∈C, we have Proposition 3.1 For any ϵ ∈C, we have XW min(,A) ⊂ϵ-XW min(,A). Proof If x /∈ϵ-XW min(,A), then there exists ¯y ∈A such that
(x, ¯y) + ϵ
∩(–intC) ̸= ∅. Thus there exists ¯z ∈(x, ¯y) such that (3.1) ¯z + ϵ ∈–intC. (3.1) ¯z + ϵ ∈–intC. Since C is a convex cone, from ϵ ∈C and (3.1) we have Since C is a convex cone, from ϵ ∈C and (3.1) we have ¯z ∈–intC – ϵ ⊂–intC – C ⊂–intC. ¯z ∈–intC – ϵ ⊂–intC – C ⊂–intC. Hence (x, ¯y) ∩(–intC) ̸= ∅, and thus x /∈XW min(,A). Then we obtain XW min(,A) ⊂ϵ-XW min(,A). □ □ Next, we show that in the proposition the relationship may be strict when ϵ ∈C \ {0}. Example 3.1 Let X = R1, A = [0,2], Y = R2, C = R2
+, and ϵ = (x0,y0) ∈C \ {0}. Let : A ×
A −→2Y be defined by (x,y) = {(p,q) : q ≥p2–x}∩([–y,y]×[0,+∞)), ∀x,y ∈A. It is obvi-
ous that XW min(,A) = {0}; however, ϵ-XW min(,A) = [0,δ], where δ = min{max{x2
0,y0},2}. Example 3.1 Let X = R1, A = [0,2], Y = R2, C = R2
+, and ϵ = (x0,y0) ∈C \ {0}. Let : A ×
A −→2Y be defined by (x,y) = {(p,q) : q ≥p2–x}∩([–y,y]×[0,+∞)), ∀x,y ∈A. It is obvi-
ous that XW min(,A) = {0}; however, ϵ-XW min(,A) = [0,δ], where δ = min{max{x2
0,y0},2}. Proposition 3.2 For any ϵ1,ϵ2 ∈C, if ϵ2 – ϵ1 ∈C, then Proposition 3.2 For any ϵ1,ϵ2 ∈C, if ϵ2 – ϵ1 ∈C, then Proposition 3.2 For any ϵ1,ϵ2 ∈C, if ϵ2 – ϵ1 ∈C, then Proposition 3.2 For any ϵ1,ϵ2 ∈C, if ϵ2 – ϵ1 ∈C, then Proposition 3.2 For any ϵ1,ϵ2 ∈C, if ϵ2 – ϵ1 ∈C, then ϵ1-XW min(,A) ⊂ϵ2-XW min(,A). Proof If x /∈ϵ2-XW min(,A), then there exists y1 ∈A such that Proof If x /∈ϵ2-XW min(,A), then there exists y1 ∈A such that
(x,y1) + ϵ2
∩(–intC) ̸= ∅.
(x,y1) + ϵ2
∩(–intC) ̸= ∅.
(x,y1) + ϵ2
∩(–intC) ̸= ∅. Thus there exists z1 ∈(x,y1) such that (3.2) z1 + ϵ2 ∈–intC. Page 6 of 17 Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181 Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181 Since C is a convex cone, from ϵ2 – ϵ1 ∈C and (3.2) we have that z1 + ϵ1 = z1 + ϵ2 – (ϵ2 – ϵ1) ∈–intC – C = –intC. Hence Hence
(x,y1) + ϵ1
∩(–intC) ̸= ∅,
(x,y1) + ϵ1
∩(–intC) ̸= ∅, and thus x /∈ϵ1-XW min(,A), so we obtain -XW min(,A) ⊂ϵ2-XW min(,A). □ □ ϵ1-XW min(,A) ⊂ϵ2-XW min(,A). In what follows, we discuss the relationship between the approximate weakly efficient
solutions and weakly efficient solutions to the set-valued vector equilibrium problems with
constraints. Proposition 3.3 We have:
ϵ∈C\{0}
ϵ-XW min(,A) = XW min(,A). Proof Firstly, we prove that Proof Firstly, we prove that XW min(,A) ⊂
ϵ∈C\{0}
ϵ-XW min(,A). From Proposition 3.1 we can see that, for any ϵ ∈C \ {0}, we have From Proposition 3.1 we can see that, for any ϵ ∈C \ {0}, we have XW min(,A) ⊂ϵ-XW min(,A). Hence XW min(,A) ⊂
ϵ∈C\{0}
ϵ-XW min(,A). Next, we prove that
ϵ∈C\{0}
ϵ-XW min(,A) ⊂XW min(,A).
ϵ∈C\{0}
ϵ-XW min(,A) ⊂XW min(,A). Suppose ¯x /∈XW min(,A). Then there exists y0 ∈A such that Suppose ¯x /∈XW min(,A). Then there exists y0 ∈A such that (¯x,y0) ∩(–intC) ̸= ∅,
(3.3) (¯x,y0) ∩(–intC) ̸= ∅,
(3.3) (3.3) (¯x,y0) ∩(–intC) ̸= ∅, and hence we can find z0 ∈(¯x,y0) such that and hence we can find z0 ∈(¯x,y0) such that and hence we can find z0 ∈(¯x,y0) such that z0 ∈–intC. Page 7 of 17 Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181 Then there exists a neighborhood U0 of 0 in Y such that z0 + U0 ⊂–intC. Choosing ϵ0 ∈(C ∩U0) \ {0}, we have Choosing ϵ0 ∈(C ∩U0) \ {0}, we have z0 + ϵ0 ∈–intC. Since z0 ∈(¯x,y0), we have
(¯x,y0) + ϵ0
∩(–intC) ̸= ∅. Hence ¯x /∈ϵ0-XW min(,A), and therefore ¯x /∈
ϵ∈C\{0}
ϵ-XW min(,A). Thus Thus
ϵ∈C\{0}
ϵ-XW min(,A) ⊂XW min(,A).
ϵ∈C\{0}
ϵ-XW min(,A) ⊂XW min(,A). From this we obtain □ 4 Kuhn–Tucker-type optimality conditions Since –intC and –intD are open sets, combining with
cl
cone
ϕ(X0) + C × D
∩
(–intC) × (–intD)
=
Since ϕ is nearly C × D-subconvexlike on X0, by
cl(cone(ϕ(X0) + C × D)) is convex. By the separation the
(s∗,k∗) ∈Y ∗× Z∗\ {(0Y∗,0Z∗)} such that
s∗,k∗
cl
cone
ϕ(X0) + C × D
≥s∗(–intC) + k∗ Proof Since ¯x is an ϵ-weakly efficient solution to the -SVEPC, we have Proof Since ¯x is an ϵ-weakly efficient solution to the -SVEPC, we have
(¯x,x) + ϵ
∩(–intC) = ∅,
∀x ∈A. (4.1) (¯x,x) + ϵ
∩(–intC) = ∅,
∀x ∈A. (4.1) (4.1) Next, we prove that Next, we prove that
cone
ϕ(X0) + C × D
∩
(–intC) × (–intD)
= ∅. (4.2) one
ϕ(X0) + C × D
∩
(–intC) × (–intD)
= ∅. (4.2) (4.2) Suppose to the contrary that there exist ˆλ ≥0 and ˆx ∈X0 such that ˆλ
(¯x, ˆx) + C + ϵ
∩(–intC) ̸= ∅ (4.3) (4.4) From 0 /∈–intD we have ˆλ > 0. Since D is a convex cone, combining with (4.4), we have
G(ˆx) ∩(–intD) ̸= ∅. G(ˆx) ∩(–intD) ̸= ∅. It is clear that 4 Kuhn–Tucker-type optimality conditions In this section, under the assumption of near C-subconvexlikeness, we establish Kuhn–
Tucker-type sufficient and necessary optimality conditions for approximate weakly effi-
cient solutions to the set-valued vector equilibrium problems, which generalize the rele-
vant results given by Gong [2] and Yang [17]. Definition 4.1 Let ¯x ∈X0, and let ϕ : X0 →2Y×Z be an ordered pair mapping defined as
ϕ(x) = ((¯x,x) + ϵ,G(x)), ∀x ∈X0. By definition, ϕ is nearly C×D-subconvexlike on X0 if and only if cl(cone(ϕ(X0)+C×D))
is convex, where ϕ(X0) =
x∈X0 ϕ(x) =
x∈X0((¯x,x) + ϵ,G(x)). Lemma 4.1 ([15]) If y∗∈C∗\ {0Y∗}, c0 ∈intC, then y∗(c0) > 0. Lemma 4.1 ([15]) If y∗∈C∗\ {0Y∗}, c0 ∈intC, then y∗(c0) > 0. Theorem 4.1 Suppose that ϕ is nearly C × D-subconvexlike on X0 and that there exists
x0 ∈X0 such that G(x0)∩(–intD) ̸= ∅. If ¯x is an ϵ-weakly efficient solution to the -SVEPC,
then there exist s∗∈C∗\ {0Y∗} and k∗∈D∗such that s∗(y) + s∗(ϵ) + k∗(z) ≥0,
∀x ∈X0,y ∈(¯x,x),z ∈G(x). alities and Applications ( 2018) 2018:181
Page 8 of 17 Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181
Page 8 of 17 Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181 Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181 Page 8 of 17 q
pp
(
)
Proof Since ¯x is an ϵ-weakly efficient solution to the -
(¯x,x) + ϵ
∩(–intC) = ∅,
∀x ∈A. Next, we prove that
cone
ϕ(X0) + C × D
∩
(–intC) × (–intD)
= ∅. Suppose to the contrary that there exist ˆλ ≥0 and ˆx ∈X
ˆλ
(¯x, ˆx) + C + ϵ,G(ˆx) + D
∩
(–intC) × (–intD
Thus,
ˆλ
(¯x, ˆx) + C + ϵ
∩(–intC) ̸= ∅
and
ˆλ
G(ˆx) + D
∩(–intD) ̸= ∅. From 0 /∈–intD we have ˆλ > 0. Since D is a convex cone
G(ˆx) ∩(–intD) ̸= ∅. It is clear that
G(ˆx) ∩(–D) ̸= ∅. Thus ˆx ∈A. Since intC ∪{0} is a cone, by (4.3) we get
(¯x, ˆx) + C + ϵ
∩(–intC) ̸= ∅. Since C is a convex cone, we have
(¯x, ˆx) + ϵ
∩(–intC) ̸= ∅,
which contradicts (4.1), and thus we obtain (4.2). It is clear that G(ˆx) ∩(–D) ̸= ∅. Thus ˆx ∈A. Since intC ∪{0} is a cone, by (4.3) we get
(¯x, ˆx) + C + ϵ
∩(–intC) ̸= ∅. Since C is a convex cone, we have
(¯x, ˆx) + ϵ
∩(–intC) ̸= ∅,
(¯x, ˆx) + ϵ
∩(–intC) ̸= ∅, which contradicts (4.1), and thus we obtain (4.2). Since –intC and –intD are open sets, combining with (4.2), we have which contradicts (4.1), and thus we obtain (4.2). Since –intC and –intD are open sets, combining with (4.2), we have
cl
cone
ϕ(X0) + C × D
∩
(–intC) × (–intD)
= ∅. (4.5) (4.5) Since ϕ is nearly C × D-subconvexlike on X0, by Definition 4.1 we can see that
cl(cone(ϕ(X0) + C × D)) is convex. By the separation theorem for convex sets, there exists
(s∗,k∗) ∈Y ∗× Z∗\ {(0Y∗,0Z∗)} such that
s∗,k∗
cl
cone
ϕ(X0) + C × D
≥s∗(–intC) + k∗(–intD). Page 9 of 17 Page 9 of 17 Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181 Since cl(cone(ϕ(X0) + C × D)) is a cone, and there is a lower bound of (s∗,k∗) on
cl(cone(ϕ(X0) + C × D)), we have
s∗,k∗
cl
cone
ϕ(X0) + C × D
≥0.
s∗,k∗
cl
cone
ϕ(X0) + C × D
≥0. Hence Hence (4.6)
s∗,k∗
ϕ(X0) + C × D
≥0. Since (0Y,0Z) ∈C × D, we have
s∗,k∗
ϕ(X0)
≥0.
s∗,k∗
ϕ(X0)
≥0. Thus s∗
(¯x,x) + ϵ
+ k∗
G(x)
≥0,
∀x ∈X0. It is clear that s∗(y) + s∗(ϵ) + k∗(z) ≥0,
∀x ∈X0,y ∈(¯x,x),z ∈G(x). (4.7) s∗(y) + s∗(ϵ) + k∗(z) ≥0,
∀x ∈X0,y ∈(¯x,x),z ∈G(x). (4.7) s∗(y) + s∗(ϵ) + k∗(z) ≥0,
∀x ∈X0,y ∈(¯x,x),z ∈G(x). (4.7) From (4.6) we get 0 ∈k∗
G(¯x)
. From 0 ∈(¯x, ¯x) and (4.14) it follows that □ Combining with (4.11), we obtain (4.10). The proof is complete. □ From (4.6) we get (4.13)
From q ∈G(¯x) we have k∗(q) ≥0. Combining with (4.12), we obtain
k∗(q) = 0. (4.13) Thus Thus (4.14) 0 ∈k∗
G(¯x)
. From (4.6) we get (4.8) Next, we prove that s∗(c) ≥0,
∀c ∈C. Suppose to the contrary that there exists c0 ∈C such that s∗(c0) < 0. When δ1 is large enough, there exist x1 ∈X0,y1 ∈(¯x,x1),z1 ∈G(x1),δ′
2 ≥0, and d1 ∈D
such that When δ1 is large enough, there exist x1 ∈X0,y1 ∈(¯x,x1),z1 ∈G(x1),δ′
2 ≥0, and d1 ∈D
such that δ1s∗(c0) < –s∗(y1 + ϵ) – k∗
z1 + δ′
2d1
, which contradicts (4.8). Hence we obtain s∗(c) ≥0,
∀c ∈C. Similarly, we get k∗(d) ≥0,
∀d ∈D. Page 10 of 17 Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181 Thus Thus s∗∈C∗,
k∗∈D∗. Then we need to prove that s∗̸= 0Y∗. Suppose to the contrary that s∗= 0Y∗. Since (s∗,k∗) ̸= (0Y∗,0Z∗), we have s∗= 0Y∗. Since (s∗,k∗) ̸= (0Y∗,0Z∗), we have k∗̸= 0Z∗. From k∗∈D∗\ {0Z∗}, s∗= 0Y∗, and (4.7) we can see that
k∗
G(x)
≥0,
∀x ∈X0. (4.9) k∗̸= 0Z∗. k∗̸= 0Z∗. From k∗∈D∗\ {0Z∗}, s∗= 0Y∗, and (4.7) we can see that
k∗
G(x)
≥0,
∀x ∈X0. From k∗∈D∗\ {0Z∗}, s∗= 0Y∗, and (4.7) we can see that From k∗∈D∗\ {0Z∗}, s∗= 0Y∗, and (4.7) we can see that k∗
G(x)
≥0,
∀x ∈X0. (4.9) (4.9) On the other hand, there exists x0 ∈X0 such that G(x0)∩(–intD) ̸= ∅, and thus there exists
p ∈G(x0) ∩(–intD), so that by Lemma 4.1 we obtain k∗(p) < 0, which contradicts (4.9). Hence s∗∈C∗\ {0Y∗}. □ Corollary 4.1 Suppose that ¯x ∈A, 0 ∈(¯x, ¯x), ϕ is nearly C × D-subconvexlike on X0, and
there exists x0 ∈X0 such that G(x0) ∩(–intD) ̸= ∅. If ¯x is a weakly efficient solution to the
-SVEPC, then there exist s∗∈C∗\ {0Y∗} and k∗∈D∗such that mink∗(G(¯x)) = 0 and min
s∗(y) + k∗(z) : x ∈X0,y ∈(¯x,x),z ∈G(x)
= 0. (4.10) (4.10) Proof In the proof of Theorem 4.1, letting ϵ = 0, we see that s∗(y) + k∗(z) ≥0,
∀x ∈X0,y ∈(¯x,x),z ∈G(x). (4.11) From ¯x ∈A we have G(¯x) ∩(–D) ̸= ∅. G(¯x) ∩(–D) ̸= ∅. Thus there exists q ∈G(¯x) such that q ∈–D, and since k∗∈D∗, we have (4.12) k∗(q) ≤0. Page 11 of 17 Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181 Letting x = ¯x in (4.11), by 0 ∈(¯x, ¯x) we have
k∗(z) ≥0,
∀z ∈G(¯x). Corollary 4.2 Assume that
(i) ¯x ∈A and s∗(y) + k∗(z) ≥0,
∀x ∈X0,y ∈(¯x,x),z ∈G(x). Then ¯x is a weakly efficient solution to the -SVEPC. Then ¯x is a weakly efficient solution to the -SVEPC. □ Proof Letting ϵ = 0 in Theorem 4.2, we get the conclusion. □ Corollary 4.3 Suppose that ¯x ∈A, 0 ∈(¯x, ¯x), ϕ is nearly C × D-subconvexlike on X0, and
there exists x0 ∈X0 such that G(x0)∩(–intD) ̸= ∅. Then ¯x is a weakly efficient solution to the
-SVEPC if and only if there exist s∗∈C∗\ {0Y∗} and k∗∈D∗such that mink∗(G(¯x)) = 0
and min
s∗(y) + k∗(z) : x ∈X0,y ∈(¯x,x),z ∈G(x)
= 0. Proof This follows directly from Corollaries 4.1 and 4.2. □ Proof This follows directly from Corollaries 4.1 and 4.2. □ □ Remark 4.1 Corollary 4.3 extends Theorem 3.1 of Gong [2] in the following aspects:
(i) The vector-valued function is extended to a set-valued function; Remark 4.1 Corollary 4.3 extends Theorem 3.1 of Gong [2] in the following aspects: (i) The vector-valued function is extended to a set-valued function; (ii) The cone-convexity of ϕ is extended to near cone-subconvexlikeness (ii) The cone-convexity of ϕ is extended to near cone-subconvexlikeness. Corollary 4.4 Suppose that ¯x ∈X0, ¯y ∈F(¯x), (F – ¯y,G) is nearly C × D-subconvexlike on
X0, and there exists x0 ∈X0 such that G(x0) ∩(–intD) ̸= ∅. If (¯x, ¯y) is a weakly efficient pair
to the (SOP), then there exist s∗∈C∗\ {0Y∗} and k∗∈D∗such that mink∗(G(¯x)) = 0 and Corollary 4.4 Suppose that ¯x ∈X0, ¯y ∈F(¯x), (F – ¯y,G) is nearly C × D-subconvexlike on
X0, and there exists x0 ∈X0 such that G(x0) ∩(–intD) ̸= ∅. If (¯x, ¯y) is a weakly efficient pair
to the (SOP), then there exist s∗∈C∗\ {0Y∗} and k∗∈D∗such that mink∗(G(¯x)) = 0 and Corollary 4.4 Suppose that ¯x ∈X0, ¯y ∈F(¯x), (F – ¯y,G) is nearly C × D-subconvexlike on
X0, and there exists x0 ∈X0 such that G(x0) ∩(–intD) ̸= ∅. If (¯x, ¯y) is a weakly efficient pair
to the (SOP), then there exist s∗∈C∗\ {0Y∗} and k∗∈D∗such that mink∗(G(¯x)) = 0 and s∗(¯y) = min
s∗
F(x)
+ k∗
G(x)
: x ∈X0
. Proof Letting (y,x) = F(x) – ¯y, it is clear that (y,x) depends only upon the second vari-
able. Theorem 4.2 Assume that
(i) ¯x ∈A and (ii) there exist s∗∈C∗\ {0Y∗} and k∗∈D∗such that
s∗(y) + s∗(ϵ) + k∗(z) ≥0,
∀x ∈X0,y ∈(¯x,x),z ∈G(x). Then ¯x is an ϵ-weakly efficient solution to the -SVEPC. Proof Suppose to the contrary that ¯x is not an ϵ-weakly efficient solution to the -SVEPC. Then we can find ˆx ∈A such that Thus there exists ˆy ∈(¯x, ˆx) such that Thus there exists ˆy ∈(¯x, ˆx) such that ˆy + ϵ ∈–intC. By s∗∈C∗\ {0Y∗} and Lemma 4.1 we have (4.15) s∗(ˆy) + s∗(ϵ) < 0. s∗(ˆy) + s∗(ϵ) < 0. Choosing ˆz ∈G(ˆx) ∩(–D), since k∗∈D∗, we have k∗(ˆz) ≤0. Combining with (4.15), we
obtain Choosing ˆz ∈G(ˆx) ∩(–D), since k∗∈D∗, we have k∗(ˆz) ≤0. Combining with (4.15), we
obtain s∗(ˆy) + s∗(ϵ) + k∗(ˆz) < 0, which contradicts (ii), and hence ¯x is an ϵ-weakly efficient solution to the -SVEPC. □ which contradicts (ii), and hence ¯x is an ϵ-weakly efficient solution to the -SVEPC. □ Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181 Page 12 of 17 Corollary 4.2 Assume that
(i) ¯x ∈A and Since ¯y ∈F(¯x), we have 0 ∈F(¯x) – ¯y, and hence 0 ∈(¯x, ¯x) and Proof Letting (y,x) = F(x) – ¯y, it is clear that (y,x) depends only upon the second vari-
able. Since ¯y ∈F(¯x), we have 0 ∈F(¯x) – ¯y, and hence 0 ∈(¯x, ¯x) and
F(x) – ¯y,G(x)
=
(¯x,x),G(x)
.
F(x) – ¯y,G(x)
=
(¯x,x),G(x)
.
F(x) – ¯y,G(x)
=
(¯x,x),G(x)
. Since (¯x, ¯y) is a weakly efficient pair to the (SOP), we can see that ¯x is a weakly efficient
solution to the -SVEPC. By Corollary 4.1 we get the conclusion. □ Since (¯x, ¯y) is a weakly efficient pair to the (SOP), we can see that ¯x is a weakly efficient
solution to the -SVEPC. By Corollary 4.1 we get the conclusion. □ Remark 4.2 From Remarks 3.1 and 3.3 in [18] we can see that if (F – ¯y,G) is generalized
C × D-subconvexlike on X0, then (F – ¯y,G) is nearly C × D-subconvexlike on X0. Thus,
Corollary 4.4 generalizes Theorem 4.2 in [17]. Remark 4.2 From Remarks 3.1 and 3.3 in [18] we can see that if (F – ¯y,G) is generalized
C × D-subconvexlike on X0, then (F – ¯y,G) is nearly C × D-subconvexlike on X0. Thus,
Corollary 4.4 generalizes Theorem 4.2 in [17]. From Theorems 4.1 and 4.2 we obtain the following result. Corollary 4.5 Suppose that ¯x ∈A, ϕ is nearly C × D-subconvexlike on X0 and that there
exists x0 ∈X0 such that G(x0) ∩(–intD) ̸= ∅. Then ¯x is an ϵ-weakly efficient solution to the
-SVEPC if and only if there exist s∗∈C∗\ {0Y∗} and k∗∈D∗such that Corollary 4.5 Suppose that ¯x ∈A, ϕ is nearly C × D-subconvexlike on X0 and that there
exists x0 ∈X0 such that G(x0) ∩(–intD) ̸= ∅. Then ¯x is an ϵ-weakly efficient solution to the
-SVEPC if and only if there exist s∗∈C∗\ {0Y∗} and k∗∈D∗such that s∗(y) + s∗(ϵ) + k∗(z) ≥0,
∀x ∈X0,y ∈(¯x,x),z ∈G(x). Page 13 of 17 Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181 5 Lagrange-type optimality conditions In this section, we present Lagrange-type sufficient and necessary optimality conditions
for approximate weakly efficient solutions to the set-valued vector equilibrium problems,
which generalize the relevant results given by Rong [15]. Theorem 5.1 Suppose that ¯x ∈A, 0 ∈(¯x, ¯x), ϕ is nearly C × D-subconvexlike on X0, and
there exists x0 ∈X0 such that G(x0) ∩(–intD) ̸= ∅. If ¯x is an ϵ-weakly efficient solution to
the -SVEPC, then there exists ¯T ∈L+(Z,Y) such that – ¯T(G(¯x) ∩(–D)) ⊂((intC ∩{0}) \
(ϵ +intC)), and ¯x is an ϵ-weakly efficient solution to the
-USVEP, where
: X0 ×X0 →2Y
is defined by (y,x) = (y,x) + ¯T
G(x)
. Proof From the proof of Theorem 4.1 we see that there exist s∗∈C∗\ {0Y∗} and k∗∈D∗
satisfying (4.7). Since s∗∈C∗\ {0Y∗}, we can find c0 ∈intC such that s∗(c0) = 1. Define the operator
¯T : Z →Y by Proof From the proof of Theorem 4.1 we see that there exist s∗∈C∗\ {0Y∗} and k∗∈D∗
satisfying (4.7). Proof From the proof of Theorem 4.1 we see that there exist s∗∈C∗\ {0Y∗} and k∗∈D∗
satisfying (4.7). Since s∗∈C∗\ {0Y∗}, we can find c0 ∈intC such that s∗(c0) = 1. Define the operator
¯T : Z →Y by Since s∗∈C∗\ {0Y∗}, we can find c0 ∈intC such that s∗(c0) = 1. Define the operator
¯T : Z →Y by ¯T(z) = k∗(z)c0,
∀z ∈Z. ¯T(z) = k∗(z)c0,
∀z ∈Z. Thus ¯T(D) = k∗(D)c0 ⊂C. It is evident that Thus ¯T(D) = k∗(D)c0 ⊂C. It is evident that ¯T ∈L+(Z,Y). Letting x = ¯x in (4.7), since 0 ∈(¯x, ¯x), we have Letting x = ¯x in (4.7), since 0 ∈(¯x, ¯x), we have Letting x = ¯x in (4.7), since 0 ∈(¯x, ¯x), we have s∗(ϵ) + k∗(z) ≥0,
∀z ∈G(¯x) ∩(–D). ( (5.1) s∗(ϵ) + k∗(z) ≥0,
∀z ∈G(¯x) ∩(–D). Noticing that z ∈–D, we obtain Noticing that z ∈–D, we obtain – ¯T(z) = –k∗(z)c0 ∈intC ∪{0}. Thus – ¯T
G(¯x) ∩(–D)
⊂intC ∪{0}. (5.2) Next, we prove Next, we prove – ¯T
G(¯x) ∩(–D)
∩(ϵ + intC) = ∅. – ¯T
G(¯x) ∩(–D)
∩(ϵ + intC) = ∅. (5.3) – ¯T
G(¯x) ∩(–D)
∩(ϵ + intC) = ∅. Theorem 5.2 Assume that ( )
(ii) there exists ¯T ∈L+(Z,Y) such that ¯x is an ϵ-weakly efficient solution to the
-USVEP, where
: X0 × X0 →2Y is defined by (y,x) = (y,x) + ¯T
G(x)
. Then ¯x is an ϵ-weakly efficient solution to the -SVEPC. 5 Lagrange-type optimality conditions (5.3) (5.3) Suppose to the contrary that there exists ˜z ∈G(¯x) ∩(–D) such that Suppose to the contrary that there exists ˜z ∈G(¯x) ∩(–D) such that – ¯T(˜z) ∈ϵ + intC. (5.4) – ¯T(˜z) ∈ϵ + intC. (5.4)
Thus – ¯T(˜z) – ϵ ∈intC. By the definition of ¯T we have
s∗
– ¯T(˜z) – ϵ
= s∗
–k∗(˜z)c0 – ϵ
= –
s∗(ϵ) + k∗(˜z)
. (5.4) – ¯T(˜z) ∈ϵ + intC. – ¯T(˜z) ∈ϵ + intC. Thus – ¯T(˜z) – ϵ ∈intC. By the definition of ¯T we have s∗
– ¯T(˜z) – ϵ
= s∗
–k∗(˜z)c0 – ϵ
= –
s∗(ϵ) + k∗(˜z)
. Page 14 of 17 Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181 Combining with (5.1), we have Combining with (5.1), we have (5.5) s∗
– ¯T(˜z) – ϵ
≤0. s∗
– ¯T(˜z) – ϵ
≤0. (5.5 On the other hand, from s∗∈C∗\ {0Y∗} and Lemma 4.1 it follows that s∗(intC) > 0, s∗(intC) > 0, which, together with (5.5), gives – ¯T(˜z) – ϵ /∈intC, which contradicts (5.4). Thus we obtain (5.3). The combination of (5.2) and (5.3) leads to which contradicts (5.4). Thus we obtain (5.3). The combination of (5.2) and (5.3) leads to which contradicts (5.4). Thus we obtain (5.3). The combination of (5.2) and (5.3) leads to – ¯T
G(¯x) ∩(–D)
⊂
intC ∩{0}
\ (ϵ + intC)
. Finally, we prove that ¯x is an ϵ-weakly efficient solution to the
-USVEP. In fact, by the definition of ¯T and (4.7) we obtain
s∗
(¯x,x) + ¯T
G(x)
+ ϵ
= s∗
(¯x,x)
+ s∗(ϵ) + k∗
G(x)
≥0,
∀x ∈X0. Since s∗(–intC) < 0, we have
(¯x,x) + ¯T
G(x)
+ ϵ
∩(–intC) = ∅,
∀x ∈X0. Consequently, Finally, we prove that ¯x is an ϵ-weakly efficient solution to the
-USVEP. In fact, by the definition of ¯T and (4.7) we obtain □ It is evident that ¯x is an ϵ-weakly efficient solution to the
-USVEP. Then ¯x is an ϵ-weakly efficient solution to the -SVEPC. Proof Since ¯x is an ϵ-weakly efficient solution to the
-USVEP, we have
x∈X0
(¯x,x) + ϵ
∩(–intC) = ∅.
x∈X0
(¯x,x) + ϵ
∩(–intC) = ∅. Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181 Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181 Page 15 of 17 Since C is a convex cone, we obtain Since C is a convex cone, we obtain Since C is a convex cone, we obtain
x∈X0
(¯x,x) + C + ϵ
∩(–intC) = ∅. (5.6) (5.6) On the other hand, for any x ∈A, we have G(x)∩(–D) ̸= ∅. Thus there exists zx ∈G(x) such
that zx ∈–D. It follows from ¯T ∈L+(Z,Y) that ¯T(zx) ∈–C, thus C – ¯T(zx) ⊂C + C ⊂C,
and hence C ⊂¯T(zx) + C; since zx ∈G(x), it is evident that C ⊂¯T(G(x)) + C. Thus x∈A
(¯x,x) + C + ϵ
⊂
x∈A
(¯x,x) + ¯T
G(x)
+ C + ϵ
⊂
x∈X0
(¯x,x) + ¯T
G(x)
+ C + ϵ
=
x∈X0
(¯x,x) + C + ϵ
. ⊂
x∈X0
(¯x,x) + ¯T
G(x)
+ C + ϵ
=
x∈X0
(¯x,x) + C + ϵ
. It follows from (5.6) that It follows from (5.6) that
x∈A
(¯x,x) + C + ϵ
∩(–intC) = ∅. Since 0 ∈C, it is evident that Since 0 ∈C, it is evident that
x∈A
(¯x,x) + ϵ
∩(–intC) = ∅. Hence ¯x is an ϵ-weakly efficient solution to the -SVEPC. □ □ Hence ¯x is an ϵ-weakly efficient solution to the -SVEPC. From Theorems 5.1 and 5.2 we obtain the following result. From Theorems 5.1 and 5.2 we obtain the following result. Corollary 5.1 Suppose that ¯x ∈A, 0 ∈(¯x, ¯x), ϕ is nearly C × D-subconvexlike on X0, and
there exists x0 ∈X0 such that G(x0) ∩(–intD) ̸= ∅. Then ¯x is an ϵ-weakly efficient solution
to the -SVEPC if and only if there exists ¯T ∈L+(Z,Y) such that ¯x is an ϵ-weakly efficient
solution to the
-USVEP, where
: X0 × X0 →2Y is defined by (y,x) = (y,x) + ¯T
G(x)
. Corollary 5.2 Suppose that (F – ¯y,G) is nearly C ×D-subconvexlike on X0 and there exists
x0 ∈X0 such that G(x0)∩(–intD) ̸= ∅. If (¯x, ¯y) is an ϵ-weakly efficient pair to the (SOP), then
there exists ¯T ∈L+(Z,Y) such that – ¯T(G(¯x) ∩(–D)) ⊂((intC ∩{0}) \ (ϵ + intC)), and (¯x, ¯y)
is an ϵ-weakly efficient pair to the (USOP) ¯T. Proof Letting (y,x) = F(x) – ¯y, since ¯y ∈F(¯x), it is evident that 0 ∈(¯x, ¯x) and (F(x) –
¯y,G(x)) = ((¯x,x),G(x)). Proof This follows directly from Corollaries 5.2 and 5.3.
□ Since C is a convex cone, we obtain Since (¯x, ¯y) is an ϵ-weakly efficient pair to the (SOP), we see that ¯x is an ϵ-weakly efficient
solution to the -SVEPC. Page 16 of 17 Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181 Thus by Theorem 5.1 there exists ¯T ∈L+(Z,Y) such that – ¯T(G(¯x) ∩(–D)) ⊂((intC ∩
{0}) \ (ϵ + intC)) and ¯x is an ϵ-weakly efficient solution to the
-USVEP, that is,
x∈X0
(¯x,x) + ¯T
G(x)
+ ϵ
∩(–intC) = ∅. Consequently, Consequently,
x∈X0
F(x) – ¯y + ¯T
G(x)
+ ϵ
∩(–intC) = ∅, which is equivalent to which is equivalent to
x∈X0
L(x, ¯T) + ϵ – ¯y
∩(–intC) = ∅. Thus Thus
L(X0, ¯T) + ϵ – ¯y
∩(–intC) = ∅.
L(X0, ¯T) + ϵ – ¯y
∩(–intC) = ∅. Hence, by Definition 2.5, (¯x, ¯y) is an ϵ-weakly efficient pair to the (USOP) ¯T. □ □ Remark 5.1 If (F,G) is C ×D-subconvexlike, then (F – ¯y,G) is nearly C ×D-subconvexlike. Thus Corollary 5.2 generalizes Theorem 3.1 in [15]. Remark 5.1 If (F,G) is C ×D-subconvexlike, then (F – ¯y,G) is nearly C ×D-subconvexlike. Thus Corollary 5.2 generalizes Theorem 3.1 in [15]. Corollary 5.3 Assume that
(i) ¯x ∈A, ¯y ∈F(¯x), and Authors’ information Authors’ information
Yihong Xu (1969-), Professor, Doctor, the major field of interest is in the area of set-valued optimization. Jian Chen, email:
1169898604@qq.com. Ke Zhang, email: 2768482283@qq.com. Authors’ information
Yihong Xu (1969-), Professor, Doctor, the major field of interest is in the area of set-valued optimization. Jian Chen, email:
1169898604@qq.com. Ke Zhang, email: 2768482283@qq.com. Corollary 5.3 Assume that (ii) there exists ¯T ∈L+(Z,Y) such that (¯x, ¯y) is an ϵ-weakly efficient pair to the (USOP) ¯T. Then (¯x, ¯y) is an ϵ-weakly efficient pair to the (SOP). Proof Letting (y,x) = F(x) – ¯y, since ¯y ∈F(¯x), it is evident that 0 ∈(¯x, ¯x) and (F(x) –
¯y,G(x)) = ((¯x,x),G(x)). Since (¯x, ¯y) is an ϵ-weakly efficient pair to the (USOP) ¯T, we see that ¯x is an ϵ-weakly
efficient solution to the
-USVEP. Combining this with Theorem 5.2, we conclude that
¯x is an ϵ-weakly efficient solution to the -SVEPC; it is clear that (¯x, ¯y) is an ϵ-weakly
efficient pair to the (SOP). □ Remark 5.2 Comparing with Theorem 3.2 in [15], this corollary is not required for the
convexity of (F,G). Corollary 5.4 Suppose that ¯x ∈A, ¯y ∈F(¯x), (F – ¯y,G) is nearly C ×D-subconvexlike on X0,
and there exists x0 ∈X0 such that G(x0) ∩(–intD) ̸= ∅. Then (¯x, ¯y) is an ϵ-weakly efficient
pair to the (SOP) if and only if there exists ¯T ∈L+(Z,Y) such that (¯x, ¯y) is an ϵ-weakly
efficient pair to the (USOP) ¯T. □ Proof This follows directly from Corollaries 5.2 and 5.3. □ Proof This follows directly from Corollaries 5.2 and 5.3. Page 17 of 17 Page 17 of 17 Chen et al. Journal of Inequalities and Applications ( 2018) 2018:181 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Received: 5 May 2018 Accepted: 22 June 2018 Received: 5 May 2018 Accepted: 22 June 2018 6 Conclusions In this paper, we discuss some relationships between approximate weakly efficient solu-
tions and weakly efficient solutions of set-valued vector equilibrium problems. We con-
clude that
ϵ∈C\{0} ϵ-XW min(,A) = XW min(,A), and hence it is really “approximate”. The
optimality conditions for set-valued vector equilibrium problems are established, and the
results we obtained generalize those of Gong[2], Yang[17], and Rong[15]. As an exten-
sive mathematical model, further research on approximate weakly efficient solutions of
set-valued vector equilibrium problems seems to be of interest and value. Competing interests Competing interests
The authors declare that they have no competing interests. Authors’ contributions
All authors contributed to each part of this work equally, and they all read and approved the final manuscript. References 1. Anh, L.Q., Duy, T.Q., Khanh, P.Q.: Continuity properties of solution maps of parametric lexicographic equilibrium
problems. Positivity 20, 1–20 (2015) p
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2. Gong, X.H.: Optimality conditions for vector equilibrium problems. J. Math. Anal. Appl. 342, 1455–1466 (2008) 2. Gong, X.H.: Optimality conditions for vector equilibrium problems. J. Math. Anal. Appl. 342, 1455–1466 (2008)
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(2001) 4. Gong, X.H.: Efficiency and Henig efficiency for vector equilibrium problems. J. Optim. Theory Appl. 108, 139–154
(2001) 5. Long, X.J., Huang, Y.Q., Peng, Z.Y.: Optimality conditions for the Henig efficient solution of vector equilibrium
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problems with constraints. Optim. Lett. 5, 717–728 (2011) 6. Qiu, Q.S.: Optimality conditions for vector equilibrium problems with constraints. J. Ind. Manag. Optim. 5, 783–790
(2017) 6. Qiu, Q.S.: Optimality conditions for vector equilibrium problems with constraints. J. Ind. Manag. Optim. 5, 783–79
(2017) 7. Luu, D.V., Hang, D.D.: Efficient solutions and optimality conditions for vector equilibrium problems. Math. Methods
Oper. Res. 79, 163–177 (2014) p
8. Luu, D.V.: Optimality condition for local efficient solutions of vector equilibrium problems via convexificators and
applications. J. Optim. Theory Appl. 171, 643–665 (2016) 9. Loridan, P.: Necessary conditions for ϵ-optimality. Math. Program. 19, 140–152 (1982) 9. Loridan, P.: Necessary conditions for ϵ-optimality. Math. Program. 19, 140–152 (1982) 10. Loridan, P.: ϵ-solutions in vector minimization problems. J. Optim. Theory Appl. 43, 265–276 (1984) 10. Loridan, P.: ϵ-solutions in vector minimization problems. J. Optim. Theory Appl. 43, 265–276 (1984) 11. Li, B.X., Li, S.J.: Continuity of approximate solution mappings for parametric equilibrium problems. J. Glob. Optim. 51
541–548 (2011) 11. Li, B.X., Li, S.J.: Continuity of approximate solution mappings for parametric equilibrium problems. J. Glob. Optim
541–548 (2011) 12. Yang, X.M., Li, D., Wang, S.Y.: Near-subconvexlikeness in vector optimization with set-valued functions. J. Optim. Theory Appl. 110, 413–427 (2001) y
13. Sach, P.H.: New generalized convexity notion for set-valued maps and application to vector optimization. J. Optim. Theory Appl. 125, 157–179 (2005) Theory Appl. 125, 157–179 (2005) 14. Funding
h Funding
This research was supported by the National Natural Science Foundation of China Grant (11461044), the Natural Science
Foundation of Jiangxi Province (20151BAB 201027), and the Educational Commission of Jiangxi Province (GJJ12010). References Xu, Y.H., Song, X.S.: The relationship between ic-cone-convexness and nearly cone-subconvexlikeness. Appl. Math. Lett. 24, 1622–1624 (2011) 15. Rong, W.D., Wu, Y.N.: ϵ-weak minimal solutions of vector optimization problems with set-valued maps. J. Optim. Theory Appl. 106, 569–579 (2000) 16. Li, Z.F., Chen, G.Y.: Lagrangian multipliers, saddle points, and duality in vector optimization of set-valued maps. J Math Anal Appl 215 279 316 (1997) 16. Li, Z.F., Chen, G.Y.: Lagrangian multipliers, saddle points, and duality in vector optimization of set-valued maps. J. Math. Anal. Appl. 215, 279–316 (1997) 16. Li, Z.F., Chen, G.Y.: Lagrangian multipliers,
J. Math. Anal. Appl. 215, 279–316 (1997) J. Math. Anal. Appl. 215, 279–316 (1997) 17. Yang, X.M., Yang, X.Q., Chen, G.Y.: The
Theory Appl. 107, 627–640 (2000) 17. Yang, X.M., Yang, X.Q., Chen, G.Y.: Theorems of the alternative and optimization with set-valued maps. J. Optim. Theory Appl. 107, 627–640 (2000) Theory Appl. 107, 627–640 (2000) 18. Sach, P.H.: Nearly subconvexlike set-valued maps and vector optimization problems. J. Optim. Theory Appl. 119,
335–356 (2003) 18. Sach, P.H.: Nearly subconvexlike set-valued maps and vector optimization problems. J. Optim. Theory Appl. 119,
335–356 (2003)
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Internet of Things on the Air Quality Monitoring System Using a Web Server
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Asian Journal of Healthcare Analytics
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Asian Journal of Healthcare Analytics (AJHA)
Vol. 1, No. 2, 2022 : 109-118 Asian Journal of Healthcare Analytics (AJHA)
Vol. 1, No. 2, 2022 : 109-118 Internet of Things on the Air Quality Monitoring System Using a
Web Server Sumardi Sadi1*, Sri Mulyati2, Perdana Bagaskara Setiawan3
Fakultas Teknik, Universitas Muhammadiyah Tangerang
Corresponding Author: Sumardi Sadi sumardiumt1@umt.ac.id A R T I C L E I N F O
Keywords: PPM, Webserver,
Atmega328p, Air Detection
Received: 03 July
Revised: 03 September
Accepted: 03 November
©2022 Sadi, Mulyati, Setiawan : This
is an open-access article distributed
under the terms of the Creative
Commons
Atribusi
4.0
Internasional. A R T I C L E I N F O
Keywords: PPM, Webserver,
Atmega328p, Air Detection
Received: 03 July
Revised: 03 September
Accepted: 03 November
©2022 Sadi, Mulyati, Setiawan : This
is an open-access article distributed
under the terms of the Creative
Commons
Atribusi
4.0
Internasional. A R T I C L E I N F O
Keywords: PPM, Webserver,
Atmega328p, Air Detection A B S T R A C T The increase in the amount of air pollution that
causes global warming and the lack of attention to
harmful gases, such as CO, PM 2.5, and NO2 can
have a negative impact, on health and can even
cause death, if it is ignored by people who breathe
it. One of the popular information media today is
the web, a tool that can monitor air quality levels
and by utilizing advances in technology this
monitoring system can be monitored via a Web
Server. In this study, using an AVR microcontroller
type ATmega 328p as a central control unit, and an
ESP
8266-01
as
a
controller
that
handles
communication between the microcontroller and
the internet network. In addition, this system uses
the C language as a configuration between the
microcontroller and the ethernet system. The level
of air pollution is measured by the MQ 07 sensor
which functions to measure CO levels, MQ 135 to
measure NO2 levels, and the Dust Sensor to
measure PM 2.5. Test results Obtain PPM (parts per
million) values for CO levels of 0.25 ppm, NO2 of
0.83 ppm, PM 2.5 of 0.10 g/m3 and the temperature
can be seen directly through the dashboard
thingboard. 109
(
DOI prefik: 10.55927
https://journal.formosapublisher.org/index.php/ajha (
DOI prefik: 10.55927
https://journal.formosapublisher.org/index.php/ajha 109 Sadi, Mulyati, Setiawan Internet pada Sistem Pemantauan Kualitas Udara Menggunakan
Server Web Sumardi Sadi1*, Sri Mulyati2, Perdana Bagaskara Setiawan3
Fakultas Teknik, Universitas Muhammadiyah Tangerang
Corresponding Author: Sumardi Sadi sumardiumt1@umt.ac.id A R T I C L E I N F O
A B S T R A K
Kata Kunci : PPM, Webserver
, Atmega328p, Deteksi Udara
Received: 03 Juli
Revised: 03 September
Accepted: 03 November
©2022 Sadi, Mulyati, Setiawan : This
is an open-access article distributed
under the terms of the Creative
Commons
Atribusi
4.0
Internasional. Peningkatan
jumlah
polusi
udara
yang
menyebabkan pemanasan global dan kurangnya
perhatian terhadap gas-gas berbahaya seperti CO,
PM 2.5, dan NO2 dapat memberikan dampak yang
negatif bagi kesehatan bahkan dapat menyebabkan
kematian jika itu diabaikan oleh orang-orang yang
menghirupnya. Salah satu media informasi yang
populer saat ini adalah web sebuah alat yang dapat
memantau tingkat kualitas udara dan dengan
memanfaatkan
kemajuan
teknologi
sistem
monitoring ini dapat dipantau melalui Web Server. Dalam
penelitian
ini,
menggunakan
mikrokontroler AVR tipe ATmega 328p sebagai
unit pusat kontrol dan sebuah ESP 8266-01 sebagai
kontroler yang menangani komunikasi antara
mikrokontroler dengan jaringan internet. Selain
itu, sistem ini menggunakan bahasa C sebagai
konfigurasi antara mikrokontroler dan sistem
ethernet. Tingkat polusi udara diukur dengan
sensor MQ 07 yang berfungsi untuk mengukur
kadar CO, MQ 135 untuk mengukur kadar NO2,
dan Dust Sensor untuk mengukur PM 2.5. Hasil
pengujian
Mendapatkan
nilai
PPM
(parts
permillion) untuk kadar CO sebesar 0.25 ppm,
NO2 sebesar 0.83 ppm, PM 2.5 sebesar 0.10 μg/m3
dan untuk temperatur bisa dilihat langsung
melalui dashboard thingboard. A B S T R A K
ver
ara
This
uted
tive
4.0
Peningkatan
jumlah
polusi
udara
yang
menyebabkan pemanasan global dan kurangnya
perhatian terhadap gas-gas berbahaya seperti CO,
PM 2.5, dan NO2 dapat memberikan dampak yang
negatif bagi kesehatan bahkan dapat menyebabkan
kematian jika itu diabaikan oleh orang-orang yang
menghirupnya. Salah satu media informasi yang
populer saat ini adalah web sebuah alat yang dapat
memantau tingkat kualitas udara dan dengan
memanfaatkan
kemajuan
teknologi
sistem
monitoring ini dapat dipantau melalui Web Server. Dalam
penelitian
ini,
menggunakan
mikrokontroler AVR tipe ATmega 328p sebagai
unit pusat kontrol dan sebuah ESP 8266-01 sebagai
kontroler yang menangani komunikasi antara
mikrokontroler dengan jaringan internet. Selain
itu, sistem ini menggunakan bahasa C sebagai
konfigurasi antara mikrokontroler dan sistem
ethernet. Tingkat polusi udara diukur dengan
sensor MQ 07 yang berfungsi untuk mengukur
kadar CO, MQ 135 untuk mengukur kadar NO2,
dan Dust Sensor untuk mengukur PM 2.5. PENDAHULUAN Internet of Thing (IoT) merupakan sebuah media untuk berkomunikasi
antara manusia dengan mesin. IoT merupakan interface modern yang dapat
menghubungkan manusia dengan mesin dengan jarak jauh (nirkabel). Aplikasi
IoT salah satunya adalah kita bisa mengembangkan tempat sampah yang
dinamakan dengan tempat sampah pintar, ini adalah salah satu penerapan dari
teknologi yang menggunakan Internet of Thing (IoT) seperti yang ditulis oleh
Ismail pada tahun 2021 (Ismail et al., 2021). Penggunaan internet of thing yang
lain, contohnya adalah pengembangan sistem pemantau konsumsi energi listrik
pada rumah tangga, dimana untuk memantau pemakaian / konsumsi energi
listrik rumah tangga bisa dipantau melalui perangkat komunikasi telepon seluler
atau ponsel. Di dalam seluler tersebut terdapat aplikasi Android yang
menghubungkan antara seluler tersebut dengan peralatan pemantauan energi
listrik. Dimana dengan pembantu alat energi tersebut dapat mengetahui
pembacaan arus dan tegangan nilai arus dan tegangan pun bisa dilihat titik
errornya, jadi yang dikembangkan oleh Hartono Budi Santoso pada tahun 2018
di mana pengembangan sistem pemantauan konsumsi energi rumah tangga
berbasis iot itu untuk pembacaan error pada arus dan tegangan(SANTOSO et al.,
2018). Contoh lainnya penerapan IoT pada Sistem Monitoring : Data,
Kelembaban dan Suhu, (Devira Ramady et al., 2019), (Siregar et al., 2021),
pendeteksi kebakaran (Sasmoko & Mahendra, 2017), kebocoran gas (Mulyati &
Sumardi, 2018), Polusi udara ditimbulkan dari hasil pembakaran yang tidak sempurna,
yang mana proses pembakaran tersebut menghasilkan gas-gas berbahaya
diantaranya yang paling banyak kita sering temukan adalah gas CO (karbon
monoksida) dan Nitrogen dioksida, dll). Sangat sulit memang untuk menekan
tingkat produksi kedua jenis gas tersebut. Hal ini dikarenakan karena kedua jenis
gas ini dihasilkan dari bahan bakar yang saat ini pemakaiannya mencakup
sangat luas. Hampir sebagian besar pada mesin-mesin industri dan kendaraan
bermotor yaitu bahan bakar bensin dan solar. Pencemaran udara diartikan
dengan turunnya kualitas udara sehingga udara mengalami penurunan mutu
dalam penggunaannya dan akhirnya tidak dapat dipergunakan lagi sebagai
mana mestinya sesuai dengan fungsinya. Polusi udara akhir-akhir ini
merupakan masalah yang banyak meresahkan masyarakat. Dampak dari polusi
udara ini sangat berbahaya bagi kesehatan. Berbagai upaya dilakukan
pemerintah untuk menanggulangi masalah ini. Internet pada Sistem Pemantauan Kualitas Udara Menggunakan
Server Web Hasil
pengujian
Mendapatkan
nilai
PPM
(parts
permillion) untuk kadar CO sebesar 0.25 ppm,
NO2 sebesar 0.83 ppm, PM 2.5 sebesar 0.10 μg/m3
dan untuk temperatur bisa dilihat langsung
melalui dashboard thingboard. 110 Asian Journal of Healthcare Analytics (AJHA)
Vol. 1, No. 2, 2022 : 109-118 Asian Journal of Healthcare Analytics (AJHA)
Vol. 1, No. 2, 2022 : 109-118 METODOLOGI Rancangan Sistem Elektronik Rancangan Sistem Elektronik g
Rancangan PCB dibuat dengan software Eagle 7. Schematik dan board
Pcb Monitoring Kualitas Udara ditunjukkan pada Gambar 3 dibawah ini. Gambar 3. Rangkaian Sistem Elektronik Gambar 3. Rangkaian Sistem Elektronik Internet of Things Internet of thing sangat diperlukan pada semua bidang teknologi,
terutama dalam hal ini bahawa dengan internet of thing semua pekerjaan yang
tadinya sulit menjadi mudah. Dalam hal ini internet of thing sebagai media yang
dapat menghubungkan antara manusia dengan mesin pada lingkungan industri
dan juga dapat digunakan pada anggota. Adapun dalam peta, contoh peralatan
yang berlandasan pada aplikasi dari internet of thing diantaranya sebagai
belikut: 111 Sadi, Mulyati, Setiawan Gambar 1. Aplikasi Internet of Things
Web Server
Gambar 2. Aplikasi Sebuah Web Server Gambar 1. Aplikasi Internet of Things Gambar 1. Aplikasi Internet of Things Gambar 1. Aplikasi Internet of Things
Web Server Gambar 1. Aplikasi Internet of Things Gambar 2. Aplikasi Sebuah Web Server Gambar 2. Aplikasi Sebuah Web Server Gambar 2. Aplikasi Sebuah Web Server 112 Asian Journal of Healthcare Analytics (AJHA)
Vol. 1, No. 2, 2022 : 109-118 Perancangan Software Setelah
melakukan
perancangan
hardware,
selanjutnya
adalah
melakukan perancangan software. Pemrograman software menggunakan
aplikasi IDE (Integrated Development Environment) arduino 1.6.9. software ini
dapat berfungsi sebagai editor dan simulator dari program yang telah dibuat
Arduino ini akan menampilkan isi dari program. Port pada saat program buatan
kita sedang dijalankan. Dengan demikian kita dapat mengetahui apakah
program yang di buat sudah benar atau salah. Jika belum benar kita dapat
memperbaiki dengan software Arduino IDE. Perancangan
software
sangat
diperlukan
dalam
penelitian
ini. Perancangan dapat dilakukan dengan menggunakan hardware ataupun
software. Untuk hardware menggunakan program Arduino sedangkan dengan
software menggunakan program proteus. Dua hal itu akan mempermudah di
dalam pengerjaan yang berkaitan dengan apapun, baik pada industri maupun
pada perikanan dan kelautan atau di seluruh bidang yang lainnya. 113 Sadi, Mulyati, Setiawan Gambar 4. Tampilan Program Sistem Monitoring Kualitas Udara Gambar 4. Tampilan Program Sistem Monitoring Kualitas Udara Diagram Alir Sistem Monitoring Kualitas Udara Diagram Alir Sistem Monitoring Kualitas Udara Diagram Alir Sistem Monitoring Kualitas Udara g
g
Pada diagram alir, dimulai dari start kemudian, diinisialisasi bagian
masukan dan bagian keluaran. Bagian maskan dari inisialisasi yaitu input sensor
MQ7, input sensor MQ135, input sensor gas, input sensor BBM180, GPS tracker. Dari masukan tersebut maka akan diproses melalui sebuah sensor. Apakah
sensornya sudah terbaca atau belum terbaca, jika belum terbaca maka akan
kembali lagi menguji bagian masukan yang telah diinisialisasi, jika terbaca oleh
sensor maka akan dilanjutkan untuk mendeteksi CO, NO2, PM, dan suhu,
masing-masing diproses lagi ke atmega328, dari atmega maka akan terbaca
tampilan di layar LCD, apa yang telah di deteksi, kemudian akan terdeteksi
melalui ESP8266-01. Jadi cara kerja untuk diagram ini merupakan gambaran
kerja dari sistem. j
Pada diagram alir dibawah ini, yang perlu diperhatikan adalah cara kerja
sensor, yaitu cara kerja sensor pada masing-masing pemasukan dan juga pada
semua sensor yang digunakan untuk pengeluaran atau sebagai output dari
sebuah sistem. Apabila semua sensor sudah bisa mendeteksi semua pemasukan
dan peneluaran, maka sistem tidak ada gangguan, maka akan mendapatkan
hasil yang lebih cepat dan akurat. 114 Asian Journal of Healthcare Analytics (AJHA)
Vol. 1, No. 2, 2022 : 109-118
Gambar 5. Diagram Alir Cara Kerja System Asian Journal of Healthcare Analytics (AJHA)
Vol. 1, No. 2, 2022 : 109-118 Gambar 5. Diagram Alir Cara Kerja System HASIL DAN PEMBAHASAN Pengujian Keseluruhan g j
Pada pengujian keseluruhan ini yaitu bertujuan untuk mengetahui kinerja
input ,output,dan proses yang digabungkan sehingga menjadi alat monitoriong
udara.pada pengujian keseluruha ini yang pertama dilakukan yaitu melihat
pembacaan akurasi sensor MQ 7 sebagai pembacaan co (karbon monoksida), MQ
135 sebagai pembacaan sensor untuk no2 (nitrogen dioksida), dust sensor
sebagai pembacaan sensor PM 2.5 untuk pendeteksi debu, bmp sebagai
penembacaan temperatur suhu , dan gps neo ublox sebagai pembacaan data
lokasi yang ke lokasi wilayah ketika alat pendeteksi udara ini di tempatkan pada
suatu daerah .terlihat pada gambar di bawah ini hasil pengujian keseluruhan
modul input pada tampilan lcd dan pada tampilan dashboard iot yang telah
dibuat dengan menggunakan cloud iot dari thingboard. 115 Sadi, Mulyati, Setiawan Gambar 6. Tampilan pada Dashboard IoT Gambar 6. Tampilan pada Dashboard IoT Selanjutnya adalah membandingkan hasil pembacaan untuk mengukur
kualitas udara ,dan pada pengukuran udara ini modul yang digunakan ini
adalah sensor MQ7 , MQ135 dan dust sensor, pada pengukuran udara ini
pembacaan sensor akan disamakan dengan table parameter yang bersumber dari
“BADAN PENGENDALI DAMPAK LINGKUNGAN 1998” parameter table
yang digunakan dalam persannan table dari sumber terlihat seperti tabel 4.1
pada di bawah ini. Tabel 1. Parameter Kualitas Udara
CO (ppm) NO2
(ppm)
PM 2.5 (
μg/m3)
Category
0.0 - 4.4
? 0.0 – 15.4
Good
4.5 – 9.4
? 15.5
–
65.4
Moderate
12.5 - 15.4
0.65
\-
1.24
100.5
–
150.4
Unhealty Tabel 1. Parameter Kualitas Udara
CO (ppm) NO2
(ppm)
PM 2.5 (
μg/m3)
Category
0.0 - 4.4
? 0.0 – 15.4
Good
4.5 – 9.4
? 15.5
–
65.4
Moderate
12.5 - 15.4
0.65
\-
1.24
100.5
–
150.4
Unhealty Dalam setiap parameter yang terdapat pada table tesebut yang akan di
pakai pada alat monitoring udara ini yaitu parameter co, no2, pm25 ,maka dalam 116 Asian Journal of Healthcare Analytics (AJHA)
Vol. 1, No. 2, 2022 : 109-118 y
( J
)
Vol. 1, No. 2, 2022 : 109-118 pengujian ini alat akan mendeteksi kualitas udara sekitar dan akan menampilkan
status keadaan udara pada tampilan lcd terlihat seperti gambar dibawah ini pengujian ini alat akan mendeteksi kualitas udara sekitar dan akan menampilkan
status keadaan udara pada tampilan lcd terlihat seperti gambar dibawah ini Gambar 7. Tampilan Hasil Deteksi Gambar 7. Tampilan Hasil Deteksi KESIMPULAN DAN REKOMENDASI Dari data hasil pengujian yang sudah dilakukan didapatkan kesimpulan
sebagai berikut: Pembuatan alat monitoring ini mendapatkan hasil gas CO
sebesar 0,25, gas NO2 sebesar 0,83 dan partikulat PM 2.5 sebesar 0.10. Alat yang
dibuat dengan konsep IoT menggunakan web server mqtt thingsboard berjalan
dengan baik secara realtime berdasarkan hasil pengiriman waktu, tempat dan
ketiga parameter yang dipakai serta suhu udara sekitar. Ketiga parameter yang
dilakukan penelitian ini dapat dinyatakan hasil gas CO dan Partikulat PM 2.5
dalam keadaan bagus untuk manusia sedangkan untuk gas NO2 dinyatakan
tidak sehat bagi manusia. PENELITIAN LANJUTAN J
Setelah
melakukan Setelah
melakukan
perancangan
hardware,
selanjutnya
adalah
melakukan perancangan software. Pemrograman software menggunakan
aplikasi IDE (Integrated Development Environment) arduino 1.6.9. software ini
dapat berfungsi sebagai editor dan simulator dari program yang telah dibuat
Arduino ini akan menampilkan isi dari program. Port pada saat program buatan
kita sedang dijalankan. Dengan demikian kita dapat mengetahui apakah
program yang di buat sudah benar atau salah. Jika belum benar kita dapat
memperbaiki dengan software Arduino IDE. 117 Sadi, Mulyati, Setiawan DAFTAR PUSTAKA Devira Ramady, G., Hidayat, R., Ghea Mahardika, A., Rahman Hakim, R., &
Tinggi Teknologi Mandala, S. (2019). Sistem Monitoring Data pada Smart
Agriculture System Menggunakan Wireless Multisensor Berbasis IoT. Prosiding Seminar Nasional Teknoka, 4(2502), E51–E58. https://doi.org/10.22236/teknoka.v Hendrawati, T. D., Wicaksono, Y. D., & Andika, E. (2018). Internet of Things:
Sistem Kontrol-Monitoring Daya Perangkat Elektronika. JTERA (Jurnal
Teknologi Rekayasa), 3(2), 177. https://doi.org/10.31544/jtera.v3.i2.2018.177-184 Ismail, M., Abdullah, R. K., & Abdussamad, S. (2021). Tempat Sampah Pintar
Berbasis Internet of Things (IoT) Dengan Sistem Teknologi Informasi. Jambura Journal of Electrical and Electronics Engineering, 3(1), 7–12. https://doi.org/10.37905/jjeee.v3i1.8099 Mulyati, S., & Sumardi. (2018). INTERNET OF THINGS (IoT) PADA
PROTOTIPE PENDETEKSI KEBOCORAN GAS BERBASIS MQ-2 dan
SIM800L. Jurnal Teknik, 7(2), 64–72. ( )
SANTOSO, H. B., PRAJOGO, S., & MURSID, S. P. (2018). Pengembangan Sistem
Pemantauan Konsumsi Energi Rumah Tangga Berbasis Internet of Things
(IoT). ELKOMIKA: Jurnal Teknik Energi Elektrik, Teknik Telekomunikasi, &
Teknik Elektronika, 6(3), 357. https://doi.org/10.26760/elkomika.v6i3.357 p
g
Sasmoko, D., & Mahendra, A. (2017). RANCANG BANGUN SISTEM
PENDETEKSI KEBAKARAN BERBASIS IoT dan SMS GATEWAY
MENGGUNAKAN ARDUINO. Simetris : Jurnal Teknik Mesin, Elektro Dan
Ilmu Komputer, 8(2), 469. https://doi.org/10.24176/simet.v8i2.1316 Siregar, M. R., Bintoro, A., & Putri, R. (2021). Sistem Monitoring Suhu dan
Kelembaban pada Penyimpanan Gabah untuk Menjaga Kualitas Beras
Berbasis Internet of Things ( IoT ). Jurnal Energi Elektrik, 10(October), 14–17. https://doi.org/10.29103/jee.v10i1.4309 p
g
j
Sulistyanto, M. P. T., Suharsono, K., & Nugraha, D. A. (2016). Monitoring dan
Kendali Peralatan Elektronik Menggunakan Logika Fuzzy Melalui Website
Dengan Protokol HTTP. Jurnal SMARTICS, 2(2), 15–20. 118
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Caracterización de la velocidad y dirección de viento en la provincia de Chimborazo
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ISSN: 2600-5859
Vol. 3, N°1.1, p. 389-407, febrero, 2020
Caracterización de la velocidad y dirección de viento en la
provincia de Chimborazo
Characterization of speed and wind direction in the province of Chimborazo
Diana Katherine Campoverde Santos. 1, Amalia Isabel Escudero Villa. 2, & Silvia Mariana Haro
Rivera.3
Recibido: 05-12-2019 / Revisado: 22-12-2019 /Aceptado: 17-01-2020/ Publicado: 07-02-2020
Abstract.
DOI: https://doi.org/10.33262/concienciadigital.v3i1.1.1156
The present research main shows the behavior of speed and direction wind in Chimborazo
province; data were taken from 2014 to 2017, it was applied in eleven weather stations located in
strategic places inside study area. Also, database was structured according to the WorPlot software
requirements for elaboration of wind roses. A descriptive statistical analysis was performed to
characterize their behavior, contrast of hypothesis was carried out to identify significant
differences on a monthly, annual and stations. Similar behaviors were determined in the wind data
between the Tunshi and Urbina stations with respect to the wind direction in months each year;
Alao, Cumandá, Espoch, Matus, Multitud, Quimiag, San Juan and Tunshi stations have wind
speeds that reached 2.0 m/s, while in Atillo, Tixán and Urbina around 3.0 m/s at 4.5 m/s,
determining that the areas with the highest availability of wind energy are located in Tixán and
Urbina with speeds exceeding 4 m/s. Finally, with the help of Google Earth, the wind roses were
visualized in each station in order to show predominant characteristics through variables studied.
Keywords: Statistical Analysis, Wind Speed, Wind Direction, Chimborazo Province.
Resumen.
La presente investigación muestra el comportamiento de la velocidad y dirección de viento en la
provincia de Chimborazo, se tomaron los datos del 2014 al 2017 de 11 estaciones meteorológicas
1Escuela
Superior Politécnica de Chimborazo, Facultad de Ciencias Pecuarias. Riobamba, Ecuador,
diana.campoverde@espoch.edu.ec
2
Escuela Superior Politécnica de Chimborazo, Facultad de Ciencias. Riobamba, Ecuador.,
aescudero@espoch.edu.ec
3Escuela Superior Politécnica de Chimborazo, Facultad de Ciencias. Riobamba, Ecuador. , sharo@espoch.edu.ec
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ubicadas en lugares estratégicos de la zona de estudio. Se estructuró una base de datos de acuerdo
con los requerimientos del software WorPlot para la elaboración de las rosas de viento. Se realizó
un análisis estadístico descriptivo para caracterizar su comportamiento, se efectuaron contrastes
de hipótesis para identificar diferencias significativas en forma mensual, anual y por estaciones.
Se determinaron comportamientos similares en los datos de viento entre las estaciones Tunshi y
Urbina respecto a la dirección de viento en todos los meses de cada año año; las estaciones Alao,
Cumandá, Espoch, Matus, Multitud, Quimiag, San Juan y Tunshi presentan velocidades de viento
que alcanzaron los 2.0 m/s, mientras que en Atillo, Tixán y Urbina de 3.0 m/s a 4.5 m/s,
determinándose que las zonas con mayor disponibilidad de energía del viento se encuentra en
Tixán y Urbina con velocidades que superan los 4 m/s. Finalmente con la ayuda de Google Earth
se visualizó las rosas de viento en cada una de las 11 estaciones con el fin de mostrar características
predominantes de las variables estudiadas.
Palabras claves: Análisis estadístico, Velocidad de viento, Dirección de Viento, Provincia
Chimborazo
Introducción.
El estudio estadístico de variables meteorológicas es de vital importancia, puesto que
permite identificar características relevantes de su comportamiento, a la vez que son útiles y
coadyuvan a estudios de diversas áreas como por ejemplo: el análisis de la relación entre
temperatura y precipitación con Aedes aegypti (Bultó et al., 2008) que es el principal vector del
dengue común y hemorrágico (Cruz, Sebrango, Hernández, Pina, Marquetti y Sánchez, 2010); la
variabilidad del cambio climático y su impacto en la salud humana (Ortíz, Pérez, Rivero, Pérez,
Cangas y Lecha, 2008), la temperatura del aire, humedad relativa, velocidad del viento, radiación
solar y precipitaciones para la estimación de la evapotranspiración de tomate usando el modelo
interacción Suelo (Olioso, Ortega, Valdés y Antonioletti, 2005); la temperatura, humedad relativa,
nubosidad, dirección y velocidad del viento para modelar la predicción de heladas mediante redes
neuronales (Ovando, Bocco y Sayago, 2005); la descripción de la variabilidad del viento, nivel del
mar y temperatura en la costa norte de Chile (Pizarro, Hormazabal, González y Yañez, 1994);
frecuencia e intensidad de viento en Zaragoza de las medias mensuales según las direcciones de
las rosas de los vientos (Navarro, 1990), el análisis de estacional característico en las turbinas en
Manisa eólicas así como el potencial energético eólico, (Tamura, 2015), entre otros.
La energía eólica se produce como resultado de la transformación de la energía cinética del aire en
movimiento, siendo aprovechada mediante el uso de dispositivos y/o aerogeneradores que operan
de forma aislada o en grupos (parques eólicos) convirtiéndola en energía de diversos tipos, siendo
la más común la energía eléctrica (De la Cruz Buelvas, Valencia y Vanegas, 2018; Quiñonez,
Huanca y Holguino, 2019).
En los últimos años a nivel mundial existe interés en buscar nuevas fuentes de energía que no
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generen contaminación en el medio ambiente (reduciendo efectos nocivos) con fuentes renovables
(energía eólica y solar) que no requieran el uso de reservas de petróleo y gas (Carta, Ramírez y
Velázquez, 2009; Watson, 2014). Köse (2004) afirma que: dado que la energía eólica es un recurso
local y de medio ambiente limpio y ecológico, es vital llevar a cabo la viabilidad técnica y
económica requerida, por lo que debería intensificarse investigaciones que viabilicen el uso de este
recurso que coadyuvará a la superación los problemas actuales de energía (p. 1632) especialmente
en aquellos que surgen por el difícil acceso, pero que sin embargo están habitadas por familias
agricultoras.
Siendo la velocidad y dirección de viento un recurso limpio y renovable del que se puede
aprovechar para cubrir necesidades de la sociedad en general , en la última década, numerosos
estudios relacionados con la caracterización del viento y potencial de energía eólica se han
realizado en diferentes países (Gökçek et al.,2007; B. Natalini y M. Natalini, 2013), la expresión
utilizada en estos estudios de energía eólica para representar las distribuciones de viento es la
distribución de Weibull.
𝑘
𝑐
𝑉 𝑘−1
𝑉 𝑘
∙ 𝑒 (− ) , donde 𝑃(𝑣):
𝑐
𝑐
𝑃(𝑣) = ( ) ( )
es la probabilidad de ocurrencia de una velocidad de
viento determinada, 𝑐: factor de escala y 𝑘: es el factor de forma (Cellura M., Cirrincioneb G., et
al, 2008; Ciampoli, Petrini, y Augusti, 2011); dichos estudios han detectado que la velocidad de
los vientos disminuye considerablemente durante los meses de invierno, estas variaciones son
causadas por las diferencias de temperatura existentes por la desigualdad del calentamiento en las
diversas zonas de la tierra (Cúneo, Cerne y Llano, 2018; Hennessey, 1977).
Sin embargo los resultados no se pueden generalizar para todos los países del mundo debido a sus
características geológicas propias, es por ello que en Ecuador se viene incentivando el estudio de
variables meteorológicas con el fin de aprovechar estos recursos naturales y con ello a aportar con
el cuidar el medio ambiente, por ello se han instalado estaciones meteorológicas en diferentes
provincias del país, entre estas en la provincia de Chimborazo, la cual se encuentra a una altura de
156 m.s.n.m. hasta los 6310 m.s.n.m.
Los asentamientos poblacionales están entre 2200 a 3300 m.s.n.m., según el Instituto Geográfico
Militar (IGM) cuenta con una extensión territorial cercana a los 6500 km2 , tiene fértiles valles
como los de Guano, Riobamba y Penipe. La provincia tiene importantes afluentes; entre los
principales están: el Chambo, Chanchán y Chimbo. Debido a la presencia de masas de aire
amazónicas y oceánicas; y sobre todo la altura, se presentan cuatro tipos de climas; a los 4.600
ms.n.m. es de tipo glacial, entre los 3.000 y 4.000 ms.n.m. es páramo, bajando; a los 2.000 ms.n.m.
el clima es mezotérmico seco; y en zonas cercanas a la costa el clima es mesotérmico húmedo y
semi-húmedo (Toainga, 2015), por esta presencia de masas se da la necesidad de analizar el
comportamiento metereológico del viento.
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En Chimborazo, un estudio realizado por Haro, Lema, Meneses y Escudero (2020), mediante el
análisis de componentes principales de velocidad de viento en el año 2015, enumeran dos tipos de
comportamientos; el primero corresponde a la región norte de la provincia y el segundo a la región
del sub trópico, la primera región se caracteriza por poseer zonas ubicadas a una altura superior
que los 1.000 ms.n.m. totalmente diferente a la segunda que tiene mayor diversidad con fauna y
flora. Estos resultados son útiles para identificar zonas con velocidades de viento con posibilidades
de producir energía eólica.
La velocidad de viento está estrechamente asociada a su dirección, es por ello que en el presente
trabajo de investigación se muestra un análisis estadístico exploratorio sobre la velocidad y
dirección del viento, representando su comportamiento mediante rosas de viento graficadas en
worplot usando google earth se las ubicó en cada zona geográfica de cada estación meteorológica
que conforma la red de estaciones del Instituto Nacional de Eficiencia Energética y Energías
Renovables y la Escuela Superior Politécnica de Chimborazo (INER – ESPOCH), con el fin de
identificar patrones de comportamientos donde las variables en estudio presenten características
similares.
La red está conformada por 11 estaciones meteorológicas, ubicadas de manera estratégica en toda
la provincia (Tabla 1 y Figura 1).
Tabla 1: Ubicación geográfica de las estaciones meteorológicas de la red INER-ESPOCH.
Coordenadas (UTM)
Longitud
Latitud
Altura
(m.s.n.m)
Alao
773499E
9793173S
3064
2
Atillo
772610E
9758048S
3467
3
Cumandá
706262E
9755580S
331
4
ESPOCH
758398E
9816965S
2754
5
Matus
777402E
9827879S
2685
6
Multitud
722699E
9764910S
1483
7
Quimiag
770084E
9816393S
2709
8
San Juan
746596E
9818849S
3232
9
Tixán
747159E
9757355S
4240
10
Tunshi
764087E
9806678S
2840
11
Urbina
754533E
9835326S
3642
No.
Nombre de la estación
1
Fuente: INER-ESPOCH-UNACH.
Elaborado por: Ramos, C., Pérez, N.
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Figura 1. Ubicación geográfica estaciones meteorológicas de la red INER-ESPOCH
Fuente: Datos INER-ESPOCH-UNACH
Elaborado por: Ramos, C., Pérez, N.
Se tomaron las velocidades de viento por hora en m/s y sus direcciones en grados registrados del
2014 al 2017 mediante el anemómetro con calibración del 5% y almacenados en Data Logger NRG
Symphonie, Los datos se analizaron de forma mensual, anual y por estaciones, de los cuales se
hallaron velocidades medias y máximas, con el fin de identificar diferencias significativas entre el
comportamiento de dichas variables con respecto al mes, año y estación, para lo cual se usó el
contraste de hipótesis t-student y para probar su normalidad el test de Shapiro Wilks, posterior a
este análisis se utilizó el software libre Worplot especialista en la construcción gráfica de rosa de
vientos para la representación gráfica de los datos de viento de cada una de las estaciones en
estudio.
Este estudio fue realizado con el fin de identificar patrones de comportamiento de las variables
estudiadas, ya sean en forma mensual, anual o por estaciones, sin embargo, se ha logrado
identificar la existencia de microclimas debido a la estructura geográfica de la zona estudiada. Los
resultados obtenidos servirán de punto de partida para realizar análisis estadísticos con técnicas
avanzadas como: análisis estadístico de datos funcionales, redes neuronales, modelación espacial
funcional para estimar datos en zonas no muestreadas, imputación de datos metereológicos
faltantes, entre otros, tanto de las variables velocidad y dirección de viento como de otras variable
meteorológicas (Rivera y Escudero, 2017; Tamura, 2015), con el fin de conocer a profundidad el
comportamiento de dichas variables en la provincia de Chimborazo.
Metodología
La transmisión de la información de las estaciones meteorológicas se la realiza automáticamente
mediante el servicio general de paquetes vía radio (General Packet Radio Service) (GPRS) en los
lugares en donde existe cobertura de la red celular, y mediante un modem GOES en donde no
existe dicha cobertura. En la figura 5 se explica el proceso de transmisión de la información hasta
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el servidor Protocolo de transferencia de archivos (en inglés File Transfer Protocol o FTP) FTP en
la ESPOCH. Para la transmisión GOES se realizaron gestiones, con el objetivo de obtener un canal
de transmisión por parte por parte del Instituto Nacional de Meteorología e Hidrología (INAMHI).
En el estudio se emplearon datos por hora, información recolectada del Data Logger y mediante
una hoja de Excel, se estructuró una base de datos de acuerdo con los requerimientos del análisis,
considerando las variables de velocidad y dirección de viento. La detección de atípicos se hizo
mediante el método de direcciones ortogonales de máxima y mínima curtosis de forma horaria por
mes, este procedimiento consiste en eliminar provisionalmente los datos extremos (sospechosos)
en estas direcciones ortogonales, luego calcular la media y la matriz de covarianza con los datos
no sospechosos y después identificar los datos atípicos (valores mayores que p+3√2p) como
aquellos que son extremos con la distancia de Mahalanobis: 𝑑𝑅2 (xi , 𝑥̅𝑅 ) = (𝑥𝑖 − 𝑥̅𝑅 )𝑆𝑅−1 (𝑥𝑖 − 𝑥̅𝑅 )´
calculada con las estimaciones sin datos atípicos, este procedimiento se lo realizó usando el
algoritmo “Mskurt” del paquete “Outliert” de Matlab (Peña, 2013)
Para la elaboración de las rosas de viento se dividieron los 360 grados en 12 sectores de 30 grados,
formando un grupo de cuatro direcciones primarias y ocho secundarias que son: Norte, Este, Sur,
Oeste, Nor-noreste, Este-noreste, Este-sureste, Sur-sureste, Sur-suroeste, Oeste-suroeste, Oestenoroeste, Nor-Noroeste. Estas direcciones están representadas por números, los cuales varían de
acuerdo con las manecillas del reloj, iniciando con 360º en el norte, teniendo el este con 90º, el sur
con 180º y el oeste con 270º. Debido a las características propias de la localidad se dividió los
datos de velocidad de acuerdo con su magnitud en 5 intervalos: de 0-3, 3-5, 5-7, 7-9 y de 9 en
adelante, los registros de 0 son reportados como calma (Bichet, Wild, Folini, y Schär, 2012). Una
vez obtenidas las distribuciones de frecuencias como tablas de contingencia en Excel de las dos
variables clasificadas, se importaron al Worplot View para construir las rosas de viento, que son
gráficas de las dos variables en coordenadas polares con barras que indican el porcentaje de
velocidad de viento alcanzado y direcciones predominantes en cada intervalo. Debido a que se
visualizó similitud en direcciones de viento entre las estaciones de Tunshi y Urbina se utilizó R
para realizar un contraste de hipótesis t-student para identificar diferencias significativas entre
dichas estaciones ya que las dos variables siguen distribución normal según el contraste de
normalidad de Shapiro. Finalmente se elaboraron mapas temáticos de la provincia de Chimborazo
según la ubicación geográfica de cada una de las estaciones meteorológicas, para facilitar la
visualización del comportamiento de las dos variables estudiadas.
Worplot View genera automáticamente una lista de horas donde existe faltantes, misma que
aparece haciendo clic en la opción Incomplete/Missing Hours de la pestaña Met Data Information,
mismos que son imputados automáticamente por el software, mediante el promedio de las cuatro
horas que rodean el dato faltante, a excepción de las dos primeras y dos últimas horas, si faltan
más de dos horas consecutivas toma los promedios de 5 o más datos a cada lado del vacío
(Atkinson y Russell, 1992).
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Resultados
Variaciones anuales: En los análisis estadísticos se obtuvieron las velocidades medias anuales
(Tabla 2), se halló que durante los cuatro años de estudio el comportamiento es similar, verificando
mayor variabilidad aproximadamente de una unidad en las estaciones de Alao, San Juan, Tixán y
Urbina. En el año 2015 se identificó mayor velocidad de viento promedio en las estaciones de
Alao, Atillo, Espoch, San Juan, Tixán y Urbina en los 4 años de estudio, mientras que, en el 2017,
la menor velocidad en 8 de las 11 estaciones meteorológicas que son: Alao, Cumandá, Espoch,
Matus, Quimiag, San Juan, Tixán y Urbina (Figura 2). La velocidad de viento incrementa en
función a la altura geográfica del lugar donde están instaladas las estaciones. Para complementar
el estudio se realizó un contraste de hipótesis t-student una vez comprobado que los datos siguen
distribución normal mediante la prueba de Shapiro Wilks, se mostró que no hay diferencias
significativas entre las velocidades de viento por año de dicha estación, al 95% de confiabilidad.
En cuanto a las demás estaciones todas tienen diferencias significativas con un valor de p igual a
0,000 con un nivel de significancia de 0,05.
Tabla 2: Promedios anuales de velocidad de viento de las 11 estaciones meteorológicas de la
Provincia de Chimborazo.
N°
1
2
3
4
5
6
7
8
9
10
11
Año
Alao
Atillo
Cumandá
Espoch
Matus
Multitud
Quimiag
San Juan
Tixán
Tunshi
Urbina
2014
1,93
3,06
0,74
1,89
0,42
1,84
1,73
3,59
1,4
3,76
2015
2,01
3,7
0,6
2,13
1,52
0,39
1,93
2,31
4,66
1,46
4,53
2016
2,01
3,37
0,48
1,98
1,68
0,2
1,95
2,13
4,24
1,47
4,11
2017
1,71
3,22
0,36
1,82
1,36
0,21
1,69
1,53
3,41
1,65
3,53
Elaborado por: Diana Campoverde
Figura 2. Velocidades de viento medios por estación meteorológica
Elaborado por: Diana Campoverde
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Para identificar el comportamiento de las direcciones de viento predominantes se construyeron
gráficos apilados (Figura 3). Multitud, Tunshi y Urbina muestran direcciones de viento
predominantes hacia el N durante el 2014; por otro lado, Atillo, Cumandá, Espoch, Multitud y
Tixán con direcciones de viento predominantes hacia el E; mientras que las estaciones Alao,
Espoch y San Juan presentan características de vientos predominantes hacia el ESE. Cumandá
presenta un viento predominante hacia el OSO y Quimiag hacia el NE (Figura 3 (a)).
Alao
Atillo
Cumandá
Espoch
Multitud
Quimiag
San Juan
Tixán
Tunshi
Urbina
DIRECCION
Figura 3. Direcciones predominantes de velocidad de viento, (a) 2014, (b) 2015, (c) 2016, y (d) 2017.
N
NNE
NE
ENE
E
ESE
SE
SSE
S
SSO
SO
OSO
O
ONO
NO
NNO
Alao
Atillo
Cumandá
Espoch
Matus
Multitud
Quimiag
San Juan
Tixán
Tunshi
Urbina
DIRECCION
(a) 2014
N
NNE
NE
ENE
E
ESE
SE
SSE
S
SSO
SO
OSO
O
ONO
NO
NNO
(b) 2015
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Alao
Atillo
Cumandá
Espoch
Matus
Multitud
Quimiag
San Juan
Tixán
Tunshi
Urbina
DIRECCION
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N
NNE
NE
ENE
E
ESE
SE
SSE
S
SSO
SO
OSO
O
ONO
NO
NNO
Alao
Atillo
Cumandá
Espoch
Matus
Multitud
Quimiag
San Juan
Tixán
Tunshi
Urbina
DIRECCION
(c) 2016
N
NNE
NE
ENE
E
ESE
SE
SSE
S
SSO
SO
OSO
O
ONO
NO
NNO
(d) 2017
Elaborado por: Diana Campoverde
En el 2015 (Figura 3 (b)) los vientos predominantes de Tunshi y Urbina se direccionan hacia el N;
Atillo, Cumandá, Espoch, Multitud y Tixán hacia el E; Alao, Espoch y San Juan hacia el ESE;
Multitud hacia S y Matus hacia el NNO. En el año 2016 (Figura 3 (c)) los vientos predominantes
de Multitud, Tunshi y Urbina son hacia el N; de Quimiag hacia el NE; de Atillo, Espoch y Tixán
hacia el E; de Alao y San Juan hacia el ESE; Atillo hacia el ONO y Matus hacia el NNO. En el
año 2017 (Figura 3 (d)) los vientos predominantes de Multitud, San Juan, Tunshi y Urbina son
hacia el N; de Atillo, Cumanda, Espoch y Tixán hacia el E; de Alao hacia el ESE; Quimiag hacia
el S y Matus hacia el NNO.
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Visualizando las gráficas notamos que las estaciones de Cumandá, Multitud, Quimiag y San Juan
son estaciones en las cuales sus direcciones de vientos predominantes no se mantienen constantes;
Cumandá durante el año 2014 su viento predomina hacia el OSO, durante el 2015 hacia el Este,
en el 2016 hacia el ONO y regresa hacia el E en el 2017; Multitud presenta direcciones de viento
predominantes hacia el S durante los años 2014-2015 y cambia de dirección durante el 2016-2017,
Quimiag presenta direcciones de viento predominantes hacia el NE durante el periodo de tiempo
del 2014 al 2016 e inciertamente el año 2017 cambia de dirección hacia el S, San Juan presenta un
caso similar a esta estación, durante los años 2014, 2015 y 2016 sus vientos predominan hacia el
ESE y en el 2017 su dirección cambia con dirección hacia el N.
Geográficamente debido a su ubicación las estaciones de Cumandá y Multitud se encuentra ubicada cerca de la cuenca del rio Chanchan (SE), estas variaciones están influenciadas por su ubicación en relación al nivel del mar y la altitud de cada estación.
Variaciones diarias: Los registros de cada estación contienen información de mediciones de 1
hora entre cada registro durante las 24 horas del día, de los 4 años de estudio, cuyo cambio típico
consiste en la velocidad del viento aumenta durante el día y desciende esa velocidad durante la
medianoche y el amanecer, estas variaciones son importantes como una característica para evaluar
ese potencial eólico.
Figura 4. Variación de la velocidad viento promedio por hora del viento durante el período 20142017 de cada una de las estaciones meteorológicas instaladas en la provincia de ChimborazoESPOCH.
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Elaborado por: Diana Campoverde.
La figura 4 presenta como característica más notable en las 11 estaciones que durante el día la
velocidad del viento es superior al medio día, es decir desde las 12:00 hasta las 18:00, luego esta
velocidad desciende hasta las 06:00, las estaciones de Cumandá y Multitud presentan un comportamiento especial, no se puede visualizar un patrón común en los 4 años de estudio.
Se construyeron rosas de viento (Figura 5) de cada una de las estaciones para los 4 años en estudio,
que asocian tanto la distribución de la velocidad como de la dirección. Las rosas de viento muestran
las direcciones predominantes y las velocidades en 5 clases de 3 𝑚/𝑠 de rango por colores: el
color verde para velocidades mayores a 9 m/s, el color azul para velocidades entre 7 y 9 m/s, el
color rojo para velocidades entre 5 y 7 m/s, el amarillo para velocidades de 3 a 5 m/s y el color
gris claro para velocidades de 0 a 3 m/s.
Finalmente se exportó las rosas de viento a Google Earth de las 11 estaciones meteorológicas con
el fin de visualizar y asociar las variables estudiadas con su posición geográfica (Figura 6).
Los resultados mensuales de velocidad y dirección de viento dieron lugar a 528 rosas de viento
(12 meses, 11 estaciones y 4 años), motivo por el cual no se las presenta en este paper, sin embargo,
en la discusión, se hace hincapié en las características encontradas en su comportamiento.
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Figura 5. Rosas de viento del comportamiento de las velocidades y direcciones de viento de cada una de las estaciones meteorológicas
instaladas en la provincia de Chimborazo-ESPOCH de 2014 al 2016.
Fuente: Datos INER-ESPOCH.
Elaborado por: Diana Campoverde.
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Figura 6. Rosas de viento de las 11 estaciones meteorológicas instaladas en la provincia de ChimborazoESPOCH de 2014 al 2017.
Elaborado por: Diana Campoverde
Conclusiones.
• El análisis correlacional de las direcciones de viento con la temperatura del aire, radiación
solar global, humedad y precipitación no mostró influencia de dichas variables en su
comportamiento, en la actualidad una de las problemáticas es el desarrollo de fuentes
energéticas a partir de recursos eólicos, esto ha motivado a realizar estudios de
caracterización que permitan evidenciar el alto potencial de energía eólica por explorar y
utilizar, que permitan seleccionar de manera decisiva la aplicación de este recurso.
• El análisis anual y mensual identificó estaciones con direcciones de viento predominantes
estables en: Alao hacia el SE, Atillo hacia el E, Espoch hacia el E, Matus hacia el NNO,
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•
•
•
Tixán hacia el E, Tunshi y Urbina hacia el N. Notándose un comportamiento diferente en
Cumandá al estar ubicada en la cuenca del rio Chanchan (SE) y el río Tixan (NE)
provocando cambios bruscos en la dirección de viento, en aproximadamente un ángulo de
135 grados. Las direcciones de viento predominantes durante los años de estudio se
clasificaron en dos grupos: de enero a junio los vientos son predominantes hacia el E en
los cuatro años de estudio y de julio a diciembre presentan vientos predominantes con
dirección hacia el SO en los años 2015 y 2016, mientras que en los años 2014 y 2017
presentó vientos predominantes hacia el NO, característica que no está estrechamente
asociado con las época cálida y lluviosa de la región costa (de junio a abril); Multitud está
influenciada por las microcuencas de los ríos Maguazo (NE) y Chimbo (SO) generando
direcciones de viento con menor estabilidad de enero a junio con una dirección de N a S
(2014, 2015, 2016 y 2017) y mayor estabilidad de julio a diciembre con vientos
predominantes al E (2014 y 2015), al SO, S y SE (2016) y al N (2017). Ligeramente se
detectaron comportamientos variantes aproximadamente cada 4 meses en ángulos de 45
hasta 180 grados, no se identificó comportamiento asociado a las épocas secas y lluviosas
de la sierra y costa; Tixán se encuentra en el centro de las microcuencas de los ríos Zula
(SE) y Sibambe (NE) provocando vientos predominantes con dirección estable hacia el E;
Espoch se halla dentro de la microcuenca del río Chibunga, la dirección predominante de
viento varía de N, NE, E, SE y S durante todo el año.
Los resultados obtenidos a partir de este estudio identificaron que las estaciones con más
altas velocidades de viento son Tixán y Urbina, ya que presentan registros de velocidades
entre los 3 y 4,0 m/s del 2014 al 2017, causando la posibilidad de aplicar modelos de
generación de energía mediante molinos de viento previo a un estudio de rentabilidad.
El comportamiento de las velocidades y direcciones de viento no presentan patrones
comunes de comportamiento, es decir, la provincia de Chimborazo presenta múltiples
ecosistemas debido a la diversidad de comportamientos de dichas variables, puesto que van
en función de la ubicación geográfica de la zona.
El software Wrtplot no destaca las características atípicas de la velocidad y dirección de
viento, los resultados no mostraron diferencias significativas entre el análisis de estas
variables con y sin un proceso previo de limpieza de datos, por lo que es una ventaja, ya
que al no poseer suficiente conocimiento estadístico para realizar dicho análisis no
imposibilita la detección de vientos predominantes. Otra ventaja del software, se destaca
al momento de realizar la imputación; Wrtplot realiza el relleno automático de datos
faltantes sin ningún problema.
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PARA CITAR EL ARTÍCULO INDEXADO.
Campoverde Santos, D. K., Escudero Villa, A. I., & Haro Rivera, S. M. (2020). Caracterización de
la velocidad y dirección de viento en la provincia de Chimborazo. ConcienciaDigital, 3(1.1), 389407. https://doi.org/10.33262/concienciadigital.v3i1.1.1156
El artículo que se publica es de exclusiva responsabilidad de los autores y no necesariamente
reflejan el pensamiento de la Revista Conciencia Digital.
El artículo queda en propiedad de la revista y, por tanto, su publicación parcial y/o total en otro
medio tiene que ser autorizado por el director de la Revista Conciencia Digital.
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A method of constructing syllable level Tibetan text classification corpus
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* Corresponding author: 1336786645@qq.com © 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/). A method of constructing syllable level Tibetan
text classification corpus j
g
Ban1,3,4
1College of Computer Science and Technology, Qinghai Normal University, Qinghai Xining, 810016,
China
2School of Computer Science and Technology, Southwest Minzu University, Sichuan Chengdu
610041, China
3Tibetan Information Processing and Machine Translation Key Laboratory of Qinghai Province,
Qinghai Xining 810008, China
4Key Laboratory of Tibetan Information Processing, Ministry of Education, Qinghai Xining 810008,
China Abstract. Corpus serves as an indispensable ingredient for statistical NLP
research and real-world applications, therefore corpus construction method
has a direct impact on various downstream tasks. This paper proposes a
method to construct Tibetan text classification corpus based on a syllable-
level processing technique which we refer as TC_TCCNL. Empirical
evidence indicates that the algorithm is able to produce a promising
performance, which may lay a starting point for research on Tibetan text
classification in the future. MATEC Web of Conferences 336, 06013 (2021)
CSCNS2020 MATEC Web of Conferences 336, 06013 (2021)
CSCNS2020 https://doi.org/10.1051/matecconf/202133606013 A method of constructing syllable level Tibetan
text classification corpus
Jizhaxi Dao1,3,4,*, Zhijie Cai1,2,3,4, Rangzhuoma Cai1,2,3,4, Maocuo San1,3,4, and Mabao
Ban1,3,4
1College of Computer Science and Technology, Qinghai Normal University, Qinghai Xining, 810016,
China
2School of Computer Science and Technology, Southwest Minzu University, Sichuan Chengdu
610041, China
3Tibetan Information Processing and Machine Translation Key Laboratory of Qinghai Province,
Qinghai Xining 810008, China
4Key Laboratory of Tibetan Information Processing, Ministry of Education, Qinghai Xining 810008,
China 2 Background Corpus is the basic resource of statistical natural language processing. Since the 13th ACL
in 1990 took the realization of large-scale real text processing as the strategic goal of
computational linguistics, large-scale corpora have been established in many countries. Tibetan corpus preprocessing generally includes removing non-Tibetan characters,
filtering out stop words, syllable segmentation, word segmentation and Part-of-speech
tagging. In 2003, Chen Yuzhong[2] and others designed a Tibetan word segmentation
algorithm (BCCF) based on case auxiliary words and continuity features by using
dictionary method, and Jiang Di [3] proposed Tibetan word segmentation method. From
2009 to 2019, Cai Zhijie et al. [4-8] studied the Tibetan word segmentation technology
based on the dictionary, and made a more comprehensive research on the design of
dictionary database, block technology, query algorithm and contraction word recognition. Tibetan corpus preprocessing generally includes removing non-Tibetan characters,
filtering out stop words, syllable segmentation, word segmentation and Part-of-speech
tagging. In 2003, Chen Yuzhong[2] and others designed a Tibetan word segmentation
algorithm (BCCF) based on case auxiliary words and continuity features by using
dictionary method, and Jiang Di [3] proposed Tibetan word segmentation method. From
2009 to 2019, Cai Zhijie et al. [4-8] studied the Tibetan word segmentation technology
based on the dictionary, and made a more comprehensive research on the design of
dictionary database, block technology, query algorithm and contraction word recognition. In 2009, sun yuan et al. [9] also studied Tibetan word segmentation technologies such as
bidirectional maximum matching method to detect intersection ambiguity and word
frequency information disambiguation based on case auxiliary word block method. In 2011,
Shi Xiaodong and Lu Yajun [10] transplanted the Chinese word segmentation system
SegTag based on HMM into Tibetan word segmentation, and designed and implemented
the Yangjin Tibetan word segmentation system; in order to make the Tibetan language
corpus standardized, unified and practical, Cai rangjia [11] and others put forward the
Tibetan word category and part of speech marker set, and established a segmentation
tagging dictionary. From 2015 to 2017, Li Yachao et al. [12-14] realized Tibetan word
segmentation system based on syllable Tagging Based on conditional random field model. Only in 2020 did Cairang Zhuoma et al. [15] propose Tibetan word segmentation strategy
and algorithm based on Part-of-Speech constraints, which can better solve the problems of
ambiguity and unknown words. 1 Introduction Corpus is a general language material stored in a computer which can be effectively
indexed, retrieved, inquired and analyzed. It is an ideal language knowledge resource. Its
construction process includes collecting and preprocessing. The scale, coverage and
preprocessing workflow of corpus determine the system performance which is modeled on
the corpus. In recent years, with the development of advanced technology in machine
learning and especially deep learning, the demand for large-scale and high-quality corpus is
growing. Corpus preprocessing should not only provide computational insights for the
language but also emphasize its application domains. From the perspective of preprocessing, Tibetan corpus can be divided into syllable level,
word level and phrase level. For a specific task, the selection of different levels of corpus
has a greater impact on its modeling domain. In the classification of English and Chinese
texts, the corpus of word level or phrase level is generally selected. Theoretically speaking,
it is more appropriate to select word level or phrase level corpus in Tibetan text
classification task, which can directly translate the relevant technologies of English and
Chinese text classification to Tibetan text classification. However, the accuracy of Tibetan
word segmentation and phrase recognition hardly meets the practical requirements, MATEC Web of Conferences 336, 06013 (2021)
CSCNS2020 https://doi.org/10.1051/matecconf/202133606013 therefore, it is difficult to establish large-scale and high-quality word level or phrase level
corpus. There are obvious boundary characters between syllables in Tibetan text, and
syllable segmentation is more convenient, so it is relatively easy to establish a syllable level
Tibetan corpus. We select 10MB corpus from the natural language processing group of
Qinghai Normal University, and observe the influence of syllable level and word level
corpus on Tibetan text classification performance. When the corpus size is the same,
syllable level and word level text classification performance are basically the same, which
indicates that syllable level corpus can be used to solve Tibetan text classification problems. Based on the analysis of the current situation of corpus construction, this paper designs a
syllable level Tibetan text classification corpus construction model, and gives the core
module text normalization algorithm TC_TCCNL, which lays the foundation for the
construction of Tibetan text classification corpus. therefore, it is difficult to establish large-scale and high-quality word level or phrase level
corpus. 2 Background Although scholars have studied the Tibetan word segmentation from various aspects,
due to the complexity of the language, the highest accuracy rate P, recall rate R and F of the
open evaluation of Tibetan word segmentation are 93.14, 92.17 and 92.66 (MLWS2017)
[16], and there is still a certain gap in meeting the actual needs. 1 Introduction There are obvious boundary characters between syllables in Tibetan text, and
syllable segmentation is more convenient, so it is relatively easy to establish a syllable level
Tibetan corpus. We select 10MB corpus from the natural language processing group of
Qinghai Normal University, and observe the influence of syllable level and word level
corpus on Tibetan text classification performance. When the corpus size is the same,
syllable level and word level text classification performance are basically the same, which
indicates that syllable level corpus can be used to solve Tibetan text classification problems. Based on the analysis of the current situation of corpus construction, this paper designs a
syllable level Tibetan text classification corpus construction model, and gives the core
module text normalization algorithm TC_TCCNL, which lays the foundation for the
construction of Tibetan text classification corpus. 3.1 TC_TCC corpus collection With the rapid development of the Internet, Tibetan text has changed from massive paper
text content to web text content. With the increasing number of Tibetan web pages, the way 2 MATEC Web of Conferences 336, 06013 (2021)
CSCNS2020 https://doi.org/10.1051/matecconf/202133606013 scholars collect corpus has shifted from traditional paper-based content manual input and
machine scanning to the most popular web crawler technology. According to the form of
information resources and the content of the text, it is a key work to choose an effective
way to collect corpus. scholars collect corpus has shifted from traditional paper-based content manual input and
machine scanning to the most popular web crawler technology. According to the form of
information resources and the content of the text, it is a key work to choose an effective
way to collect corpus. In this paper, the strategy of crawler based and manual input supplemented is used to
collect Tibetan text corpus of news, novel, scripture and medicine. The main sources of the
corpus are China Tibet network, China Tibetan Netcom and Qiongmai literature network. Among them, the collection of news and fiction corpus is completed by crawler, while the
acquisition of scripture and medical corpus adopts the method of manual input (due to the
lack of text corpus of scripture and medical web page). A total of 153.1MB Tibetan text
corpus is collected in this paper, which contains a total of 13760350 Tibetan syllables. The
source, size and number of syllables are shown in Table 1. Table 1. TC_ TCCsource distribution of corpus. Serial
number
source
data
size(MB)
Number of
syllables
1
Qiongmai Literature Network(http:
//www.tibetcm.com/)
15.89
1350133
2
China Tibetan Netcom(http: //www.tibet3.com/)
71.75
5731600
3
China Tibet Online(http://tb.tibet.cn/)
1.12
90196
4
Manual entry
64.34
6588416
total
153.1
13760350 Table 1. TC_ TCCsource distribution of corpus. Table 1. TC_ TCCsource distribution of corpus. 3.2 TC_TCC build model With the rapid development of computer technology, deep learning has become the
mainstream technology of natural language processing. However, deep learning requires
the scale and quality of corpus, so it is very important to construct large-scale and high-
quality corpus. q
y
p
Text classification is one of the most classic scenarios in the field of natural language
processing. It uses computers to automatically classify text according to a certain
classification system or standard. Since Maron published his first paper on automatic text
classification in Journal of ASM in 1960, scholars have studied text classification
technology from different perspectives and put forward many classical mathematical
models for text classification. Text classification has experienced knowledge-based method,
traditional machine learning method and the most popular deep learning method. These
approaches establish classifiers according to the knowledge formation rules provided by
experts or train the formation rules on the pre-classified corpus, so as to automatically
classify the samples of unknown categories. Knowledge based text classification method
needs to design rules manually, so it is difficult to construct classifier. The text
classification method based on machine learning can automatically obtain features from the
pre classified text, and it is convenient to construct a classifier, which has become the
mainstream method of text classification. When using machine learning based text
classification method, the granularity setting and preprocessing of corpus features are
extremely important. According to the analysis in the introduction section in this paper, it is
more appropriate to construct a syllable level Tibetan text classification corpus under the
current technical conditions. The syllable level Tibetan text classification corpus is called
the syllable level Tibetan text classification corpus (Tibetan Characters Text Classification
Corpus, TC_TCC) . Build TC_ TCC time, First, we should collect and acquire Tibetan students' corpus, and
then preprocess the raw materials. TC_ TCC construction model is shown in Figure 1. 3 MATEC Web of Conferences 336, 06013 (2021)
CSCNS2020 https://doi.org/10.1051/matecconf/202133606013 Fig. 1. TC_ TCC construction model. Syllable
processing
Module
Syllable
correction
Syllable
mentation
Raw corpus
Normalization
module
Text
classification
corpus
Text
unification
Category
labeling
Noise
reduction
module
Text
cleaning
Remove
stop words Noise
reduction
module Normalization
module Text
cleaning Text
unification Text
classification
corpus Fig. 1. TC_ TCC construction model. TC_ TCC construction model consists of three modules, namely, syllable processing,
noise reduction and normalization annotation. The syllable processing module includes two
parts: syllable segmentation and syllable correction. 3.2 TC_TCC build model There is an obvious syllable separator
"་" between Tibetan syllables, which can be used as a segmentation point to obtain high-
quality training corpus and improve the performance of text classification. 3.2 TC_TCC build model The main task of syllable segmentation
is to use the Tibetan syllable separator "་" as the cut-off point to segment the syllabic of
corpus, such as sentence "ང་ནི་ʃོབ་ཡིན"(I am a student), according to the syllable
separator"་"cut into"ང/ནི/ʃོབ/ཡིན/"; the main task of syllable correction It is to correct the
errors caused by the missing syllable separator "་", such as "བǦཤིས" (Zhaxi) to "བǦ་ཤིས". The
noise reduction module includes two parts: text cleaning and removing stop words. The
main function of text cleaning is to replace single vertical character, double vertical
character and four vertical character at the end of Tibetan sentence with single vertical
character; the other is to replace non Tibetan characters such as numbers, English, Chinese
characters and punctuation marks with "N" to ensure that the text structure will not be
affected; the main function of text cleaning is to replace single vertical character, double
vertical character and four vertical character at the end of Tibetan sentence according to the
stop list, the Tibetan function words are eliminated. The normalization annotation module
consists of two parts: text normalization and category annotation. The main function of text
normalization is to normalize the text size to ensure that each text size is consistent; the
main function of category annotation is to label the text category. Using TC_TCC to classify Tibetan text has the following advantages: Using TC_TCC to classify Tibetan text has the following advantages: 1）The vector dimension can be reduced. The total number of Tibetan syllables is far
less than the total number of Tibetan words. When training the language model and other
downstream tasks, the vector dimension can be reduced and the calculation amount of the
model can be reduced. 1）The vector dimension can be reduced. The total number of Tibetan syllables is far
less than the total number of Tibetan words. When training the language model and other
downstream tasks, the vector dimension can be reduced and the calculation amount of the
model can be reduced. 2）High quality training corpus can be obtained. There is an obvious syllable separator
"་" between Tibetan syllables, which can be used as a segmentation point to obtain high-
quality training corpus and improve the performance of text classification. 2）High quality training corpus can be obtained. 3.3 TC_TCC normalization method Because there are obvious separators between syllables in Tibetan text, syllable
segmentation is relatively simple. Syllable correction mainly corrects the non-true word
errors in the text, which can be corrected according to the method in the literature [17]. In
the process of text noise reduction, stop words and non-Tibetan character table can be
directly replaced with "N", and single vertical character, double vertical character and four
vertical character can also be replaced with single vertical character. Tibetan language
corpus is mainly obtained by crawler and manual input, so the length of the corpus is
inconsistent. In the text classification model training based on machine learning, text
normalization is an indispensable basic work, which will directly affect the results of model
learning. There are a series of problems such as too much calculation and loss of
information when training the language model for long texts. The ideal model can be 4 MATEC Web of Conferences 336, 06013 (2021)
CSCNS2020 https://doi.org/10.1051/matecconf/202133606013 obtained by training the corpus with the same size. Therefore, the core of text classification
corpus is to normalize the text. Text category annotation is carried out at the same time of
normalization. obtained by training the corpus with the same size. Therefore, the core of text classification
corpus is to normalize the text. Text category annotation is carried out at the same time of
normalization. The goal of TC_TCC text normalization is to keep the text size consistent. Text
normalization can use text size (the number of bytes of text) or the number of syllables
contained in the text as the standard for cutting. It is difficult to ensure that the cutting point
is the end of the sentence (single vertical character) or adjust the position of the cutting
point. In this case, the text content will be incomplete. Taking the number of syllables in the
text as the standard cutting can not guarantee the cutting The cutting point is a sentence
ending character, but it is easy to find the sentence ending character nearest to the tangent
point. The sentence ending character found can be used as the cutting point, which can not
only maintain the consistency of the text size, but also ensure the integrity of the text
content. 4. Conclusion Corpus serves as the most important ingredient of statistics and machine learning, and the
quality and distribution of corpus have a great impact on the further research. From the
perspective of constructing corpus, Tibetan corpus can be divided into syllable level, word
level and phrase level. Due to the restriction of word segmentation and phrase technology,
Tibetan word level and phrase level corpus construction technology can not meet the
specifications in real-world applications. Based on the analysis of the current situation of
Tibetan corpus construction, this paper studies the construction method of Tibetan text
classification corpus, and proposes a syllable level Tibetan text classification corpus
construction method, including the syllable level Tibetan text classification corpus
construction model and the core algorithm TC_TCCNL. Experimental data show that the
algorithm achieves the expected effect, which lays the foundation for the construction of
Tibetan text classification corpus. In the future, the Tibetan text classification technology
based on syllable level could be studied on the basis of this achievement. The National Natural Science Foundation of China (618666032,61966031), Projects funded by the
Department of science and technology of Qinghai Province (2019-SF-129), innovation team funding
project of "Yangtze River scholars and innovation team development plan" (IRT1068), Qinghai
Provincial Key Laboratory Project (2013-Z-Y17, 2014-Z-Y32, 2015-Z-Y03), Key Laboratory of
Tibetan information processing and machine translation (2013-Y-17). MATEC Web of Conferences 336, 06013 (2021)
CSCNS2020 https://doi.org/10.1051/matecconf/202133606013 As can be seen from the table above, the scripture text size is 12.00MB and contains
1,200,000 Tibetan syllables. When the normalization parameter λ=6, a total of 2,000
segmented texts are obtained, and the text size is between 5.7-6.2KB; The class text size is
6.31MB and contains 630848 Tibetan syllables. When the normalization parameter λ=6, a
total of 1052 segmented texts are obtained, of which the size of the first 1051 texts is
between 5.8-6.3KB, and the 1052th text The size of the news text is 4KB; the size of the
news text is 9.14MB, containing 914,121 Tibetan syllables. When the normalization
parameter λ=6, a total of 1524 segmented texts are obtained, of which the size of the first
1523 texts is between 5.8-6.2KB The size of the 1524th text is 2KB; the size of the novel
text is 8.41MB, containing 841,026 Tibetan syllables, when the normalization parameter
λ=6, a total of 1402 segmented texts are obtained, of which the size of the first 1401 texts
Between 5.9-6.3KB, the size of the 1402th text is 4KB. Experimental data shows that the
TC_TCCNL algorithm can normalize a given text to a specified size. When the size of the
source text is not an integer multiple of the parameter λ, the size of the last text is
inconsistent with the size of the previous text. 3.3 TC_TCC normalization method The size of text is usually described by the number of bytes, and it is more
appropriate to use the size of text when the number of syllables is used as the standard for
cutting text. In order to reveal the relationship between the size of the text and the number
of Tibetan syllables contained in the text, we examine the relationship between the size of
the text and the number of Tibetan syllables contained in the text. According to the
statistics of 30512.8KB corpus, it is found that there is a relationship between the size of
Tibetan text and the number of syllables it contains. There are about 100 Tibetan syllables
in 1KB text. The relationship between the size of the text and the number of syllables is
shown in Table 2. Table 2. Relationship between Tibetan text size and syllable number. Table 2. Relationship between Tibetan text size and syllable number. Text name
Size(KB)
Number of syllables
Syllable/1KB
Sakya motto
150.6
15213
101.02
Kanjur
2750
280130
101.87
Script
326.6
33022
101.11
News
3700
366961
99.18
Literary review
5700
565983
99.30
Academic
5400
537383
99.52
Fiction
12200
1231509
100.94
Common sense
115.5
11682
101.14
History
170.1
16910
99.41
Average value
3390.31
339865.89
100.25 From the above analysis, TC_TCC normalization algorithm(TC_TCCNL, Tibetan
characters Text classification corpus Normalization) can be obtained. The basic idea is
calculating the position of the cutting point according to the given file size λ. If the cutting
point is at the end of the sentence, the text will be segmented by the cutting point, otherwise,
the closest sentence ending character to the cutting point will be found to segment the text. g
g
g
We used the TC_TCCNL algorithm to perform a normalization experiment on a
35.86MB Tibetan text classification corpus containing 3585995 syllables for scriptures,
medicine, news, and novels, and achieved the expected results. The text normalized data is
shown in Table 3. Table 3. TC_TCCNL experimental data table(λ=6). Serial number Text type Text size(MB) Number of syllables contained Number of split text
1
Scripture
12.00
1,200,000
2000
2
Medicine
6.31
630,848
1052
3
News
9.14
914,121
1524
4
Fiction
8.41
841,026
1402
total
35.86
3,585,995
5978 5 MATEC Web of Conferences 336, 06013 (2021)
CSCNS2020 References 1. Ouzhu, zhaxiga. Tibetan computational linguistics [M]. Southwest Jiaotong University
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https://www.nature.com/articles/s41419-019-1988-0.pdf
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English
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The desmosomal cadherin desmoglein-3 acts as a keratinocyte anti-stress protein via suppression of p53
|
Cell death and disease
| 2,019
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cc-by
| 10,209
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© The Author(s) 2019
OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction
in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if
changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If
material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Abstract Finally, we evaluated whether this
pathway was operational in the autoimmune disease PV in which Dsg3 serves as a major antigen involved in blistering
pathogenesis We
nco ered increased p53
ith diff se c toplasmic and/or n clear staining in the oral m cosa of A R T I C L E O p e n A c c e s s The desmosomal cadherin desmoglein-3 acts as
a keratinocyte anti-stress protein via suppression
of p53 Ambreen Rehman1, Yang Cai2, Christian Hünefeld3,6, Hana Jedličková4, Yunying Huang1,2, Muy Teck Teh
1,
Usama Sharif Ahmad
1, Jutamas Uttagomol1, Ying Wang2, Angray Kang1, Gary Warnes5, Catherine Harwood5,
Daniele Bergamaschi
5, Eric Kenneth Parkinson1, Martin Röcken3 and Hong Wan
1 Rehman et al. Cell Death and Disease (2019) 10:750
https://doi.org/10.1038/s41419-019-1988-0 Rehman et al. Cell Death and Disease (2019) 10:750
https://doi.org/10.1038/s41419-019-1988-0 Cell Death & Disease Cell Death & Disease Abstract Abstract
Desmoglein-3 (Dsg3), the Pemphigus Vulgaris (PV) antigen (PVA), plays an essential role in keratinocyte cell–cell
adhesion and regulates various signaling pathways involved in the progression and metastasis of cancer where it is
upregulated. We show here that expression of Dsg3 impacts on the expression and function of p53, a key transcription
factor governing the responses to cellular stress. Dsg3 depletion increased p53 expression and activity, an effect
enhanced by treating cells with UVB, mechanical stress and genotoxic drugs, whilst increased Dsg3 expression
resulted in the opposite effects. Such a pathway in the negative regulation of p53 by Dsg3 was Dsg3 specific since
neither E-cadherin nor desmoplakin knockdown caused similar effects. Analysis of Dsg3−/−mouse skin also indicated
an increase of p53/p21WAF1/CIP1 and cleaved caspase-3 relative to Dsg3+/−controls. Finally, we evaluated whether this
pathway was operational in the autoimmune disease PV in which Dsg3 serves as a major antigen involved in blistering
pathogenesis. We uncovered increased p53 with diffuse cytoplasmic and/or nuclear staining in the oral mucosa of
patients, including cells surrounding blisters and the pre-lesional regions. This finding was verified by in vitro studies
where treatment of keratinocytes with PV sera, as well as a characterized pathogenic antibody specifically targeting
Dsg3, evoked pronounced p53 expression and activity accompanied by disruption of cell–cell adhesion. Collectively,
our findings suggest a novel role for Dsg3 as an anti-stress protein, via suppression of p53 function, and this pathway is
disrupted in PV. Abstract
Desmoglein-3 (Dsg3), the Pemphigus Vulgaris (PV) antigen (PVA), plays an essential role in keratinocyte cell–cell
adhesion and regulates various signaling pathways involved in the progression and metastasis of cancer where it is
upregulated. We show here that expression of Dsg3 impacts on the expression and function of p53, a key transcription
factor governing the responses to cellular stress. Dsg3 depletion increased p53 expression and activity, an effect
enhanced by treating cells with UVB, mechanical stress and genotoxic drugs, whilst increased Dsg3 expression
resulted in the opposite effects. Such a pathway in the negative regulation of p53 by Dsg3 was Dsg3 specific since
neither E-cadherin nor desmoplakin knockdown caused similar effects. Analysis of Dsg3−/−mouse skin also indicated
an increase of p53/p21WAF1/CIP1 and cleaved caspase-3 relative to Dsg3+/−controls. Introduction Cell Death and Disease (2019) 10:750 Dsg3 is upregulated in cancer with its exact role remains
uncertain1. In vitro gain-of-function experiments support
its pro-cancerous role; overexpression of Dsg3 elicited
pronounced membrane protrusions and augmented cell
migration via activation of various pathways1,3–5,18. Con-
versely, Dsg3 depletion resulted in inhibition of tumor
growth and metastasis19. To further evaluate the specificity of the Dsg3-p53 path-
way, we performed similar knockdown experiments for
desmoplakin, a marker of desmosomes and E-cadherin, a
classical cadherin in adherens junctions and found that
neither desmoplakin nor E-cadherin depletion evoked
comparable effects (Fig. 1g, h, see below). Because of the highly dynamic nature of p5329, we were
concerned that moderate changes in p53 in knockdown
cells might be masked partially by its rapid turnover. Hence, we treated the siRNA transfected cells with MG-
132 (25 µM, 3 h) before protein extraction and a greater
increase of both p53 and p21WAF1/CIP1 was detected in
Dsg3 knockdown cells (Fig. 2a). Similar results were
observed in lentivirus shRNA Hit-1 cells compared to
controls (Fig. 1e). We also monitored p53 protein turn-
over in cells treated with cycloheximide (30 µg/ml) by
extracting protein at various time points for up to 6 h. As
expected, a delayed reduction of p53, accompanied by
stabilization of MDM2, a key negative regulator of p5330,
was found in Dsg3-depleted cells compared to controls
(Fig. 2b). p53 half-life calculations indicated the protein
existed approximately 2-fold longer in knockdown cells
than in control cells (~120 min in RNAi vs ~50 min in
control). To test whether the regulation of p53 by Dsg3
had functional consequences we monitored whether there
was an increased expression of cleaved/active caspase-3,
an established specific marker of epithelial apoptosis31. A
FACS based Zombie NIR-caspase-3 assay32 was per-
formed in siRNA pre-treated cells grown to confluent and
sub-confluent conditions that detected a marked increase
of positive caspase-3 events (Zombie NIR−ve/Caspase-
3+ve) in Dsg3 knockdown cells as compared to the
respective controls; the effects were seen particularly in
sub-confluent culture (Fig. 2c). Furthermore, we also
detected a reduction of PCNA and Cyclin A that regulate
cell cycle progression, in cells with Dsg3 knockdown (see
below in Fig. 3d). Collectively, these findings are con-
sistent with our hypothesis that Dsg3 restrains p53
expression and activity. Introduction skin, Dsg3 is largely restricted to the basal layer of the
epidermis, while in oral mucosa uniform expression
occurs across stratified squamous epithelia8,9. Why these
different distribution patterns exist is unknown. Desmoglein-3 (Dsg3), a cadherin superfamily member,
is an adhesion protein in desmosomes. Recent evidence
suggests that Dsg3 acts as a regulator of various pathways
governing cell adhesion, proliferation, differentiation,
morphogenesis, and migration1–7. However, the function
of non-junctional Dsg3 remains poorly understood. In the Dsg3 is down-regulated in PV where Dsg3 serves as a
major antigen (PVA) for autoantibodies, causing disrup-
tion of cell–cell cohesion and pemphigus acantholysis in
Dsg3-expressing
tissues10–14. However,
other
studies
suggest PV is caused by autoantibodies that target non-
Dsg receptors, triggering intracellular signaling and con-
sequently cell apoptosis, leading to blistering14–16. Despite
numerous studies, the pathogenesis of PV remains an
issue of debate15,17. Correspondence: Hong Wan (h.wan@qmul.ac.uk)
1Centre for Oral Immunobiology and Regenerative Medicine, Institute of
Dentistry, Barts and The London, School of Medicine and Dentistry, Queen
Mary University of London, London, UK
2CB Joint MHNCRL, Hospital and School of Stomatology, Guizhou Medical
University, Guiyang, China
Full list of author information is available at the end of the article. Edited by A. Stephanou Correspondence: Hong Wan (h.wan@qmul.ac.uk)
1Centre for Oral Immunobiology and Regenerative Medicine, Institute of
Dentistry, Barts and The London, School of Medicine and Dentistry, Queen
Mary University of London, London, UK
2CB Joint MHNCRL, Hospital and School of Stomatology, Guizhou Medical
University, Guiyang, China
Full list of author information is available at the end of the article. Edited by A. Stephanou © The Author(s) 2019
OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction
in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if
changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If
material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Page 2 of 14 Rehman et al. Cell Death and Disease (2019) 10:750 Rehman et al. Dsg3 depletion induces p53 in keratinocytes g
y
To investigate our hypothesis, we performed an RNAi
study in NTERT keratinocytes harboring wild type p53
(wtp53). Knockdown of Dsg3 caused no apparent changes
in other junctional proteins including Dsg2 (not shown). Immunofluorescence indicated a significant induction of
nuclear p53 in cells with Dsg3 knockdown compared to
controls (Fig. 1a). Western blotting detected only a
moderate but significant increase of p53 with an increase
of its targets p21WAF1/CIP1/Bax (Fig. 1b). Lysates of the
nuclear and cytoplasmic fractions of the siRNA treated
cells were extracted and subjected to Western blotting
analysis. Increased p53/p21WAF1/CIP1 was evident in the
nuclear fraction of RNAi treated cells relative to control
(Fig. 1c), confirming augmented p53 levels in cells with
Dsg3 depletion. To determine the specificity of Dsg3
RNAi mediated p53 induction, we performed double
knockdown for Dsg3/p53 that demonstrated the induc-
tion of p53 is mediated by Dsg3 depletion since cells with
double knockdown showed attenuation of enhanced
p21WAF1/CIP1 expression (Fig. 1d). In parallel, we per-
formed Dsg3 knockdown in NTERTs with the lentiviral
shRNAs (Dharmacon, USA) targeting three different
regions in the Dsg3 gene. We found that one hit rendered
Dsg3
knockdown
coupled
with
induction
of
p53/
p21WAF1/CIP1 relative to non-target control whereas the
other two hits evoked no Dsg3 knockdown and sig-
nificantly no p53 induction (Fig. 1e). In line with these
data, immunofluorescence also detected a significant
increase of p53 in both nucleus and cytoplasm in
knockdown cells compared to controls (Hit-1 in Fig. 1f). Introduction Our
preliminary
observation,
made
in
MDCK
(Madin–Darby canine kidney) cells, showed that dome
formation, marking the initiation of epithelial cell dif-
ferentiation20–22 was suppressed by Dsg3 overexpression. Furthermore, overexpression of Dsg3 resulted in the
suppression of p53/p21WAF1/CIP1, suggesting that Dsg3
could act as an anti-stress protein through negative
regulation of p5323. p53 is found to be upregulated in
some epidermal pathologies, such as psoriasis24,25 and
lichen planus (LP)26–28, though whether any alteration of
p53 in PV currently remains unknown. Here, we inves-
tigate the hypothesis that Dsg3 counteracts p53 in ker-
atinocytes and explore its potential contribution to the
pathogenesis of PV. Official journal of the Cell Death Differentiation Association Dsg3 overexpression causes suppression of p53 and
activity Cells treated without and with UVB irradiation were shown here (see Dsg3 KD + UV in Fig. 3a below). (mean ± s.d., *p < 0.05, **p < 0.01, ***p < 0.001) explore the broad role of p53 in cell biology, the influence
of Dsg3 modulation (knockdown and ectopic over-
expression) on the keratinocyte differentiation program
was examined by a series of qPCR analyses for various
genes involved in early and late differentiation programs
in keratinocytes. To this end, we observed a general
inverse relationship between Dsg3 expression and key
keratinocyte differentiation markers (Fig. S1). Thus,
Dsg3 silencing caused their enhanced expression [pre-
mature cell differentiation] whereas the inverse result was
detected in Dsg3 overexpressing cells (cell dedifferentia-
tion). These results suggest that the Dsg3-p53 pathway
has some influence, at least in part, on the keratinocyte
differentiation program. by dampening the p53 response. This hypothesis was
supported by additional mechanical loading experiments
in which siRNA treated cells were challenged with
equiaxial cyclic strain (FX-5000: 1 Hz, 20%) for 4 h. Lysates extracted either immediately after strain (0 h) or 2
and 24 h later, respectively, followed by Western blotting
analysis. It showed increased p53/p21WAF1/CIP1/Bax in
Dsg3 knockdown cells, particularly at 0 h and 2 h in post-
strained cells (Fig. 3c). Thus the loss of Dsg3 affected
p53 stabilization in response to mechanical stress rein-
forcing its ability to counterbalance p53 response to
mechanical stress. To explore the upstream components involved in p53
activation in our knockdown system, we analyzed total
and phosphorylated protein expression of ATM serine/
threonine protein kinase, as well as one of its targets
CHK2, which is activated by DNA double-strand breaks33. Cells were subjected to UV or no UV exposure for 1 day
before Western blotting analysis (Fig. 3e). Although the
effects were modest both phospho-ATM (pATM) and
-CHK2 (pCHK2) showed elevated levels as well as total
p53/phospho-p53-S20 in Dsg3 depleted cells, with further
enhancement by UVB. Both total proteins also showed a
similar
expression
pattern
with
increased
levels
in
knockdown cells compared to controls, regardless of UV. These results suggest that the DNA double-strand break
induced activation of pATM is involved in the upregula-
tion of the p53 pathway in Dsg3 depleted cells. Dsg3 overexpression causes suppression of p53 and
activity To confirm our findings, an alternative approach with
gain-of-function was conducted in a cutaneous cell line
T8 (p53 null) expressing low endogenous Dsg3. T8 cells
with transduction of pBABE-hDsg3.myc to overexpress
Dsg3 (D3) or pBABE-puro empty vector (Vect), were
transiently transfected with a wtp53 plasmid for 2d prior
to analysis of p53 expression. We observed marked sup-
pression of p53/p21WAF1/CIP1 in D3 line, compared to
Vect control, at both the transcript and protein levels (Fig. 2d, e). The p53 transcriptional activity was confirmed by a
p53 luciferase assay (Fig. 2f). These results, again, support
our hypothesis that Dsg3 negatively regulates p53. To Official journal of the Cell Death Differentiation Association Rehman et al. Cell Death and Disease (2019) 10:750 Page 3 of 14 Fig. 1 (See legend on next page.) Official journal of the Cell Death Differentiation Association Rehman et al. Cell Death and Disease (2019) 10:750 Page 4 of 14 (
g
p
p g )
Fig. 1 Dsg3 depletion in human keratinocytes enhances p53 expression and activity. a Immunofluorescence in NTERTs transiently transfected
with Dsg3 specific or scrambled siRNA for 2d showed significantly increased nuclear p53 relative to control (n = 7, pooled from 2 independent
experiments). Scale bars, 20 µm. b Western blotting for p53 and its targets, p21WAF1/CIP1/Bax, in NTERTs with Dsg3 knockdown indicated a moderate
but significant increase of p53 (n = 3–4). c Biochemical fractionation of NTERTs with or without Dsg3 knockdown (RNAi). Increased p53 and p21WAF1/CIP1
in RNAi samples, especially in the nuclear fraction compared to control. d Western blots for the indicated antibodies in lysates with single (Dsg3) and
double (Dsg3/p53) knockdown. e Western blotting analysis of NTERT cell lines with transduction of GIPZ Lentiviral shRNAs, including non-target (NT)
and three hits targeting different regions in the Dsg3 gene. Cells with transduction of one (Hit-1), among three hits, exhibited Dsg3 knockdown with
concomitant induction of p53/ p21WAF1/CIP1 without and with either MG-132 (25 µM for 3 h) or UVB irradiation, as compared to NT and negative Hit
controls. f Immunofluorescence analysis indicated a marked increase of p53 in both the nucleus and cytoplasm in cells with transduction of Hit-1
compared to NT and other negative Hit controls. g, h Western blotting analysis in NTERTs with Dp or E-cadherin knockdown shows distinct protein
expression profiles for p53/p21WAF1/CIP1/Bax. Dsg3 overexpression causes suppression of p53 and
activity To con-
firm the presence of DNA double-strand breaks in these
cells we performed immunofluorescence for 53BP1 and
measured the nuclear foci in both control and Dsg3
depleted cells with and without UVB. Increased number
of 53BP1 foci indicated enhanced DNA double-strand
breaks, in particular in Dsg3 depleted cells exposed to
UVB which were significantly different from scrambled
siRNA controls (Fig. 3f). Finally, cells were treated in the
presence and absence of KU55933 (ATM inhibitor) and
SB203580 (p38 MAPK inhibitor) and both drugs inhib-
ited the enhanced CHK2 phosphorylation in Dsg3
depleted
cells
relative
to
corresponding
scrambled
siRNA controls in parallel with a strong reduction in Dsg3 constrains p53 in response to stress g
Dsg3 expressing tissues, e.g. skin and oral mucosa, are
exposed daily to various stresses that could induce p5323. Thus, we challenged cells with various stresses, i.e. UV
exposure and mechanical stretching, before analyzing p53. Cells
subjected
to
UVB
irradiation
(10–30 mJ/cm2)
showed a trend of elevated p53/p21WAF1/CIP1 after 1d. This effect was enhanced markedly in Dsg3 depleted cells,
indicating Dsg3’s ability to antagonize UV induced p53
expression (Figs. 1e, 3a). Reduction in cell cycle regulators
PCNA and Cyclin A was demonstrated by Western
blotting analysis in knockdown cells in both the presence
and absence of UV (Fig. 3d). Moreover, Bax immunos-
taining indicated enhanced signals in both the cyto-
plasmic and nuclear compartments in RNAi treated cells
(Fig. S4a). Consistently, overexpression of Dsg3 resulted
in suppression of p53/p21WAF1/CIP1 after UV (Fig. S2a). Moreover, these cells were highly resistant to UV-induced
cell death relative to control cells. Similar findings were
made in A2780 and HCT116 (wtp53) (Fig. S2b). Addi-
tionally, cells treated with genotoxic drugs, such as acti-
nomycin D (Act D) or mitomycin C (MMC), also showed
a similar effect with strong induction of p53/p21WAF1/CIP1
in Dsg3 depleted cells compared to cells without knock-
down (Fig. 3b). These data suggest that the expression of
Dsg3 protects cells against various environmental insults Dsg3 expressing tissues, e.g. skin and oral mucosa, are
exposed daily to various stresses that could induce p5323. Thus, we challenged cells with various stresses, i.e. UV
exposure and mechanical stretching, before analyzing p53. Cells
subjected
to
UVB
irradiation
(10–30 mJ/cm2)
showed a trend of elevated p53/p21WAF1/CIP1 after 1d. This effect was enhanced markedly in Dsg3 depleted cells,
indicating Dsg3’s ability to antagonize UV induced p53
expression (Figs. 1e, 3a). Reduction in cell cycle regulators
PCNA and Cyclin A was demonstrated by Western
blotting analysis in knockdown cells in both the presence
and absence of UV (Fig. 3d). Moreover, Bax immunos-
taining indicated enhanced signals in both the cyto-
plasmic and nuclear compartments in RNAi treated cells
(Fig. S4a). Consistently, overexpression of Dsg3 resulted
in suppression of p53/p21WAF1/CIP1 after UV (Fig. S2a). Moreover, these cells were highly resistant to UV-induced
cell death relative to control cells. Similar findings were
made in A2780 and HCT116 (wtp53) (Fig. S2b). Dsg3 constrains p53 in response to stress Addi-
tionally, cells treated with genotoxic drugs, such as acti-
nomycin D (Act D) or mitomycin C (MMC), also showed
a similar effect with strong induction of p53/p21WAF1/CIP1 Official journal of the Cell Death Differentiation Association Rehman et al. Cell Death and Disease (2019) 10:750 Page 5 of 14 Fig. 2 The p53 suppression by Dsg3 was further supported by the inhibition of protein degradation and overexpression of Dsg3. a Western
blots of siRNA-transfected cells with and without treatment of MG-132 (25 µM) for 3 h. GAPDH and HSC70 were the loading controls. b Protein turnover
analysis for p53, as well as MDM2, indicated reduced p53 turnover accompanied by MDM2 stabilization in Dsg3 depleted cells. The above is the timeline
of the experiment. The band density for each blot was normalized against the loading control in each sample and then against the one at 0 min time
point in each condition. The calculated half-life for p53 and MDM2 were shown in the graphs. c Flow cytometric analysis of cell viability with live cells
(Zombie NIR−ve/Caspase-3−ve), Zombie NIR−ve/Caspase-3+ve, both positive (Zombie NIR+ve/Caspase-3+ve) and Zombie NIR+ve/Caspase-3−ve in NTERTs
with and without Dsg3 knockdown, grown at 100% or ~40% confluences (the represented data of 3 independent attempts). d Protein expression in
cutaneous keratinocytes T8 (p53 null, with p53 transfection) Vect control and Dsg3 overexpression (D3) that showed suppression of p53/p21WAF1/CIP1 in
D3 cells compared to Vect cells. e RT-qPCR analysis of p53 expression (mean ± s.e.m.) in T8 cell lines (n = 3 independent assays of duplicate in each test). f p53 luciferase assay (mean ± s.d.) in T8 cell lines (n = 3, a representative of two independent experiments). The comparison was via unpaired two-sided
student t-test. (**p < 0.01, **p < 0.01, ***p < 0.001) Fig. 2 The p53 suppression by Dsg3 was further supported by the inhibition of protein degradation and overexpression of Dsg3. a Western
blots of siRNA-transfected cells with and without treatment of MG-132 (25 µM) for 3 h. GAPDH and HSC70 were the loading controls. b Protein turnover
analysis for p53, as well as MDM2, indicated reduced p53 turnover accompanied by MDM2 stabilization in Dsg3 depleted cells. The above is the timeline ig. 2 The p53 suppression by Dsg3 was further supported by the inhibition of protein degradation and overexpressio on by Dsg3 was further supported by the inhibition of protein degradation and overexpression of Dsg3. Increased p53 expression and activity in Dsg3−/−mouse
skin in vivo Having confirmed that Dsg3 negatively regulates p53 in
keratinocytes, we then asked whether alteration of this
pathway is detectable in Dsg3 knockout mice. Mice with
targeted ablation of Dsg3 exhibit runting and wave-pattern
hair loss, accompanied by oral and skin lesions, after
weaning34,35. Increased expression of p53/p21WAF1/CIP1/
cleaved caspase-3 was detected in Dsg3−/−hair follicles in
dorsal skin samples from such mice, but not in Dsg3+/−
littermates (Fig. 4). This result confirmed that Dsg3
expression is associated with the prevention of p53 acti-
vation in mouse skin in vivo. Dsg3 constrains p53 in response to stress a Western blots of siRNA-transfected cells with and without treatment of MG-132 (25 µM) for 3 h. GAPDH and HSC70 were the loading controls. b Protein turnover
analysis for p53, as well as MDM2, indicated reduced p53 turnover accompanied by MDM2 stabilization in Dsg3 depleted cells. The above is the timeline
of the experiment. The band density for each blot was normalized against the loading control in each sample and then against the one at 0 min time
point in each condition. The calculated half-life for p53 and MDM2 were shown in the graphs. c Flow cytometric analysis of cell viability with live cells
(Zombie NIR−ve/Caspase-3−ve), Zombie NIR−ve/Caspase-3+ve, both positive (Zombie NIR+ve/Caspase-3+ve) and Zombie NIR+ve/Caspase-3−ve in NTERTs
with and without Dsg3 knockdown, grown at 100% or ~40% confluences (the represented data of 3 independent attempts). d Protein expression in
cutaneous keratinocytes T8 (p53 null, with p53 transfection) Vect control and Dsg3 overexpression (D3) that showed suppression of p53/p21WAF1/CIP1 in
D3 cells compared to Vect cells. e RT-qPCR analysis of p53 expression (mean ± s.e.m.) in T8 cell lines (n = 3 independent assays of duplicate in each test). f p53 luciferase assay (mean ± s.d.) in T8 cell lines (n = 3, a representative of two independent experiments). The comparison was via unpaired two-sided
student t-test. (**p < 0.01, **p < 0.01, ***p < 0.001) Official journal of the Cell Death Differentiation Association Official journal of the Cell Death Differentiation Association Rehman et al. Cell Death and Disease (2019) 10:750 Page 6 of 14 Fig. 3 (See legend on next page.) Official journal of the Cell Death Differentiation Association Rehman et al. Cell Death and Disease (2019) 10:750 Page 7 of 14 (see figure on previous page)
Fig. 3 Dsg3 depletion causes further induction of p53 expression and activity in response to stress signals. a Western blotting of siRNA pre-
treated NTERT cells with and without UVB irradiation for the indicated proteins with the quantitation shown on the right (n = 3 biologically independent
samples, *p < 0.05). b Western blotting for p53/p21WAF1/CIP1 in cells treated with or without actinomycin D (Act D, 5 nM) and mitomycin C (MMC, 5 µg/ml)
for 24 h, respectively. The quantitation data are shown on the right. Enhanced expression of p53/p21WAF1/CIP1 was shown in cells with Dsg3 knockdown
and treated with drugs. Dsg3 constrains p53 in response to stress c Mechanical stretching induced increased expression of p53 and p21WAF1/CIP1/ Bax in Dsg3 KD cells. The siRNA pre-treated cells
were seeded at confluent density in BioFlex plates and then subjected to cyclic strain (TX-5000, 20% amplitude, 1 Hz) for 4 h the following day. Lysates
were extracted either immediately after strain or 2 h and 24 h later, respectively, after transferring to a stationary state, along with static control cells. d Western blotting analysis for PCNA and cyclin A in siRNA treated cells with and without UV. e Western blots for the indicated proteins upstream of p53
as well as phospho-p53-S20 in siRNA pre-treated cells with and without UVB (30 mJ/cm2). f Qnatitation of 53BP1 nuclear staining (n = 10, mean ± S.D.,
**p < 0.01, ***p < 0.001). g Western blotting in siRNA transfected cells treated in the presence and absence of ATM inhibitor KU55933 (20 µM) and p38
MAPK inhibitor SB203580 (20 µM), respectively. All cells were exposed to UVB (30 mJ/cm2) in this case. Cells were treated with drugs 1 h before UV and
were grown overnight before lysate extraction. h The expression of p53 in the same samples as phosphorylated ATM and CHK2 (the last two corrected for
total protein) following the indicated drug treatments in Dsg3-depleted cells. The data are expressed as the band intensities in the Dsg3-depleted cells
relative to the corresponding scrambled siRNA controls, which were normalized to 1 oral tissue biopsies from 25 patients and found a
remarkable increase of p53, in both cytoplasm and
nucleus across almost the entire stratified epithelial layer,
in 12 PV cases (~50%), especially in cells surrounding or
in the clusters within blisters. Normal samples showed
only a few p53 nuclear-positive cells located in the basal
and suprabasal layers (Fig. 5a). Cells immunopositive for
cleaved caspase-3 in PV were also positive for p53, indi-
cating activation of the p53 pathway in PV (Fig. 5b). Notably, positive staining for both proteins was observed
in non-lesional areas in PV (Case-3 in Fig. 5). These
results are indicative of alterations of the Dsg3-p53
pathway in PV that lead to caspase-3 activation as
reported previously36. total p53/phospho-p53-S20 (Fig. 3g, h) and this was
particularly marked in the case of SB203580 (Fig. 3h
right panel). Dsg3 constrains p53 in response to stress These findings suggest that, in the presence
of DNA damage, Dsg3 depletion further potentiates
DNA double-strand breaks, which are involved in acti-
vation of ATM and its downstream targets CHK2 and
p53, leading to cell cycle arrest and pre-apoptosis. Again, the knockdown studies for desmoplakin and E-
cadherin were performed in conjunction with UV but no
comparable results were obtained, albeit the UV exposure
induced p53 (Fig. 1g, h). Only a small reduction of p53/
p21WAF1/CIP1/Bax was detected in E-cadherin knockdown. No apparent changes of p53 were shown in desmoplakin
knockdown while it had no effect on p21WAF1/CIP1 and
only caused a marginal increase in Bax. Collectively, these
data suggest that the regulation of p53 by Dsg3 likely is
independent of desmoplakin and E-cadherin, implying
that this pathway may be mediated by extra-desmosomal
Dsg317. Since activation of p53 occurs in other diseases as
described above, it is important to determine the speci-
ficity of this Dsg3-p53 pathway in PV. To evaluate our
in vivo finding in PV, next, in vitro studies were per-
formed with PV sera collected from a different cohort of
17 patients, and more specifically, with AK23, a well-
characterized pathogenic monoclonal antibody targeting
the adhesion site at Dsg3 N-terminus37. Confluent cells
were treated with PV sera or AK23 (40% PV sera,
1–100 µg/ml AK23) before immunostaining for p53/Dsg3
(Fig. 6a–e). In controls treated with normal sera, nuclear
p53 was predominant, with limited cytoplasmic staining. In contrast, cells treated with PV sera (for 24 h) exhibited
an augmentation of both nuclear and cytoplasmic p53
(Fig. 6a–c). In some samples, membranous and cyto-
plasmic staining was evident. Intriguingly, the membrane
distribution of p53 showed co-localization with Dsg3
where there was severe membrane disruption (Fig. 6a
arrows in the inserts). The enhanced cytoplasmic p53 in
PV serum-treated cells may indicate the enhanced protein
synthesis (Fig. 6c). In parallel, treatment with AK23 mir-
rored increased nuclear p53, in a time and dose-
dependent manner (Fig. 6d, e). As expected, disruption
of junctions was apparent in cells with membrane dis-
tribution of p53 (arrows in Fig. 6e). Some difference in Discussion p53, a “guardian of the genome”, is a central player in
cell responses to environmental stress23,29. Here we pro-
vide the first evidence that Dsg3 acts as an anti-stress
protein by restricting p53 responses to stress signals in
keratinocytes (Fig. 6f). Knockdown of Dsg3 in vitro or its
ablation in vivo caused elevated expression/stabilization
of p53, coupled with decreased PCNA and Cyclin A, and
increased activation of caspase-3, indicative of early
apoptosis31,36. Our in vitro studies suggest an involvement
of at least ATM and CHK2 activation upstream of p53
upregulation, following Dsg3 depletion. This effect was
more pronounced in cells exposed to various stresses. Conversely, overexpression of Dsg3 resulted in the
opposite effect with marked suppression of p53. Finally,
we showed that this pathway seemed to be altered in PV,
at least in a subset of patients, suggesting that the anti-
bodies which mediated Dsg3 disruption induces p53
activation. This finding underscores the possibility that
failure in this anti-stress pathway contributes to PV
acantholysis. Fig. 4 Increased expression of p53, p21WAF1/CIP1 and cleaved
caspase-3 is observed in the back skin of Dsg3 knockout mice. /
+/ Fig. 4 Increased expression of p53, p21WAF1/CIP1 and cleaved
caspase-3 is observed in the back skin of Dsg3 knockout mice. /
+/ Fig. 4 Increased expression of p53, p21WAF1/CIP1 and cleaved
caspase-3 is observed in the back skin of Dsg3 knockout mice. /
/ caspase 3 is observed in the back skin of Dsg3 knockout mice. a Immunofluorescent staining in the back skin of Dsg3−/−and Dsg3+/−
(heterozygous littermate) mice showed elevated signals for the
indicated proteins in the hair follicles of Dsg3−/−mice compared to
heterozygous littermate, though no positive staining was observed in
the epidermis (n = 2 mice per group, aged 8–12 weeks). Some
fibroblasts in the dermis were also shown positive staining of p53. Epi:
epidermis, HF: hair follicle. The inset in the top right panel highlight
cells with double positive staining for p53 and active caspase-3 in
Dsg3 null skin. Scale bar, 20 µm. b Tables summarize the scores of
positive hair follicle staining for p53/active caspase-3 and p53/
p21WAF1/CIP1, respectively. Each hair follicle containing one or more
positively stained keratinocytes was scored positive PV is caused by autoantibodies targeting Dsgs that lead
to defects in cell–cell adhesion, however, the precise
molecular mechanism is still not fully understood. Enhanced p53 expression in PV and in keratinocyte
cultures treated with PV sera as well as a characterized
pathogenic antibody To explore whether our identified pathway is operative
in PV, we performed immunohistochemistry for p53 in Official journal of the Cell Death Differentiation Association Page 8 of 14 Rehman et al. Cell Death and Disease (2019) 10:750 Page 8 of 14 Fig. 4 Increased expression of p53, p21WAF1/CIP1 and cleaved
caspase-3 is observed in the back skin of Dsg3 knockout mice. a Immunofluorescent staining in the back skin of Dsg3−/−and Dsg3+/−
(heterozygous littermate) mice showed elevated signals for the
indicated proteins in the hair follicles of Dsg3−/−mice compared to
heterozygous littermate, though no positive staining was observed in
the epidermis (n = 2 mice per group, aged 8–12 weeks). Some
fibroblasts in the dermis were also shown positive staining of p53. Epi:
epidermis, HF: hair follicle. The inset in the top right panel highlight
cells with double positive staining for p53 and active caspase-3 in
Dsg3 null skin. Scale bar, 20 µm. b Tables summarize the scores of
positive hair follicle staining for p53/active caspase-3 and p53/
p21WAF1/CIP1, respectively. Each hair follicle containing one or more
positively stained keratinocytes was scored positive background in p53 staining was shown in cases, p53
knockdown rendered significant reduction of p53 (Fig. 6a
right panel, Figure S3). Additionally, Bax staining showed
enhanced cytoplasmic signals in cells treated with PV sera
(Fig. S4b). For Dsg3 staining, two Dsg3 antibodies were
used. While Dsg3 staining with rabbit antibody (H145)
that binds to its C-terminus exhibited broad variations,
another mouse antibody (5H10) that binds the N-
terminus at the extracellular domain of Dsg3 showed
marked depletion of Dsg3 from the surface in cells treated
with PV sera (Fig. 6a, b). Drastic disruption/reduction of
Dsg3 at the junctions was detected with H145 in cells
treated with PV sera and to a lesser extent, with AK23
(arrowhead in Fig. 6a, e). Some PV sera samples even
showed a marked increase accompanied with pronounced
Dsg3 disruption at the junctions and its aggregates in the
cytoplasm (PV serum-12, Fig. 6a). Taken together, these
in vitro findings demonstrate that treatment of keratino-
cytes with PV autoantibodies and pathogenic antibody
evoked marked disruption/depletion of Dsg3 from the
plasma membrane, leading to induction of p53 and
activation. Discussion Pre-
vious studies indicated that apoptosis and activation of
caspases are involved in PV pathogenesis with the hypoth-
esis that an activated death signaling could be the under-
lying mechanism in PV-IgG induced acantholysis38–41. Increased FasL/FasR were detected in keratinocyte and skin
organ cultures treated with PV-IgG39–41. In addition, there
is evidence suggesting that blistering is associated with p53 staining was observed between AK23 and PV serum-
treated cells suggesting variations between monoclonal
antibody and patient sera with polyclonal antibodies. The
specificity of enhanced p53, induced by PV sera, was
confirmed by p53 knockdown experiments where cells
were transfected with p53 siRNA alongside with control
siRNA for 1d before treated with PV or control sera. p53
knockdown almost completely abolished p53 signals in
control of serum-treated cells (Fig. S3). However, in the
PV serum treated samples, although some non-specific Official journal of the Cell Death Differentiation Association Rehman et al. Cell Death and Disease (2019) 10:750 Page 9 of 14 Fig. 5 Enhanced p53 and cleaved Caspase-3 expression is shown in clinical PV patient samples and also in keratinocyte cultures treated
with PV sera. p53 a and cleaved Caspase-3 b immunohistochemistry in oral mucous tissues from PV patients. Significantly enhanced p53 staining
was detected in 48% of patients (arrowheads indicate positive nuclear staining whereas arrows indicate predominant cytoplasmic staining),
compared to normal controls. Oral mucous cancer was used as positive control here. Asterisks indicate the areas of the blisters. Positive staining of
active caspase-3 was also detected in PV patients with the positive p53 staining in oral mucous tissues, especially in the basal and immediate
suprabasal layers of stratified squamous epithelium. The positive staining was also detected in cells located in sub-mucous connective tissue (right) Fig. 5 Enhanced p53 and cleaved Caspase-3 expression is shown in clinical PV patient samples and also in keratinocyte cultures treated
with PV sera. p53 a and cleaved Caspase-3 b immunohistochemistry in oral mucous tissues from PV patients. Significantly enhanced p53 staining
was detected in 48% of patients (arrowheads indicate positive nuclear staining whereas arrows indicate predominant cytoplasmic staining),
compared to normal controls. Oral mucous cancer was used as positive control here. Asterisks indicate the areas of the blisters. Positive staining of
active caspase-3 was also detected in PV patients with the positive p53 staining in oral mucous tissues, especially in the basal and immediate
suprabasal layers of stratified squamous epithelium. Official journal of the Cell Death Differentiation Association Discussion The positive staining was also detected in cells located in sub-mucous connective tissue (right) surface receptors other than Dsgs16,17,42. Thus, the direct
link between Dsg3 and the p53 pathway is unclear. In this
study, we provide evidence of a potential link between Dsg3
and p53, indicating that disruption of Dsg3 by PV IgG may
cause p53 induction leading to caspase-3 activation. Our
in vitro study demonstrated that Dsg3 depletion causes
enhanced p53 with elevated expression of its targets
p21WAF1/CIP1, resulting in a reduction of PCNA and Cyclin surface receptors other than Dsgs16,17,42. Thus, the direct
link between Dsg3 and the p53 pathway is unclear. In this
study, we provide evidence of a potential link between Dsg3
and p53, indicating that disruption of Dsg3 by PV IgG may
cause p53 induction leading to caspase-3 activation. Our
in vitro study demonstrated that Dsg3 depletion causes
enhanced p53 with elevated expression of its targets
p21WAF1/CIP1, resulting in a reduction of PCNA and Cyclin
A coupled with elevated Bax/cleaved (activated) caspase-3. We also showed that such responses were accelerated when
cells were exposed to stresses that trigger a p53 activation. These in vitro findings were supported by an in vivo study
in Dsg3 null mice, as well as by the gain-of-function
studies that caused marked suppression of p53 and its
transcription activity. These findings collectively support
the hypothesis that Dsg3 dampens the stress response
pathway by negatively regulating p53. Notably, we showed that this pathway is altered in ~50% PV patient samples
with enhanced p53 and caspase-3 that was not only found
in cells surrounding the blisters but also in pro-lesions
regions, indicating that activation of the p53 pathway
occurs early prior to the event of pemphigus acantholysis. The apparent heterogeneity of p53 elevation in PV
patients could well reflect variations of clinical activities/
treatment status of the disease and/or be related to the
transient response of p53 to cellular stress. It is well-
known that drugs which caused DNA damage initially
induce high levels of p53, as well as p21WAF1/CIP1, but
these changes decline over time43. It is worth noting that
the specificity of p53 induction caused by antibody tar-
geting Dsg3 was verified by our in vitro studies with PV
sera (samples 17 cases) and a well-characterized specific
pathogenic monoclonal antibody, AK23 targeting the
Dsg3 adhesion site. Thus, our findings in PV may indicate A coupled with elevated Bax/cleaved (activated) caspase-3. Discussion d Treatment
of NTERTs with the pathogenic monoclonal antibody AK23 targeting Dsg3 N-terminus, caused p53 induction, in a time and dose-dependent manner
(n = 11 fields per condition, pooled from 2 independent experiments). For the dose-response experiment, cells were treated with AK23 for 6 h. e Confocal images of cells with triple staining, treated in the presence and absence of AK23. Disruption of F-actin along with Dsg3 (arrowheads) was
readily detectable in cells exposed to AK23. Increase p53 expression was detected predominantly in the nucleus and also was observed at the plasma
membrane where it showed colocalization with Dsg3 and F-actin (arrows) in cells treated with AK23. The membrane distribution of p53 was not
detectable in control cells. The protein colocalization of the dotted line box is shown at the bottom. (*p < 0.05, **p < 0.01, ***p < 0.001). Scale bar,
10 µm. f Schematic model of Dsg3 in the suppression of p53 in keratinocyte response to stresses. Disruption of Dsg3 by RNAi or PV autoantibodies
evokes p53 induction caspase-3 in PV seems to be a consensus finding and
inhibition of caspase-3 has been shown to be effective in
preventing blister formation in both in vitro and in vivo
PV models. Thus, the anti-Dsg3 antibody mediated acti-
vation of p53 and its regulated target caspase-3 could well
be the key factor to cause PV acantholysis. In support of
this notion, activation of caspase-3 has been reported to
be a causative factor for the rapid disruption of tight
junctions in endothelial cells46. Furthermore, the staur-
osporine induces activation of caspase-3 is accompanied
by disturbance of adherens junctions accompanied by a
significant increase in cell permeability which can be
inhibited
by
pretreatment
with
caspase-3
specific
inhibitor47. a specific p53 induction associated with the PV IgG
induced Dsg3 disturbance since treatment of cells with
AK23 caused augmented p53 in a time and dose-
dependent manners. Taken together, these results sug-
gest that activation of the Dsg3-p53 pathway may con-
tribute, at least in part, to PV pathology36,39,40. gy
Activation of p53 also occurs in other chronic or
inflammatory conditions including LP26–28, psoriasis24,25
and Rheumatoid arthritis44 in which the role of Dsg3 has
not yet been defined. Thus, disruption of Dsg3 may be one
mechanism by which p53 can be activated in human skin
diseases. Discussion We also showed that such responses were accelerated when
cells were exposed to stresses that trigger a p53 activation. These in vitro findings were supported by an in vivo study
in Dsg3 null mice, as well as by the gain-of-function
studies that caused marked suppression of p53 and its
transcription activity. These findings collectively support
the hypothesis that Dsg3 dampens the stress response
pathway by negatively regulating p53. Notably, we showed Official journal of the Cell Death Differentiation Association Rehman et al. Cell Death and Disease (2019) 10:750 Page 10 of 14 Fig. 6 (See legend on next page.) Fig. 6 (See legend on next page.) gend on next page.) Fig. 6 (See legend on next page.) Official journal of the Cell Death Differentiation Association Rehman et al. Cell Death and Disease (2019) 10:750 Page 11 of 14 (see figure on previous page)
Fig. 6 Altered p53 expression and distribution were detected in keratinocyte cultures treated with PV sera and pathogenic antibody. a Confocal microscopy of NTERTs treated with the PV sera, dual labeled for Dsg3 and p53. Cells were seeded at confluent densities in KGM for
overnight before being treated with PV sera (at 40% concentration in KGM) from a different cohort of PV patients (n = 17), for 24 h. Disruption or
depletion of Dsg3 at the plasma membrane accompanied with marked increases in p53 was observed in PV serum-treated cells that were abolished
by p53 knockdown, compared to controls exposed to sera of healthy individuals that displayed, predominantly, nuclear p53 signals. Of note, p53 also
showed distribution at the membrane where it colocalized with the fragmented Dsg3 (arrows in the inserts). Additional data for p53 knockdown was
shown in Supplementary material Fig. S5. The image magnification in PV serum-2 was doubled, relative to other panels, to highlight the disruption of
junctions and peripheral distribution of p53. b Scatter and whisker plots of Dsg3 and p53 cellular and subcellular expression (n = 16 for PV serum
samples, n = 6 for control samples). Student t-test and the Wilcoxon–Mann–Whitney Rank Test were used for statistically significant analysis here and
gave similar results. c The relative ratio of p53 nuclear versus cytoplasmic cellular distribution in controls and 16 PV sera treated NTERTs. Discussion As p53 is a key factor in cell cycle control, dif-
ferentiation and apoptosis as well as a valuable biomarker
for prediction of malignant transformation, it is not sur-
prising to see the altered p53 expression in other condi-
tions although the molecular basis underlying the p53
activation may differ. Elevated p53 in psoriasis was
thought to be associated with cell proliferation24,25
whereas its detection in LP might be due to p53 gene
mutation26–28. Furthermore, an activated p53 pathway
can elicit pro-apoptotic/apoptotic events through multi-
ple mechanisms depending on the location and levels of
its expression. The cytoplasmic p53 can mediate apoptosis
by directly interacting with Bcl-2 family members45, while
nuclear p53 can bind to DNA and activate pro-apoptotic
gene expression, which ultimately contributes to disease
pathology. PV is a complex autoimmune disease with Dsg3 being a
central player in pemphigus acantholysis that likely is
triggered by a collection of signaling pathways, including
Src, p38 MAPK, EGFR, c-Myc, and Rho GTPases,
downstream of PV-IgG targeting Dsg3 disruption4,17. We
now report a potential involvement of abnormal p53
activation in PV caused by PV IgG targeting Dsg3 which
acts as an anti-stress protein in counterbalancing p53 in
the maintenance of normal epithelial homeostasis. Official journal of the Cell Death Differentiation Association Materials and methods
Cell lines, animal and clinica Cell lines, animal and clinical patient oral mucosal samples
Various epithelial cell lines derived from skin and other
tissues were used in the study, i.e. NTERT immortalized
skin keratinocytes (wild-type p53: wtp53) maintained in
keratinocytes serum-free medium (KSFM) (17005042,
Thermo Scientific); T8 cutaneous squamous cell carci-
noma cell line with a frameshift mutation at amino acid
91 of TP53 resulting in a truncated protein and making it
essentially p53 null (gift from Prof. Catherine Harwood),
and they were cultured in complete keratinocyte growth The finding of increased caspase-3 in cells with Dsg3
knockdown may indicate early apoptosis, however, our
FACS analysis for Annexin V did not detect any evident
apoptosis in Dsg3 knockdown cells. A recent study has
highlighted that a transient, modulated activation of
caspase-3 is triggered by antibody-mediated Dsg3 dis-
ruption in PV, but this event is uncoupled from the
classical
apoptotic
pathways36. Nevertheless,
active Official journal of the Cell Death Differentiation Association Rehman et al. Cell Death and Disease (2019) 10:750 Page 12 of 14 medium KGM containing Dulbecco’s Modified Eagle
Medium (DMEM) (12–604F, Lonza):Ham’s 12 (11765054,
Thermo Scientific) in the ratio of 3:1 supplemented with
10% fetal calf serum (FCS) (Biosera), epidermal growth
factor (EGF) (13247-051, Invitrogen), Insulin human
solution (19278, Sigma), cholera toxin (C8052, Sigma),
and hydrocortisone (H4001, Sigma). MDCK (Madin
Darby canine kidney) cells (wt p53) are the simple epi-
thelial cell line, which is derived from canine kidney
tubule epithelium; A431 cell line (mutant p53-R273H) is
derived from vulva squamous cell carcinoma; A2780
ovarian cancer cell line (wt p53) and HCT116 colorectal
carcinoma cell line (wt p53). All these cell lines were
maintained in DMEM (12–604 F, Lonza) supplemented
with 10% FCS (Biosera, UK). Due to the low levels of
endogenous Dsg3 expression, these cell lines were used
for the gain-of-function studies by transduction of retro-
viral construct pBABE-hDsg3.myc along with the empty
vector control3,48 namely FL Dsg3 and Vect Ct cells,
respectively3. Cells were incubated at 37 °C in a humidi-
fied atmosphere of 95% air and 5% CO2. The medium was
changed on alternate days and cells were subjected to
subculture routinely once they reached to about 70–80%
confluence. 3-phosphate dehydrogenase (GAPDH) rAb (14c10, Cell
Signaling); HSC70 mAb (B6:sc-7298, Santa Cruz); β-actin
mAb (8H10D10, Cell Signaling); anti-53BP1 (05–726,
Upstate); anti-ATM (phospho S1981) (ab81292, Abcam);
anti-ATM (ab32420, Abcam); anti-CHK2 (phospho T68)
(ab32148, Abcam); anti-CHK2 (ab109413, Abcam). Materials and methods
Cell lines, animal and clinica The
anti-ATM antibodies were validated on Western blots of a
range of normal and neoplastic oral keratinocytes lines
and ATM-deficient human epidermal keratinocytes from
an Altaxia Telangiectasia patient, the last of which showed
no ATM protein expression (K.Ng and E.K.Parkinson -
manuscript in preparation). Antibodies The following mouse (m) and rabbit (r) monoclonal/
polyclonal antibodies (Abs) were used: Dsg3 mAb against
the N-terminus (5H10) (sc-23912, Santa Cruz); Dsg3 rAb
against the C- terminus (H145) (sc-20116, Santa Cruz);
p53 mAb (DO-1) (ab1101, Abcam); p53 rAb (C-19) (sc-
1311-R, Santa Cruz); MDM2 rAb (EP16627) (ab178938,
Abcam); phospho MDM2 rAb (S166) (ab131355, Abcam);
p21WAF1/CIP1 rAb (C-19) (sc-397, Santa Cruz); Bax mAb
(sc-20067, Cell Signaling); Caspase3 rAb (clone C92-605,
RUO) (14C10, BD Biosciences); Caspase3 rAb (9662 S,
Cell Signaling); active Caspase3 rAb (ab49822, Abcam);
Desmoplakin rAb (sc-33555, Santa Cruz); Plakoglobin
mAb (PG51, Progen); Dsc2 rAb (610120, Progen); Dsg2
mAb (33-3D) was kindly received from Prof. David Gar-
rod; E-Cadherin mAb (HECD-1) (ab1416, Abcam); H-
432, rabbit Ab to Cyclin A (sc-751, Santa Cruz); PC10,
mouse Ab to PCNA (sc-56, Santa Cruz); Glyceraldehyde- Treatments with ultraviolet (UV) B, drugs, and mechanical
stretching The siRNA treated cells were seeded at confluence
densities in 6-well plates before irradiation of UVB
(10–30 mj/cm2) using a CL-1000 Ultraviolet Crosslinker
(Ultra-Violet Products, CA) or treatment with actinomy-
cin D (Act-D, 5 nM) and mitomycin C (MMC, 5 ug/ml)
for 24 h, respectively. Protein lysates were extracted for
Western blotting analysis. The regimen for the cyclic strain was adapted from a
previous publication50. Briefly, cells were plated at con-
fluent densities and grown for 1–2 days on collagen-
coated BioFlex 6-well plates (Flexcell® International
Corporation) prior to equiaxial cyclic stretching (20%
amplitude with 1 Hz, FX-5000 Tension System (Flexcell
International, Burlington, NC) for 4 h). Control cells were
seeded in the same plates without strain. Lysates were
extracted either immediately after strain or transferred to
the static state in an incubator and harvested later for the
indicated time points. Mouse back skin samples from Dsg3 null (Dsg3−/−) and
heterozygous
control
littermates
(Dsg3+/−)
were
obtained, as described previously49. PV sera (anonymous,
17 cases) were received from our collaborator based in
First Department of Dermatovenerology,
St. Anne’s
Faculty Hospital, Brno, Czech Republic, and oral tissue
samples of PV patients (25 PV cases and 10 normal
healthy tissue controls as well as 3 cancer patient samples)
were obtained from our collaborator in Guiyang Medical
University, China; all with informed patient consent and
ethical approval. The details of siRNA/plasmid transfection/transduc-
tion, immunofluorescence, immunohistochemistry in PV
specimens, nuclear extraction, Western blotting, lucifer-
ase assay, FACS based Cell Viability-Caspase-3 assay and
RT-qPCR,
etc. were
described
in
Supplementary
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providing reagents and assistance with imaging work. The study was
supported by the Barts and The London School of Medicine and Dentistry and
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1 1Centre for Oral Immunobiology and Regenerative Medicine, Institute of
Dentistry, Barts and The London, School of Medicine and Dentistry, Queen
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School of Stomatology, Guizhou Medical University, Guiyang, China. 3Department of Dermatology, Eberhard Karls University, Tübingen, Germany. 4First Department of Dermatovenerology, St. Anne’s Faculty Hospital, Brno,
Czech Republic. 5Centre for Cell Biology and Cutaneous Research, Blizard
Institute, Barts and The London, School of Medicine and Dentistry, Queen Mary
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https://openalex.org/W4288684795
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https://amb-express.springeropen.com/counter/pdf/10.1186/s13568-022-01442-6
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English
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Characterization of a novel chitinolytic Serratia marcescens strain TC-1 with broad insecticidal spectrum
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AMB express
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cc-by
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Key points • A new Serratia marcescens strain TC -1 showed high
insecticidal activity against several phytophagous
insect species and nematocidal activity against Cae- *Correspondence: silianghuang@aliyun.com
School of Life Science and Agricultural Engineering, Nanyang Normal
University, Nanyang 473061, China School of Life Science and Agricultural Engineering, Nanyang Normal
University, Nanyang 473061, China © 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/. Abstract The Gram-negative rod-shaped bacterium Serratia marcescens is an opportunistic pathogen of many organisms,
including insects. We report the identification and optimal in vitro chitinase production conditions of a novel chi‑
tinolytic S. marcescens strain TC-1 isolated from a naturally infected white grub (Anomala corpulenta) collected from
a peanut field at Nanyang city, Henan province, China. Strain identification was conducted by morphological, physi‑
ological, biochemical and molecular analyses. The amplified 16S rRNA gene of TC-1 showed a similarity greater than
99% with multiple strains of S. marcescens. Based on Neighbor-joining phylogenetic tree analysis of bacterial 16S rRNA
gene sequences, TC-1 formed a clade with S. marcescens, clearly separated from other Serratia spp. The strain TC-1
showed larvicidal activities against five insect species (A. corpulenta, Plutella xylostella, Spodoptera exigua, Helicoverpa
armigera, Bombyx mori) and the nematode Caenorhabditis elegans, but not against S. litura. The operating parameters
of chitinase production by TC-1 were optimized by response surface methodology using a three-factor, three-level
Box-Behnken experimental design. The effects of three independent variables i.e. colloidal chitin concentration
(7–13 g l−1), incubation time (24–72 h) and incubation temperature (24–32 °C) on chitinase production by TC-1 were
investigated. A regression model was proposed to correlate the independent variables for an optimal chitinase activ‑
ity predicted as 20.946 U ml−1, using a combination of colloidal chitin concentration, incubation time and incubation
temperature of 9.06 g l−1, 63.83 h and 28.12 °C, respectively. The latter agreed well with a mean chitinase activity of
20.761 ± 0.102 U ml−1 measured in the culture supernatants of TC-1 grown under similar conditions with a colloidal
chitin concentration, incubation time and incubation temperature of 9 g l−1, 64 h and 28 °C, respectively. Our study
revealed the S. marcescens strain TC-1 with potential as a biocontrol agent of insect pests and nematodes and demon‑
strated the proposed regression model’s potential to guide chitinase production by this strain. Keywords: Serratia marcesecens, Insect pathogen, Anomala corpulenta, White grub, Biological control, Chitinase,
Response surface methodology norhabditis elegans.h Tao et al. AMB Express (2022) 12:100
https://doi.org/10.1186/s13568-022-01442-6 Tao et al. AMB Express (2022) 12:100
https://doi.org/10.1186/s13568-022-01442-6 Open Access Characterization of a novel chitinolytic
Serratia marcescens strain TC‑1 with broad
insecticidal spectrum Aili Tao, Tan Wang, Fahu Pang, Xueling Zheng, Camilo Ayra‑Pardo, Siliang Huang* , Ruxin Xu, Fengqin Liu,
Jiakang Li, Yibin Wei, Zhiqing Wang, Qiuhong Niu and Dandan Li Aili Tao, Tan Wang, Fahu Pang, Xueling Zheng, Camilo Ayra‑Pardo, Siliang Huang* , Ruxin Xu, Fengqin Liu,
Jiakang Li, Yibin Wei, Zhiqing Wang, Qiuhong Niu and Dandan Li norhabditis elegans.
• The larvae of S. litura differed in their susceptibility to
TC-1 compared to other Lepidoptera insect species.
• A regression model correlated well with the variables
(chitin concentration, time and temperature) for high
chitinase production by TC -1. Introduction production by a bacterium is mainly influenced by the
culture conditions (Gutiérrez-Román et al. 2012), which
must be optimized for the individual bacterial strains.h Serratia marcescens is a Gram-negative bacillus that
occurs naturally in soil, water, foodstuff and animals
(Hejazi and Falkiner 1997). The bacterium produces a red
pigment, named prodigiosin, though this ability is absent
in some strains isolated from humans (Gargallo et al. 1987; Grimont and Grimont 1978). p
The metallic green beetle Anomala corpulenta Mots-
chulsky (Coleoptera: Scarabaeidae: Rutelinae) is an
economically important insect pest throughout Asia
including China. The larvae (white grubs) and adults of
this insect pest commonly infest the underground and
aboveground parts of multiple plant species, respectively. The adults feed preferably on the leaves of apple, pear,
grape, peanut, soybean, poplar and elm (Ji et al. 2017; Li
et al. 2009), resulting in significant damage to agriculture,
forestry and urban greening in the severe cases. The pri-
mary control methods for A. corpulenta rely heavily on
conventional pesticide sprays (Gong et al. 2016; Ji et al. 2017), which is a concern for the environment, biodi-
versity and human health. Microbe-based biopesticides
can provide an economical, ecofriendly and sustainable
approach to insect pest management. Considerable diversity has been found among S. marc-
escens populations. Many strains of the bacterium are
known to be saprophytic in soil (Nawani and Kapadnis
2001) or endophytic in plants (Gyaneshwar 2001), but
some are notorious for causing diseases on plants (Bru-
ton et al. 1995, 2003; Lukezic et al. 1982; Sears et al. 1975;
Wang et al. 2015) and animals (Quesenberry and Short
1983) as well as on humans (Whalen 1970; Zipper et al. 1996). Several studies have been focused on the beneficial
functions of some S. marcescens strains, such as biodeg-
radation and bioremediation potential of environmen-
tal pollutants (Abo-Amer 2011; Cycoń et al. 2012); as a
bio-collector for hematite flotation (Yang et al. 2014a, b);
plant-growth-promoting potential (George et al. 2013;
Lavania et al. 2006; Selvakumar et al. 2008); the biocon-
trol of insect pests (Deng et al. 2008; Fu et al. 2021; King
et al. 1975; Podgwaite and Cosenza 1976; Qi et al. 2004;
Sikorowski et al. 2001; Wang et al. 2010; Yang et al. 2014a,
b; Zhao et al. 2017; Zhang et al. 2011, 2021), plant path-
ogens (Ahmed 2010; Dong et al. 2016; Feng et al. 2018;
Someya et al. Introduction 2001), plant diseases (Ordentlich et al. 1987
and 1988; Someya et al. 2000; Wei et al. 1996) and weeds
(Yang et al. 2015a, b; Li et al. 2021). In 2013, we isolated a new chitinolytic S. marcescens
strain TC-1 from a naturally infected white grub (A. cor-
pulenta) and found that its culture supernatants (CS)
could cause the death of second- and third-instar larvae
of Spodoptera exigua (Additional file 1: Table S1). Iso-
electric point screening tests of proteins in TC-1’s CS
revealed that the protein with the highest chitinolytic
activity precipitated at pH 6.7 (Additional file 1: Tables S2
and S3). Furthermore, we used the protein precipitated at
pH 6.7 as a crude chitinase and confirmed its larvicidal
activity against S. exigua (Additional file 1: Table S4). In
the present work, we aimed to identify and characterize
the strain TC-1 by evaluating its pathogenicity against
larvae of six phytophagous insect species and one nema-
tode, and optimizing its chitinase production conditions
using response surface methodology (RSM). The virulence of S. marcescens on both insects and
fungi is due in part to its chitinase enzymes, which hydro-
lyze chitin—the second most abundant natural biopoly-
mer after cellulose that constitutes the major structural
component of certain rigid structures in invertebrates
(e.g. the insects’ exoskeleton) and the cell walls of fungi
(Shahidi et al. 1999; Nawani and Kapadnis 2001; Mer-
zendorfer and Zimoch 2003). Chitin metabolism is an
essential life sustaining activity of phytophagous insects,
phytopathogenic fungi and parasitic nematodes, which
are the major limiting factors of the agricultural produc-
tion system (Subbanna et al. 2018). For this reason, the
breakdown of chitin or inhibition of chitin metabolism
can lead to the death of these agricultural pests. There-
fore, isolation and characterization of chitinolytic S. marcescens strains are considered crucial for the devel-
opment of efficient biocontrol agents against insect pests
(Chen et al. 2001; Li 1982; Liu et al. 2019; Lysenko 1976;
Parani et al. 2011; Sezen et al. 2001; Yang et al. 2015a,
b; Yin et al. 2004) and plant pathogenic fungi (Babash-
pour et al. 2012; Gutiérrez-Román et al. 2012; Kobayashi
et al. 1995; Moon et al. 2017; Oppenheim and Chet
1992; Parani et al. 2011; Someya et al. 2005). Chitinase Tao et al. AMB Express (2022) 12:100 Page 2 of 13 Page 2 of 13 Tao et al. AMB Express (2022) 12:100 Characterization and identification of the isolated strainh Characterization and identification of the isolated strain
The morphological analysis was carried out by record-
ing the phenotypic features of TC-1 colonies grown on
BPYDA plates for 48 h at 28 °C. For the TC-1’s physiolog-
ical and biochemical characterizations, 28 reactions i.e. oxidase, Gram-staining, phenylalanine deaminase, lysine
decarboxylase, ornithine decarboxylase, DNase, arginine
decarboxylase, urease, motility, productions of H2S and
indole, acid production from glucose, Voges-Proskauer
(V.-P.), methyl red, utilization of the carbon sources
(mannitol, melibiose, sucrose, sorbitol, lactose, adonitol,
xylose, raffinose, bile esculin, and L-arabinose), gelatin
liquefaction, Simmons citrate, malonate, and reduction
of nitrate, were performed using routine bacteriologi-
cal methods (Buchanan and Gibbon 1984; Dong and Cai
2001). Four TC-1’s infectious doses were prepared for white
grub bioassays by mixing the bacterial suspension
with sterilized soil (i.e. 1 × 109, 5 × 108, 2.5 × 108, and
1.25 × 108 CFU g−1). The amount of soil was based on the
numbers of white grubs tested (Xu et al. 2009). The white
grubs were placed individually in feeding boxes contain-
ing potato slices and the soil with the different TC-1’s
doses. The mortality rate was scored every three days. A
larva was considered dead if no movement was detected
after being stimulated with a blunt-ended tip. Each treat-
ment consisted of 20 white grubs with three independent
replicates. The soil treated with sterilized water alone was
used as control (CK). A corrected mortality rate (RMR)
was calculated as RMR = (MRT-MRCK)/(1-MRT) × 100,
where MRT and MRCK represent the mortality rate of
treatment and the mortality rate of CK, respectively. For the molecular identification, the TC-1’s genomic
DNA was extracted using a procedure previously
described by Tao et al. (2014), and used for PCR of the
16S rRNA gene sequence with universal primers 27F
(5´-AGAGTTTGATCATGGCTCAG-3´)
and
1492R
(5´-TACGGTTACCTTGTTACGACTT-3´) in a 50-μl
reaction mixture (Sambrook and Russell 2001). The PCR
reaction was run for 30 cycles of DNA denaturation for
60 s at 94 °C, annealing for 30 s at 53 °C, and extension for
60 s at 72 °C. The amplified product was visualized fol-
lowing electrophoresis in 1.0% agarose gels stained with
GelRed (Biotium) and sent for bi-directional sequenc-
ing using primers 27F and 1492R. Sanger sequences In the bioassays with B. Bacterial strain isolation were generated at Shanghai Bajun Biological Technology
Co., Ltd. (China). Generated sequences were converted
to Fasta format and compared with the bacterial 16S
rRNA gene sequences deposited in GenBank using the
algorithm BLAST (https://blast.ncbi.nlm.nih.gov/Blast.
cgi). A Neighbor-joining phylogenetic tree of the TC-1’s
16S rRNA sequence with other bacterial 16S rRNA
gene sequences retrieved from the NCBI GenBank was
reconstructed using the Molecular Evolutionary Genet-
ics Analysis version 7.0 (MEGA7) under 1000 bootstrap
replicates. In August 2013, a naturally infected white grub (A. cor-
pulenta) was collected from a peanut field located at the
western campus of Nanyang Normal University (112°
28′ 44"N, 32°58′ 34"E, 131 m above sea level). The insect
body was 1.5 cm in length and showed clear symptoms of
disease with a pale violet red color. Surface disinfection
was carried out as follows: The affected insect was dipped
in 70% ethanol for 10 s, followed by 4 min in 0.1% HgCl2,
and five rinses with sterile water. The surface-disinfected
insect was homogenized in five ml of sterilized water
with a sterilized pestle to produce a bacterial suspension
that was plated (200 μl per plate) on beef-peptone-yeast-
dextrose agar (BPYDA: beef extract, 3 g l−1; peptone,
5 g l−1; yeast extract, 1 g l−1; dextrose, 10 g l−1; agar,
15 g l−1; pH 7.0). The plates were incubated at 28 °C for
24 h to obtain bacterial colonies that were further puri-
fied in three rounds of single-colony isolation on BPYDA. A representative strain (TC-1) was randomly selected
and identified through morphological, physiological, bio-
chemical, and molecular methods. The strain TC-1 was
deposited at the China Center for Type Culture Collec-
tion (CCTCC) with serial number M2015634. Materials and methods
Insects and nematodeh The following insect larvae were used in this study: A. corpulenta (white grub), Bombyx mori (silkworm), Heli-
coverpa armigera (cotton bollworm), Plutella xylostella
(diamondback moth), S. exigua (beet armyworm) and S. litura (common cutworm). The larvae of A. corpulenta and
B. mori were reared at our university on potato slices and
mulberry leaves, respectively, at 25 °C ± 1 °C and a photo-
period of 16/8 h (light/dark). The insects of H. armigera,
P. xylostella, S. exigua and S. litura in the second and third
larval instars and their artificial diets were purchased from
Henan Baiyun Industrial Co., Ltd. (China). A population of
nematode Caenorhabditis elegans was maintained in the
laboratory of microbiology of the Nanyang Normal Uni-
versity using the standard technique (Stiernagle 2005). Tao et al. AMB Express (2022) 12:100 Tao et al. AMB Express (2022) 12:100 Page 3 of 13 Pathogenicity testsh The TC-1’s virulence against six insect species (A. corpu-
lenta, B. mori, H. armigera, P. xylostella, S. exigua, and
S. litura) and the nematode C. elegans was investigated
through bioassays. To prepare the inocula, a loopful of
TC-1 colony grown on a BPYDA slant was inoculated
into a BPYDB liquid medium (the same components as
BPYDA except agar) and grown overnight at 28 °C with
vigorous shaking (180 rpm). One ml of this culture was
then used to inoculate 250-ml BPYDB and allowed to
grow in a rotary incubator (180 rpm) at 28 °C for 48 h. Bacterial cells were harvested by centrifugation (3000 × g,
10 min), washed twice, and re-suspended with sterilized
distilled water to a final concentration of 1 × 109 CFU
(colony forming unit) ml−1. Characterization and identification of the isolated strainh mori, fresh mulberry leaves
were washed with tap water, air-dried, sprayed with
1 × 109 CFU ml−1 of a TC-1’s suspension, and used for
feeding larvae from second- and third-instar stages. Mor-
tality rates were scored daily. Leaves sprayed with water
alone were used as CK. Each treatment consisted of 20
larvae with four independent replicates.h The virulence of strain TC-1 to H. armigera, P. xylos-
tella, S. exigua and S. litura was tested by feeding sec-
ond- and third-instar larvae with artificial diet portions Tao et al. AMB Express (2022) 12:100 Page 4 of 13 Page 4 of 13 supernatant was determined by measuring the absorb-
ance at 540 nm using an UV spectrophotometer (PRE-
SEE ANALYTICS, TU-1901). The DNS reagent was
prepared by dissolving 36.4 g of Seignette salt in 100 ml
of distilled water and heating until it was completely
dissolved. Then, DNS (1.26 g), sodium hydroxide
(4.20 g), and crystal phenol (1 g) were dissolved sepa-
rately into the Seignette salt solution, and the volume
was made up to 200 ml with distilled water. A stand-
ard curve of NAG was constructed using the method
described by Hu et al. (2016) with the regression equa-
tion Y = 0.1366X-0.1724, where “Y” and “X” represent
OD540 and NAG concentration (mg l−1), respectively
(Additional file 1: Fig. S1). The correlation coefficient
(r) of the equation was 0.9971, showing a highly posi-
tive relationship between the NAG concentration
in the solution and the OD540 value. One unit of chi-
tinase activity per milliliter (U ml−1) was defined as the
amount of the enzyme required for producing 1 µmol
of NAG from chitin. (approximately 0.2 × 0.2 × 0.2 cm3) that were previously
dipped in a TC-1’s suspension (1 × 109 CFU ml−1). Mor-
tality rates were scored daily. Diets dipped in sterilized
water alone were used as CK. Each treatment consisted
of 20 larvae with four independent replicates. The larvae
of H. armigera, S. exigua and S. litura were reared indi-
vidually to prevent cannibalism. y
p
The virulence of strain TC-1 on C. elegans was tested
using the method previously described by Niu et al. (2010) with a slight modification. Autoclaved cello-
phane paper was used to cover the PBA medium (pep-
tone, 10 g l−1; beef extract, 3 g l−1; dextrose, 10 g l−1;
NaCl, 5 g l−1; agar, 16 g l−1). Characterization and identification of the isolated strainh A TC-1’s suspension
(106 CFU ml−1) was spread on the cellophane paper and
the plates incubated at 28 °C for 3 d. One drop (50 μl) of
a C. elegans suspension containing approximately 1000
worms from second- and third-instars was placed on
the TC-1 lawn. The number of dead worms was counted
under a stereomicroscope at the three incubation times
(36, 48 and 72 h). The nematodes were considered dead
if no movement was detected after being stimulated gen-
tly with a stick. The experiment was independently repli-
cated three times. Optimization of culture conditions for chitinase production
The effects of four carbon sources (colloidal chitin,
powdered chitin, starch, and cellulose) on chitinase
production by the strain TC-1 were tested. Ten gram of
each carbon source were added separately to 1000 ml
of PBB liquid medium (the same components as PBA,
except agar) without dextrose. Strain TC-1 was inocu-
lated into each medium containing different carbon
sources and allowed to grow in a rotary incubator
(180 rpm) at 28 °C for 60 h. The chitinase activities in
the culture supernatants were separately determined
using the method as described above. Three replicates
were set up for each treatment.hf Preparation of colloidal chitin Colloidal chitin was prepared by adding concentrated
hydrochloric acid (36–38%, 100 ml) to 15 g of pow-
dered chitin (Beijing Solarbio Science & Technology Co.,
Ltd.) followed by continuous stirring at 4 °C. After stir-
ring for 20 min, the chitin was precipitated as a colloi-
dal suspension by slowly adding 2 l of distilled water at
4 °C. The precipitate was collected and treated again with
concentrated hydrochloric acid as described above. The
resultant precipitate was re-suspended in 100 ml of dis-
tilled water, mixing carefully to produce a solution with
butyrous consistency containing 1.5% (m/v) colloidal chi-
tin. The pH of the colloidal chitin was adjusted to 7 using
a sodium hydroxide solution. The effects of four nitrogen sources (peptone, ammo-
nium chloride, ammonium sulfate, and beef extract) on
chitinase production by the strain TC-1 were tested. For
the ammonium chloride and ammonium sulfate tests,
both beef extract and peptone in the PBB liquid medium
were replaced by a test nitrogen source. For the peptone
test, the PBB liquid medium free of beef extract was used. For the beef extract test, the PBB liquid medium free of
peptone was used. In all cases, the concentration of the
nitrogen source was 10 g l−1. The strain TC-1 was inoc-
ulated into a test medium with a single nitrogen source
and allowed to grow in a rotary incubator (180 rpm)
at 28 °C for 60 h. The chitinase activities in the culture
supernatants were determined separately using the
method described above. Three replicates were set up for
each treatment. Chitinase activity assayh The chitinase activity was tested by detecting N-acetyl-
glucosamine (NAG) as the final product using a routine
method (Moon et al. 2017; Abudunasier et al. 2019)
with a slight modification. Briefly, 1-ml of the bacterial
suspension was centrifuged (10,000 × g, 10 min) and
0.4 ml of the culture supernatant mixed with an equal
amount of 1.5% (m/v) colloidal chitin solution followed
by incubation at 30 °C in a water bath for 30 min. Then,
0.6 ml of distilled water and 3,5-dinitrosalicylic acid
(DNS) reagent were added separately to stop the reac-
tion, followed by heating at 100 ºC for 5 min. After cen-
trifugation (10,000 × g, 10 min), reducing sugar in the Tao et al. AMB Express (2022) 12:100 Tao et al. AMB Express (2022) 12:100 Page 5 of 13 Colloidal chitin concentration time, and 26 °C, 28 °C and 30 °C for the incubation tem-
perature, respectively (Additional file 1: Table S5). Strain TC-1 was inoculated into the PBB liquid medium
in which dextrose was substituted with colloidal chitin at
different concentrations (7, 8, 9, 10, 11, 12, and 13 g l−1)
and allowed to grow in a rotary incubator (180 rpm)
at 28 °C for 60 h. The chitinase activities in the culture
supernatants were determined separately using the
method described above. Three replicates were set up for
each treatment. Incubation temperature Strain TC-1 was inoculated in PBB liquid medium and
allowed to grow in a rotary incubator (180 rpm) at dif-
ferent temperatures (24, 26, 28, 30, and 32 °C) for 60 h. The chitinase activities in the culture supernatants were
determined separately using the method described above. Three replicates were set up for each treatment. Morphological, physiological, and biochemical properties
of strain TC‑1 As shown in Fig. 1b and c, TC-1 was a Gram-negative,
short rod-shaped, non-sporulating bacterium with peri-
trichous flagella and fluorochrome. When grown on
BPYDA media at 28 °C for 48 h, the resultant colonies
were round, rose red, 2–3 mm in diameter, with a moist
glassy surface. Incubation time Strain TC-1 was inoculated in PBB liquid medium and
allowed to grow in a rotary incubator (180 rpm) at 28 °C
for 24, 30, 36, 54, 60, 66, and 72 h, respectively. The chi-
tinase activities in the culture supernatants were deter-
mined separately using the method described above. Three replicates were set up for each treatment. g
y
The results of the physiological and biochemical char-
acterization of TC-1 are summarized in Table 1. Of the
28 physiological and biochemical items tested, positive Table 1 Physiological and biochemical characteristics of strain
TC-1
Note: “ + ” and “−” represent positive and negative reactions, respectively
Trait
Response
Trait
Response
Oxidase
−
Phenylalanine deaminase
−
Gram staining
−
Lysine decarboxylase
+
Motility
+
Ornithine decarboxylase
+
H2S production
−
Arginine decarboxylase
+
Mannitol
+
Bile esculin
+
Urease
−
Methyl red
−
Indole production
−
Sucrose
+
Melibiose
−
Acid from glucose
+
Sorbitol
+
Voges-Proskauer
+
DNase
+
Raffinose
−
Lactose
−
L-Arabinose
−
Adonitol
+
Gelatin liquefaction
+
Xylose
+
Simmons citrate
+
NO3− → NO2−
+
Malonate
− Table 1 Physiological and biochemical characteristics of strain
TC-1
Note: “ + ” and “−” represent positive and negative reactions, respectively
Trait
Response
Trait
Response
Oxidase
−
Phenylalanine deaminase
−
Gram staining
−
Lysine decarboxylase
+
Motility
+
Ornithine decarboxylase
+
H2S production
−
Arginine decarboxylase
+
Mannitol
+
Bile esculin
+
Urease
−
Methyl red
−
Indole production
−
Sucrose
+
Melibiose
−
Acid from glucose
+
Sorbitol
+
Voges-Proskauer
+
DNase
+
Raffinose
−
Lactose
−
L-Arabinose
−
Adonitol
+
Gelatin liquefaction
+
Xylose
+
Simmons citrate
+
NO3− → NO2−
+
Malonate
− Table 1 Physiological and biochemical characteristics of strain
TC-1 Response surface designh The chitinase production by strain TC-1 was optimized
with response surface methodology (RSM) using a three-
factor (colloidal chitin concentration, incubation time
and incubation temperature), three-level Box-Behnken
experimental design. The three factors were selected
based on single-factor experiments. The three levels were
8.2 g l−1, 8.95 g l−1 and 9.7 g l−1 for the colloidal chitin
concentration, and 58 h, 64 h and 70 h for the incubation Note: “ + ” and “−” represent positive and negative reactions, respectively Note: “ + ” and “−” represent positive and negative reactions, respectively Fig. 1 Symptoms of infection by S. marcescens TC-1 and morphological characteristics of strain TC-1. a Symptoms of TC-1 infection in white grub; b
TC-1 bacterial cells (scale bar = 10 μm); c TC-1 rose red colonies grown on a BPYDA plate (7 mm in diameter) after 48 h at 28 ℃ ig. 1 Symptoms of infection by S. marcescens TC-1 and morphological characteristics of strain TC-1. a Symptoms of TC-1 infec
C-1 bacterial cells (scale bar = 10 μm); c TC-1 rose red colonies grown on a BPYDA plate (7 mm in diameter) after 48 h at 28 ℃ Fig. 1 Tao et al. AMB Express (2022) 12:100 Page 6 of 13 Page 6 of 13 the inoculum could be isolated from surface-disinfected
dead grubs, confirming strain TC-1 as a pathogen of
A. corpulenta based on Koch’s postulates. The bacte-
rial concentration significantly influenced the mortal-
ity rate of white grubs in the soil. The highest mortality
rate (91.7%) was observed at the higher concentration of
the inoculum (1 × 109 CFU ml−1) (Table 2). A regression
equation modeling the relationship between grub mor-
tality rate (Y) and TC-1 concentration (X) was obtained
as Υ = 5.5287 + 1.3010Χ, with correlation coefficient (r)
equal to 0.9619. reactions were observed in the 16 items (motility, utili-
zation of mannitol, sorbitol, adonitol, xylose and sucrose,
acid production from glucose, DNase, lysine decarboxy-
lase, ornithine decarboxylase, arginine decarboxylase,
bile esculin, V.-P., gelatin liquefaction, Simmons citrate,
reduction of NO3
− to NO2). The remaining 12 items
(oxidase, urease, phenylalanine deaminase, methyl red,
production of H2S and indole, utilization of melibiose,
lactose, raffinose and L-arabinose and malonate) were
negative. Molecular identification and phylogenetic analysis of strain
TC‑1 More than 95% of B. mori larvae fed with TC-1-sprayed
mulberry leaves showed anorectic behavior, and became
inactive and insensitive to external stimulation within
the first 24 h. During the next 24 h, most larvae stopped
feeding completely. After 48 h, successive deaths were
recorded in treated larvae. B. mori larval mortality rate
reached 88.6% and 100% after 96 and 144 h of exposure
to TC-1-sprayed mulberry leaves, respectively (Table 3). However, no bacterial culture with TC-1’s phenotypic
features could be re-isolated from the surface-disinfected
B. mori cadavers. The amplified sequence of TC-1’s 16S rRNA gene was
1448 base pairs in length and was deposited in GenBank
(accession number KF700093). BLAST analysis showed
a sequence identity greater than 99% to multiple strains
of S. marcescens. In the MEGA 7.0-reconstructed Neigh-
bor-joining phylogenetic tree of bacterial 16S rRNA gene
sequences, strain TC-1 clustered with S. marcescens
(GenBank acc. no. NR041980) at 100% bootstrap level,
clearly separated from other Serratia spp. (Fig. 2). The
molecular data conclusively supported strain TC-1 as a
member of S. marcescens. H. armigera, P. xylostella, S. exigua, and S. litura larval
mortality rates were investigated between 24 and 144 h
of TC-1 exposure. During this time, successive mortality
was recorded in H. armigera, P. xylostella and S. exigua
larvae, with corrected mortality rates of 83.8%, 72.2% and
94.6%, respectively (Table 3). The peak mortality rates
for P. xylostella and H. armigera occurred within 76 h
and 144 h of TC-1 exposure, respectively. P. xylostella
larvae that survived bioassays started premature pupa-
tion after 76 h of TC-1 exposure. No death due to TC-1 Virulence of strain TC‑1 against insects and nematodeh Virulence of strain TC‑1 against insects and nematode
The white grubs (A. corpulenta) began to die 48 h after
exposure to the bacterium TC-1, with mortality peaking
at 84 h. The infected insects turned inactive and stopped
feeding. The white grub cadavers turned reddish-brown
and gradually softened, losing all their elasticity (Fig. 1a). Bacterial cultures with similar phenotypic features to Fig. 2 The MEGA7.0-constructed Neighbor-joining phylogenetic tree based on 16S rRNA sequences showing the genetic relationship between
strain TC-1 and other Serratia spp. retrieved from the GenBank database. The numbers in parentheses represent the accession numbers in
GenBank. The values (from 1000 replicates) are indicated at the branch nodes as the percentages supported by bootstrap. Pseudomonas fluorescens
(JX514415) was used to root the phylogenetic tree. The scale bar represents a genetic distance of 0.02 substitutions per nucleotide position Fig. 2 The MEGA7.0-constructed Neighbor-joining phylogenetic tree based on 16S rRNA sequences showing the genetic relationship between
strain TC-1 and other Serratia spp. retrieved from the GenBank database. The numbers in parentheses represent the accession numbers in
GenBank. The values (from 1000 replicates) are indicated at the branch nodes as the percentages supported by bootstrap. Pseudomonas fluorescens
(JX514415) was used to root the phylogenetic tree. The scale bar represents a genetic distance of 0.02 substitutions per nucleotide position Fig. 2 The MEGA7.0-constructed Neighbor-joining phylogenetic tree based on 16S rRNA sequences showing the genetic relationship between
strain TC-1 and other Serratia spp. retrieved from the GenBank database. The numbers in parentheses represent the accession numbers in
GenBank. The values (from 1000 replicates) are indicated at the branch nodes as the percentages supported by bootstrap. Pseudomonas fluorescens
(JX514415) was used to root the phylogenetic tree. The scale bar represents a genetic distance of 0.02 substitutions per nucleotide position Page 7 of 13 Tao et al. RSM optimization of TC‑1 culture conditions for chitinase
production Based on the results of single-factor experiments, the
three most significant factors for chitinase production by
TC-1, i.e. colloidal chitin concentration, incubation time
and incubation temperature, were selected for further
optimization using a three-factor, three-level RSM design
(Table 5). The response of chitinase production (Y) by the
strain TC-1 could be expressed by the following quad-
ratic regression model: Virulence of strain TC‑1 against insects and nematodeh litura
Test
0
0
0
0
0
0
CK
0
0
0
0
0
0
0 Data with different lowercases in each column indicate significant difference at P < 0.05 level Table 3 Virulence of strain TC-1 to insect larvae and Caenorhabditis elegans
Note: “ + ” and “−” represent “pupation” and “termination of observation”, respectively
Target organism
Treatment
Mortality (%)
96 h
Corrected
mortality (%)
24 h
48 h
72 h
96 h
120 h
144 h
C. elegans
Test
20.0
72.2
93.3
100
−
−
100
CK
1
2.0
4.0
4
4
4
B. mori
Test
0
18.3
41.2
86.6
98.3
100
86.4
CK
0
1.6
1.6
1.6
1.6
5.0
P. xylostella
Test
20.0
48.9
71.2 ( +)
75.3 ( +)
+
+
72.2
CK
1.7
6.3
6.3
11.3
+
+
S. exigua
Test
15.0
92.5
95.0
95.0
95.0
95.0
94.6
CK
5.0
7.5
7.5
7.5
7.5
7.5
H. armigera
Test
3.8
26.3
60.0
85.0
90.0
92.5
83.8
CK
0
0
2.5
7.5
11.3
13.5
S. litura
Test
0
0
0
0
0
0
CK
0
0
0
0
0
0
0 Table 3 Virulence of strain TC-1 to insect larvae and Caenorhabditis elegans highest chitinase activity (20.79 U ml−1) was recorded
with 9.5 g l−1 of colloidal chitin compared to the other
concentrations tested (7, 8, 8.5, 9, 10, and 10.5 gl−1). was recorded in S. litura larvae within 144 h of TC-1
exposure. C. elegans was highly susceptible to the strain TC-1 and
its larval mortality rate reached 100% after 96 h of expo-
sure to the bacterium (Table 3). Some worm cadavers
turned reddish-brown, while others displayed no signifi-
cant change in body color. Virulence of strain TC‑1 against insects and nematodeh AMB Express (2022) 12:100 Table 2 Virulence of strain TC-1 to white grubs (Anomala corpulenta) Table 2 Virulence of strain TC-1 to white grubs (Anomala corpulenta)
Data with different lowercases in each column indicate significant difference at P < 0.05 level
Concentration of
strain TC-1 (108 CFU
ml−1)
Number of white
grub tested
Number of dead white grub
Mortality (%)
Corrected
mortality (%)
LogC(Y)
Fatal
probability(X)
Test1
Test2
Test3
10 ×
20
18
19
18
91.7 a
91.1 a
9
6.3469
5 ×
20
16
16
15
78.3 b
76.7 b
8.699
5.7290
2.5 ×
20
12
11
14
61.7 c
58.9 c
8.3979
5.2250
1.25 ×
20
10
9
10
48.3 d
44.6 d
8.0969
4.8642
0.625 ×
20
8
10
10
46.7 d
42.9 d
7.7959
4.8211
CK
20
1
1
2
6.7 e g
(
p
)
Data with different lowercases in each column indicate significant difference at P < 0.05 level
Concentration of
strain TC-1 (108 CFU
ml−1)
Number of white
grub tested
Number of dead white grub
Mortality (%)
Corrected
mortality (%)
LogC(Y)
Fatal
probability(X)
Test1
Test2
Test3
10 ×
20
18
19
18
91.7 a
91.1 a
9
6.3469
5 ×
20
16
16
15
78.3 b
76.7 b
8.699
5.7290
2.5 ×
20
12
11
14
61.7 c
58.9 c
8.3979
5.2250
1.25 ×
20
10
9
10
48.3 d
44.6 d
8.0969
4.8642
0.625 ×
20
8
10
10
46.7 d
42.9 d
7.7959
4.8211
CK
20
1
1
2
6.7 e
Table 3 Virulence of strain TC-1 to insect larvae and Caenorhabditis elegans
Note: “ + ” and “−” represent “pupation” and “termination of observation”, respectively
Target organism
Treatment
Mortality (%)
96 h
Corrected
mortality (%)
24 h
48 h
72 h
96 h
120 h
144 h
C. elegans
Test
20.0
72.2
93.3
100
−
−
100
CK
1
2.0
4.0
4
4
4
B. mori
Test
0
18.3
41.2
86.6
98.3
100
86.4
CK
0
1.6
1.6
1.6
1.6
5.0
P. xylostella
Test
20.0
48.9
71.2 ( +)
75.3 ( +)
+
+
72.2
CK
1.7
6.3
6.3
11.3
+
+
S. exigua
Test
15.0
92.5
95.0
95.0
95.0
95.0
94.6
CK
5.0
7.5
7.5
7.5
7.5
7.5
H. armigera
Test
3.8
26.3
60.0
85.0
90.0
92.5
83.8
CK
0
0
2.5
7.5
11.3
13.5
S. Effects of culture conditions on chitinase production
by strain TC‑1 A total of four variables (carbon source, nitrogen source,
incubation time, and incubation temperature) were ana-
lyzed regarding their effects on chitinase production by
the strain TC-1 (Table 4). The results revealed peak chi-
tinase activities of 18.89 U ml−1 when colloidal chitin
was used as the sole carbon source, 17.8 U ml−1 when
peptone was used as the sole nitrogen source, 17.44 U
ml−1 at 60 h of incubation time, and 17.33 U ml−1 at the
incubation temperature of 28 °C, respectively. Regard-
ing the colloidal chitin concentration in the medium, the g
Y = 20.90 + 0.73A-0.12B + 0.25C − 0.26AB + 0.14AC −
0.13BC − 2.55A2 − 2.82B2 − 2.36C2, where A is the colloi-
dal chitin concentration, B is the incubation time; and C
is the incubation temperature. The analysis of variance (ANOVA) of the quadratic
regression model was performed (Table 6). The Pvalues Tao et al. AMB Express (2022) 12:100 Tao et al. AMB Express (2022) 12:100 Page 8 of 13 Page 8 of 13 for the model (< 0.0001) and "lack-of-fit" (0.0579), sug-
gested that the established regression model was appro-
priate without significant deviation. The greater the F
value was from unity and the lower the P-value was, the
greater the effect of the tested factor on chitinase pro-
duction by TC-1. The P-value of A (< 0.0001) indicated
the effect of colloidal chitin concentration on chitinase
production by the bacterium was highly significant. The
P-value of C (0.016) indicated that the influence of tem-
perature on chitinase production by the bacterium was
significant. Consequently, the order of influence of the
three factors on chitinase production, from the largest
to the smallest, was colloidal chitin concentration (A),
incubation temperature (C) and incubation time (B) in
sequence. The P-values of the three factors’ combina-
tions AB AC and BC
ere 0 0568 0 2512 and 0 2669
Table 4 Effects of culture conditions on chitinase production by
strain TC-1
The analysis of variance for each item was independently tested. Effects of culture conditions on chitinase production
by strain TC‑1 Data with
different capitals and lowercases in the same item represent significant
differences at P < 0.01 and P < 0.05 levels, respectively
Items tested
Chitinase activity (U ml−1)
Carbon source
Coloidal chitin
18.89 ± 0.04 Aa
Chitin
12.69 ± 0.04 Bb
Starch
3.09 ± 0.06 Cd
Celullose
3.28 ± 0.03 Cc
Nitrogen source
Peptone
17.8 ± 0.17 Aa
Ammonium sulphate
9.49 ± 0.14 Cc
Yeast extract
15.37 ± 0.25 Bb
Beef extract
15.16 ± 0.19 Bb
Incubation time (h)
24
7.75 ± 0.24 Gh
30
8.2 ± 0.11 Fg
36
8.45 ± 0.11 Fg
42
9.98 ± 0.23 Ef
48
10.44 ± 0.05 De
54
12.81 ± 0.13 Cc
60
17.44 ± 0.07 Aa
66
14.69 ± 0.28 Bb
72
12.47 ± 0.19 Cd
Incubation temperature (°C)
24
10.55 ± 0.06 Cc
26
14.91 ± 0.09 Bb
28
17.33 ± 0.17 Aa
30
14.92 ± 0.15 Bb
32
9.69 ± 0.08 Cd
Colloidal concentration (g l-1)
7.5
13.08 ± 0.25 Ee
8.0
14.39 ± 0.16 Dd
8.5
18.51 ± 0.09 Bb
9.0
18.68 ± 0.08 Bb
9.5
20.79 ± 0.19 Aa
10.0
16.43 ± 0.10 Cc
10.5
10.91 ± 0.10 Ff
Table 5 The chitinase activities in the culture supernatants
of strain TC-1 under different combinations of three factors at
different levels
The three factors “A”, “B” and “C” represent chitin concentration, incubation time
and incubation temperature, respectively. Effects of culture conditions on chitinase production
by strain TC‑1 “Y” represents chitinase activity
Serial number
A (g l−1)
B (h)
C (°C)
Y (U ml−1)
1
9.70
64.00
26.00
16.43
2
8.95
64.00
28.00
20.72
3
8.95
58.00
30.00
16.13
4
9.70
70.00
28.00
15.70
5
8.95
64.00
28.00
20.88
6
8.95
70.00
30.00
15.97
7
8.20
58.00
28.00
14.87
8
8.95
58.00
26.00
15.21
9
9.70
64.00
30.00
17.06
10
8.20
64.00
30.00
15.28
11
9.70
58.00
28.00
16.79
12
8.95
64.00
28.00
21.06
13
8.95
70.00
26.00
15.59
14
8.95
64.00
28.00
20.97
15
8.95
64.00
28.00
20.88
16
8.20
70.00
28.00
14.80
17
8.20
64.00
26.00
15.21 The three factors “A”, “B” and “C” represent chitin concentration, incubation time
and incubation temperature, respectively. “Y” represents chitinase activity Table 6 Analysis of variance for the regression model of
chitinase activity
Source of variation
Degree of
freedom
F-value
P-value
Significance
of difference
Model
9
220.03
< 0.0001 **
A
1
84.49
< 0.0001 **
B
1
2.20
0.1812
C
1
9.98
0.0160 *
AB
1
5.19
0.0568
AC
1
1.56
0.2512
BC
1
1.45
0.2669
A2
1
544.65
< 0.0001 **
B2
1
666.29
< 0.0001 **
C2
1
468.37
< 0.0001 **
Residual
7
Lack-of-fit
3
6.01
0.0579
Pure error
4
Cor total
16 Table 6 Analysis of variance for the regression model of
chitinase activity The analysis of variance for each item was independently tested. Data with
different capitals and lowercases in the same item represent significant
differences at P < 0.01 and P < 0.05 levels, respectively The analysis of variance for each item was independently tested. Data with
different capitals and lowercases in the same item represent significant
differences at P < 0.01 and P < 0.05 levels, respectively for the model (< 0.0001) and "lack-of-fit" (0.0579), sug-
gested that the established regression model was appro-
priate without significant deviation. The greater the F
value was from unity and the lower the P-value was, the
greater the effect of the tested factor on chitinase pro-
duction by TC-1. The P-value of A (< 0.0001) indicated
the effect of colloidal chitin concentration on chitinase
production by the bacterium was highly significant. The
P-value of C (0.016) indicated that the influence of tem-
perature on chitinase production by the bacterium was significant. Effects of culture conditions on chitinase production
by strain TC‑1 Data with
different capitals and lowercases in the same item represent significant
differences at P < 0.01 and P < 0.05 levels, respectively
Items tested
Chitinase activity (U ml−1)
Carbon source
Coloidal chitin
18.89 ± 0.04 Aa
Chitin
12.69 ± 0.04 Bb
Starch
3.09 ± 0.06 Cd
Celullose
3.28 ± 0.03 Cc
Nitrogen source
Peptone
17.8 ± 0.17 Aa
Ammonium sulphate
9.49 ± 0.14 Cc
Yeast extract
15.37 ± 0.25 Bb
Beef extract
15.16 ± 0.19 Bb
Incubation time (h)
24
7.75 ± 0.24 Gh
30
8.2 ± 0.11 Fg
36
8.45 ± 0.11 Fg
42
9.98 ± 0.23 Ef
48
10.44 ± 0.05 De
54
12.81 ± 0.13 Cc
60
17.44 ± 0.07 Aa
66
14.69 ± 0.28 Bb
72
12.47 ± 0.19 Cd
Incubation temperature (°C)
24
10.55 ± 0.06 Cc
26
14.91 ± 0.09 Bb
28
17.33 ± 0.17 Aa
30
14.92 ± 0.15 Bb
32
9.69 ± 0.08 Cd
Colloidal concentration (g l-1)
7.5
13.08 ± 0.25 Ee
8.0
14.39 ± 0.16 Dd
8.5
18.51 ± 0.09 Bb
9.0
18.68 ± 0.08 Bb
9.5
20.79 ± 0.19 Aa
10.0
16.43 ± 0.10 Cc
10.5
10.91 ± 0.10 Ff
Table 5 The chitinase activities in
of strain TC-1 under different comb
different levels
The three factors “A”, “B” and “C” represent chiti
and incubation temperature, respectively. “Y” r
Serial number
A (g l−1)
B (h)
1
9.70
64.00
2
8.95
64.00
3
8.95
58.00
4
9.70
70.00
5
8.95
64.00
6
8.95
70.00
7
8.20
58.00
8
8.95
58.00
9
9.70
64.00
10
8.20
64.00
11
9.70
58.00
12
8.95
64.00
13
8.95
70.00
14
8.95
64.00
15
8.95
64.00
16
8.20
70.00
17
8.20
64.00
Table 6 Analysis of variance for
chitinase activity
Source of variation
Degree of
freedom
F-val
Model
9
220.0
A
1
84.4
B
1
2.2
C
1
9.9
AB
1
5.1
AC
1
1.5
BC
1
1.4
A2
1
544.6
B2
1
666.2
C2
1
468.3
Residual
7
Lack-of-fit
3
6.0
Pure error
4 Table 5 The chitinase activities in the culture supernatants
of strain TC-1 under different combinations of three factors at
different levels of strain TC-1 under different combinations of three factors at
different levels
The three factors “A”, “B” and “C” represent chitin concentration, incubation time
and incubation temperature, respectively. Effects of culture conditions on chitinase production
by strain TC‑1 “Y” represents chitinase activity
Serial number
A (g l−1)
B (h)
C (°C)
Y (U ml−1)
1
9.70
64.00
26.00
16.43
2
8.95
64.00
28.00
20.72
3
8.95
58.00
30.00
16.13
4
9.70
70.00
28.00
15.70
5
8.95
64.00
28.00
20.88
6
8.95
70.00
30.00
15.97
7
8.20
58.00
28.00
14.87
8
8.95
58.00
26.00
15.21
9
9.70
64.00
30.00
17.06
10
8.20
64.00
30.00
15.28
11
9.70
58.00
28.00
16.79
12
8.95
64.00
28.00
21.06
13
8.95
70.00
26.00
15.59
14
8.95
64.00
28.00
20.97
15
8.95
64.00
28.00
20.88
16
8.20
70.00
28.00
14.80
17
8.20
64.00
26.00
15.21
Table 6 Analysis of variance for the regression model of
chitinase activity
Source of variation
Degree of
freedom
F-value
P-value
Significance
of difference
Model
9
220.03
< 0.0001 **
A
1
84.49
< 0.0001 **
B
1
2.20
0.1812
C
1
9.98
0.0160 *
AB
1
5.19
0.0568
AC
1
1.56
0.2512
BC
1
1.45
0.2669
A2
1
544.65
< 0.0001 **
B2
1
666.29
< 0.0001 **
C2
1
468.37
< 0.0001 **
Residual
7
Lack-of-fit
3
6.01
0.0579
Pure error
4
Cor total
16 for the model (<0 0001) and "lack of fit" (0 0579) sug
Table 4 Effects of culture conditions on chitinase production by
strain TC-1
The analysis of variance for each item was independently tested. Effects of culture conditions on chitinase production
by strain TC‑1 Consequently, the order of influence of the
three factors on chitinase production, from the largest
to the smallest, was colloidal chitin concentration (A),
incubation temperature (C) and incubation time (B) in
sequence. The P-values of the three factors’ combina-
tions AB, AC and BC were 0.0568, 0.2512 and 0.2669, Tao et al. AMB Express (2022) 12:100 Page 9 of 13 striolata (Yang et al. 2014a, b), S. exigua (Qi et al. 2004;
Yang et al. 2015a, b; Zhao et al. 2017), S. litura (Niu et al. 2015), Heortia vitessoides (Tan and Zhang 2005), Rhyn-
chophorus ferrugineus (Zhang et al. 2011) and Orthaga
achatina (Guan et al. 2018). In the present study, strain
TC-1 was isolated from a naturally infected larva (white
grub) of the underground pest species (A. corpulenta). striolata (Yang et al. 2014a, b), S. exigua (Qi et al. 2004;
Yang et al. 2015a, b; Zhao et al. 2017), S. litura (Niu et al. 2015), Heortia vitessoides (Tan and Zhang 2005), Rhyn-
chophorus ferrugineus (Zhang et al. 2011) and Orthaga
achatina (Guan et al. 2018). In the present study, strain
TC-1 was isolated from a naturally infected larva (white
grub) of the underground pest species (A. corpulenta). respectively, indicating that the difference of recipro-
cal effects of the three factors A, B and C on chitinase
production by TC-1 was not significant. The P-values of
A2, B2 and C2 were all less than 0.0001, indicating that
the quadratic terms of the three factors A, B and C had
a significant influence on chitinase production by the
bacterium. The credibility analysis of the regression model was
performed (Additional file 1: Table S6). The standard
deviation and the coefficient of variation of the regres-
sion model were 0.22 and 1.3, with PRESS (prediction
sum of squares) and “signal-to-noise” ratio being 4.69
and 36.269, respectively. The multiple correlation coef-
ficient, the predicted correlation coefficient and the cor-
rected correlation coefficient were 0.9965, 0.9529 and
0.9919, respectively, indicating high fit of the constructed
regression model. Based on the regression analysis and
fitness of the constructed regression model, three RSM
diagrams were obtained (Fig. 3). The chitinase activity
peaked at the appropriate combination of colloidal chitin
concentration and incubation time (Fig. 3a), colloidal chi-
tin concentration and incubation temperature (Fig. 3b),
or incubation time and incubation temperature (Fig. 3c). Effects of culture conditions on chitinase production
by strain TC‑1 Based on the analyses using software Design-Expert 8.0
(Stat-Ease Inc., Minneapolis, MN, USA), the predicted
optimal chitinase activity was 20.946 U ml−1 at a combi-
nation of colloidal chitin concentration, incubation time,
and incubation temperature of 9.06 g l−1, 63.83 h, and
28.12 °C, respectively. The mean chitinase activity meas-
ured under laboratory conditions was 20.761 ± 0.102 U
ml−1 at a combination of colloidal chitin concentration,
incubation time, and incubation temperature of 9 g l−1,
64 h and 28 °C, respectively, for three replicates. No
significant differences were detected between the pre-
dicted optimal chitinase activity and the actual activity
measured in the laboratory, indicating that the proposed
regression model is reliable. Both the physiochemical and molecular data conclu-
sively supported strain TC-1 as a member of S. marc-
escens. The bacterial isolates with the ability to ferment
glucose, liquefy gelatin rapidly, oxidase and phenylala-
nine-deaminase negative and cannot acidify arabinose
and raffinose in peptone water under aerobic condi-
tions are generally accepted as S. marcescens (Anderhub
et al. 1977). The physiochemical reactions of strain TC-1
agreed well with these S. marcescens-specific features. In
the Neighbor-joining phylogenetic tree of bacterial 16S
rRNA gene sequences, the strain TC-1 formed a clade
with S. marcescens, clearly separated from other Serratia
spp. Previously, Niu et al. (2015) reported an S. marcescens
strain S-JS1 virulent against two lepidopteran species
S. exigua and S. litura. In our study, pathogenicity tests
revealed the broad insecticidal spectrum of S. marces-
cens TC-1, including an underground Coleoptera pest A. corpulenta and four species of Lepidoptera (B. mori, P. xylostella, S. exigua, and H. armigera). Interestingly, TC-1
showed no toxicity to another Spodoptera species (S. litura), suggesting the existence of host species-specific
interactions between the virulence factors in TC-1 and
the insects’ innate immune system. Further comparison
between the compatible S. exigua-TC-1 and incompatible
S. litura-TC-1 interactions at the molecular level could
provide valuable insights into the mechanism(s) of viru-
lence of the strain TC-1. Little is known about S. marc-
escens strains with the potential to control underground
insect pests. This study is the first on a S. marcescens
strain with the potential to control the larvae of A. cor-
pulenta, which damage the root of many crops. The lar-
vicidal activity of S. marcescens TC-1 against C. elegans
also shows the potential of this bacterium as a biocontrol
agent against plant diseases caused by nematodes. Availability of data and materials The raw sequencing data of the 16S rRNA gene of strain TC-1 have been
submitted to the NCBI, and the sequence read archive number (accession
number) was KF700093. The other datasets generated during and/or analyzed
during the study are available from the corresponding author on reasonable
request. Huang et al. (2010) reported RSM-optimized culture
conditions for chitinase production by S. marcescens
strain S418 based on a combination of 0.2% colloidal chi-
tin, 1% peptone and 0.05% KH2PO4. In our study, a con-
centration of 9 g l−1 of colloidal chitin was optimal for
strain TC-1. This concentration is significantly higher
than the reported by Huang et al. (2010) for strain S418
and could be associated with strain-specific differences
in the efficiency of utilizing colloidal chitin as a carbon
source. Funding g
The study has been financed in parts by grants from the Special Project
(No.16105) of Henan Province Scientific Research Service Platform and the
Henan Fungus Food Engineering and Technology Research Center. CA-P
was supported by the Henan Science and Technology Department, grant
HNGD2021049. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript. Discussion litura may be due to the
fact that the chitinase isoforms in this strain differ from
those of the S. litura-toxic S. marcescens S-JS1 isolate
reported by Niu et al. (2015). Author contributions ALT and SLH conceived and designed the study. ALT, TW, FHP, XLZ, RXX,
FQL, JKL, YBW and ZQW conducted experiments and contributed to data
analysis. ALT, SLH and CA-P wrote and revised the manuscript. QHN and DDL
provided the nematode Caenorhabditis elegans and the insect Bombyx mori,
respectively. In our study, an intriguing issue was the nature of the
bacterial factor(s) that caused a high prevalence (95%) of
TC-1-induced anorexia in B. mori larvae during the first
stage of the insect death. However, the impossibility of
re-isolating the strain TC-1 from exposed insects’ cadav-
ers suggests no multiplication of TC-1 within the insect
body. We speculated that strain TC-1 could directly
produce volatiles that induced anorexia in B. mori lar-
vae and/or produce substance(s) that trigger the release
of volatiles from the mulberry leaves that induced ano-
rexia in the insect larvae. The absence of TC-1 in B. mori
cadavers could be reasonably explained as a consequence
of the insect’s anorexia. Acknowledgements The authors thank the editor and anonymous reviewers for useful comments. Consent for publication Consent for publication
Not applicable. Competing interests
The authors declare that they have no competing interest. Competing interests
The authors declare that they have no competing interest. Supplementary Information The online version contains supplementary material available at https://doi.
org/10.1186/s13568-022-01442-6. Additional file 1: Table S1. Effects of two levels (A and B) of chitinase-
containing culture supernatants (CCS) of strain TC -1 on death rates of
Spodoptera exigua larvae. Table S2. A broad-spectrum screening assay
of primary chitinase with the highest chitinolytic activity in the culture
supernatant of the strain TC-1 by isoelectric precipitation. Table S3. A
narrow-spectrum screening assay of primary chitinase with the highest
chitinolytic activity in the culture supernatant of the strain TC-1 by iso‑
electric precipitation. Table S4. Toxicity of the pH 6.7-precipitated crude
chitinase from the culture supernatant of strain TC-1 against the second-
and third-instar larvae of Spodoptera exigua1. Table S5. Factors and levels
of response surface design for optimization of chitinase production condi‑
tions of strain TC-1. Table S6. Credibility analysis of the regression model. Figure S1. The standard curve of N-acetylglucosamine. Discussion Biological control of insect pests represents an important
eco-friendly agronomic measure for sustainable agricul-
ture and forestry. Successful biocontrol of an insect pest
depends largely on the acquisition of microbial strains
with high insecticidal activity. Many S. marcescens strains
can colonize the alimentary canal of insects and induce
their death by septicemia. Infections of insects by S. marcescens have been reported in numerous pest species
that attack aboveground parts of crops, such as H. armig-
era (Chen et al. 2005; Shi et al. 2003), Oedaleus inferna-
lis (Feng et al. 2002; Jin et al. 2005), Diatraea saccharalis
(King et al. 1975), Heliothis virescens (Sikorowski et al. 2001), Lymantria dispar (Podgwaite and Cosenza 1976),
Anoplophora glabripennis (Deng et al. 2008), Phyllotreta Chitinase is an important enzyme for the control of
insect pests as well as fungal plant pathogens (Oppen-
heim and Chet 1992). Previous studies have shown that
S. marcescens produced multiple chitinase isozymes. Horn et al. (2006) reported that S. marcescens could pro-
duce several chitinolytic enzymes, including chitinases
A, B and C, which enable the bacterium to degrade the
insoluble chitin polymer efficiently. Someya et al. (2001)
discovered four chitinolytic enzymes among the extracel-
lular proteins produced by S. marcescens strain B2, and
found both chitinolytic enzymes and prodigiosin could Tao et al. AMB Express (2022) 12:100 Page 10 of 13 nse surface and counter plots of the interactive effects on chitinase production by the strain TC-1 between colloidal chitin
and incubation time (a), colloidal chitin concentration and incubation temperature (b), and incubation time and incubation
c) Fig. 3 Response surface and counter plots of the interactive effects on chitinase production by the strain TC-1 between colloidal chitin
concentration and incubation time (a), colloidal chitin concentration and incubation temperature (b), and incubation time and incubation
temperature (c) Fig. 3 Response surface and counter plots of the interactive effects on chitinase production by the strain TC-1 between colloidal chitin
concentration and incubation time (a), colloidal chitin concentration and incubation temperature (b), and incubation time and incubation
temperature (c) Tao et al. AMB Express (2022) 12:100 Page 11 of 13 that our RSM-developed quadratic regression model has
the potential to guide chitinase production by strain TC-1
under laboratory conditions. Further studies and revisions
of the regression model may be required for large-scale chi-
tinase production by TC-1. act synergistically against the grey mold pathogen, Bot-
rytis cinerea. Watanabe et al. Discussion (1997) reported four chi-
tinases, A, B, C1, and C2 in the culture supernatant of S. marcescens 2170. Fuchs et al. (1986) found that S. marc-
escens produced five unique chitinolytic proteins with
subunit molecular masses of 21, 36, 48, 52, and 57 kDa. Jones et al. (1986) characterized the genes encoding two
chitinase enzymes of S. marcescens. Having established
that the isoelectric point of the primary chitinase pro-
duced by the strain TC-1 was pH 6.7 (Additional file 1:
Tables S2 and S3), we applied selective isoelectric point
precipitation to CS of TC-1. In initial tests, we then con-
firmed that this chitinase-enriched CS showed larvicidal
activity against S. exigua (Additional file 1: Table S4). Further purification and enzymatic characterization of
the chitinase(s) produced by the strain TC-1 is required
for a full understanding of its virulence against the insect
species tested and C. elegans. Although the number of
chitinases produced by strain TC-1 remains to be speci-
fied, we speculate that the lack of insecticidal activity of
S. marcescens TC-1 against S. litura may be due to the
fact that the chitinase isoforms in this strain differ from
those of the S. litura-toxic S. marcescens S-JS1 isolate
reported by Niu et al. (2015). act synergistically against the grey mold pathogen, Bot-
rytis cinerea. Watanabe et al. (1997) reported four chi-
tinases, A, B, C1, and C2 in the culture supernatant of S. marcescens 2170. Fuchs et al. (1986) found that S. marc-
escens produced five unique chitinolytic proteins with
subunit molecular masses of 21, 36, 48, 52, and 57 kDa. Jones et al. (1986) characterized the genes encoding two
chitinase enzymes of S. marcescens. Having established
that the isoelectric point of the primary chitinase pro-
duced by the strain TC-1 was pH 6.7 (Additional file 1:
Tables S2 and S3), we applied selective isoelectric point
precipitation to CS of TC-1. In initial tests, we then con-
firmed that this chitinase-enriched CS showed larvicidal
activity against S. exigua (Additional file 1: Table S4). Further purification and enzymatic characterization of
the chitinase(s) produced by the strain TC-1 is required
for a full understanding of its virulence against the insect
species tested and C. elegans. Although the number of
chitinases produced by strain TC-1 remains to be speci-
fied, we speculate that the lack of insecticidal activity of
S. marcescens TC-1 against S. Declarations Ethics approval and consent to participate
Not applicable. Consent for publication
Not applicable. Competing interests
The authors declare that they have no competing interest. Received: 6 January 2022 Accepted: 22 July 2022 Ethics approval and consent to participate
Not applicable. Consent for publication
Not applicable. Competing interests
The authors declare that they have no competing interest. Received: 6 January 2022 Accepted: 22 July 2022 Ethics approval and consent to participate Ethics approval and consent to participate
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multiple correlation coefficient and a corrected correla-
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ference between the predicted chitinase activity (20.946
U ml−1) and that actually measured in the laboratory
(20.761 ± 0.102 U ml−1) was only 0.201 U ml−1, revealing Received: 6 January 2022 Accepted: 22 July 2022 Received: 6 January 2022 Accepted: 22 July 2022 Tao et al. AMB Express (2022) 12:100 Tao et al. AMB Express (2022) 12:100 Page 12 of 13 Page 12 of 13 References Acta Agric Zhejiangensis 28:252–258 Lukezic FL, Hildebrand DC, Schroth MN, Shinde PA (1982) Association of Ser-
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B.
C.
D.
Time (Min):
0
15
30
45
60
75
90
105
0
15
30
45
60
75
135
150
0
75
90
105
120
150
225
270
0
15
30
150
210
305
410
1,010
Exit &
Divide
Exit &
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Exit &
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siNS
siLIN9
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0.5
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Relative LIN37
*
*
NS LIN37 NS LIN37
Sen.
Res.
siRNA:
1
10
100
1000
0.00
0.25
0.50
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[Docetaxel, nM]
Relative Cell Number
Sen. siNS
Sen. siLIN9
Res. siNS
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*
* * **
*
#
#
#
#
1
10
100
1000
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[Paclitaxel, nM]
Relative Cell Number
Sen. siNS
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Res. siNS
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ABCB1 ABCC2 ABCC3 ABCG2
0.0
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n.s.
n.s.
n.s.
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n.s.
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n.s.
n.s.
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231
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1.0
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n.s.
n.s.
Sen Res Sen Res
231
468
H. B. C. D. Time (Min):
0
15
30
45
60
75
90
105
0
15
30
45
60
75
135
150
0
75
90
105
120
150
225
270
0
15
30
150
210
305
410
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Exit &
Divide
Exit &
Divide
Exit &
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siNS
siLIN9
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H. E. F. Supplemental Figure 1
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Minimally invasive surgery of no tumor containment for cervical cancer surgery
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Min Hu Min Hu Jinhua Maternity and Child Health Care Hospital, Jinhua Women's and Children's Hospital
Limei Ji
Jinhua Hospital of Zhejiang University
Lanying Jin
Jinhua Hospital of Zhejiang University
Mingjun Shao
Jinhua Hospital of Zhejiang University
Xiujuan Fu
Pan'an County People's Hospital
Baomei Shi
XiYu Central Hospital
Article
Keywords: Cervical cancer, Disease-free survival rate, Minimally invasive surgery, Overall survival rate
Posted Date: April 5th, 2024
DOI: https://doi.org/10.21203/rs.3.rs-4165320/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Additional Declarations: No competing interests reported. DOI: https://doi.org/10.21203/rs.3.rs-4165320/v1 Additional Declarations: No competing interests reported. Page 1/11 Abstract The aim of this study is to evaluate the oncologic outcomes of minimally invasive radical hysterectomy
with no tumor containment. Between January 2017 and June 2020, we recruited 350 women with early
stage of cervical cancer, all eligible patients were informed of the potential complications and benefits of
abdominal radical hysterectomy and laparoscopic radical hysterectomy. All patients in our study were
refrain from using uterine manipulator, myoma drill or uterus suture combined with protective vaginal
closure of the colpotomy such as clamp, vaginal cuff or vaginal suture instead, put the specimen in a
collection bag, and extraction of the collection bag via the vagina. This research was approved by the
Ethics Committee of Jinhua Municipal Central Hospital (2020 − 268). Over a median follow-up of 51
(range 30–72) months period, there were 5 patients lost to follow-up, and 3 patients refused to treatment
after surgery. A total of 342 women with cervical cancer were ultimately analyzed in our study. Initial
International Federation of Gynecology and Obstetrics (FIGO) 2018 stage was IA1 lymphovascular space
invasion (LVSI+) 22 (6.29%), IA2 36 (10.29%), IB1 137 (39.14%), IB2 126 (36%), IIA1 14 (4%) and IIIC1p 15
(4.29%) respectively, and histology was squamous cell in 269 (76.86%), adenocarcinoma in 75 (21.43%),
and adenosquamous in 6 (1.71%) patients. Lymphovascular invasion was confirmed in 80 (22.86%)
patients. Lymph nodes were tumor-free in 325 (92.86%) patients. After radical hysterectomy, 83 (23.71%)
patients underwent adjuvant chemoradiation or/and radiation. After a median follow-up of 51 (range 30–
72) months, we noted the disease-free survival and overall survival rate were 95.71% (335/350) and
98.86% (346/350) respectively. The technique of minimally invasive surgery using maneuvers to avoid
peritoneal contamination during surgery provides excellent oncologic outcome for patients with early
cervical cancer. Our retrospective data suggest that laparoscopical surgery may be oncologically safe
and may be validated in further randomized trials. Patient selection This multiplecener study involved the multiplecenter retrospective analysis of cervical cancer who were
treated at the hospital located in Jin hua area from January 2017 and June 2020. We included the
patients who with the stage of Ia1 LVSI (+) to IIa1 cervical cancer (FIGO 2018), who underwent
laparoscopic radical hysterectomy (Type B-C). We excluded the case with tumor size larger than 4 cm; a
past history of cervical conization within 2 years; previous chemotherapy or radiation; conversion from
MIS to laparotomy. A total of 350 patients were identified and all underwent laparoscopical radical
hysterectomy with complete pelvic lymphadenectomy. All eligible patients were informed of the potential
complications, benefits and risks of abdominal radical hysterectomy and laparoscopic radical
hysterectomy before they were assigned into our study. This research was approved by the Ethics
Committee of Jinhua Municipal Central Hospital (2020 − 268) and all patients provided written informed
consent before recruitment. The operating time was determined from the first skin incision to closure. The diameter of tumor was
determined by pelvic MRI. Operative blood loss was estimated by gathering the blood volume in suction
bottles during the surgery. Introduction Combined with our cervical cancer patients who underwent laparoscopical
radical surgery with decreased recurrence rate since 2017, we give a detailed description of oncologic
outcomes of the patients with early cervical cancer undergoing laparoscopic surgery with protective
methods to avoid tumor spread, and give a median follow-up of 51 (range 30–72) months after surgery. following period, the number of patients with central recurrence has decreased significantly. In 2018, the
Laparoscopic Approach to Cervical Cancer (LACC) trial examined oncological safety of minimally
invasive surgery (MIS) for early stage cervical cancer, and abdominal approach conferred a survival
advantage over MIS.[4] Following these unexpected findings of LACC trial, the majority of European and
American gynecologic oncology centers changed their clinical practice in the management of women
with early-stage cervical cancer, reversing from the minimally invasive technique to the previous practice
of abdominal approach.[5–7] In conventional MIS, the vagina is opened laparoscopically above the
manipulator and tumor cells may be exposure to the peritoneal cavity by insufflating carbon dioxide, and
tumor spill into the peritoneal cavity may an etiology for the inferior outcomes of MIS. However, based on
the advantages of laparoscopic surgery, many patients may still choose the MIS when choosing the
mode of the operation. Combined with our cervical cancer patients who underwent laparoscopical
radical surgery with decreased recurrence rate since 2017, we give a detailed description of oncologic
outcomes of the patients with early cervical cancer undergoing laparoscopic surgery with protective
methods to avoid tumor spread, and give a median follow-up of 51 (range 30–72) months after surgery. Introduction Cervical cancer is the one of the most frequent cause of death from all female cancers worldwide and
the majority of deaths are from low-resource countries or from people who live in socioeconomically
weaker areas, and the disease threatens women’s health seriously [2]. Surgery by radical hysterectomy
and systematic pelvic lymphadenectomy represents the standard of treatment for the early stage for
cervical cancer. Over the last decades, minimal invasive approach has been widely practiced in early-
stage gynecologic cancers broadly adopted throughout the world. Several studies have previously
compared the safety, morbidity and survival outcomes of abdominal radical hysterectomy (ARH) and
laparoscopic radical hysterectomy (LRH), and showed short-term less morbidity and similar survival
outcomes compared with open abdominal surgery [3]. Philp et al comparing abdominal and minimally
invasive approaches demonstrated the result that the advantages of minimally invasive radical
hysterectomy over abdominal radical hysterectomy in both recovery, complications and survival. [22] We
used minimally invasive methods with uterine manipulator to perform radical surgery for cervical cancer
in our department before 2016. However, during the following period, we encountered some cases with
short-term central-recurrence, and we thought deeply about it and modified the surgical approach. Since
2017, we have abandoned the uterine manipulator, myoma drill or vaginal cuff instead. In the subsequent Page 2/11 Page 2/11 following period, the number of patients with central recurrence has decreased significantly. In 2018, the
Laparoscopic Approach to Cervical Cancer (LACC) trial examined oncological safety of minimally
invasive surgery (MIS) for early stage cervical cancer, and abdominal approach conferred a survival
advantage over MIS.[4] Following these unexpected findings of LACC trial, the majority of European and
American gynecologic oncology centers changed their clinical practice in the management of women
with early-stage cervical cancer, reversing from the minimally invasive technique to the previous practice
of abdominal approach.[5–7] In conventional MIS, the vagina is opened laparoscopically above the
manipulator and tumor cells may be exposure to the peritoneal cavity by insufflating carbon dioxide, and
tumor spill into the peritoneal cavity may an etiology for the inferior outcomes of MIS. However, based on
the advantages of laparoscopic surgery, many patients may still choose the MIS when choosing the
mode of the operation. Surgical technique All of the cases recruited in this study received surgery by five proficient gynecologists. The patients
were placed in the Trendelenburg position at 30 degrees. After general anesthesia, we created the
vaginal cuff in about the upper third of the vaginal in exogeneitic cervical cancer (tumor size > 2cm),
then, we performed the laparoscopic procedure with 5 trocars. Page 3/11 Page 3/11 At the first period of this study, we applied a myoma drill (Fig. 1a ) to grip the uterus to manipulate the
uterine, however, as to prevent tear the uterus and urologic and vascular complications by myoma drill,
we modified this approach to the thread fixing in the uterus (Fig. 1b ) and manipulated the uterus
laparoscopically. We refrain from using uterine manipulator, and the remaining procedure of the MIS has
been used widely. During surgery, we put the removed specimens such as lymphonodus into a collection
bag. In the final step before removal of the uterus, a special clamp was used to clamp (Fig. 1c-1e) the
vaginal tube in the non-exogeneitic cervical cancer (tumor size ≤ 2cm). As the clamp may fall off during
surgery, we fixed in the both ends of the clamp. Meanwhile, in the later period of this process, we
performed vaginal suture (Fig. 1f ) before colpotomy in patients with smaller tumors (≤ 2cm) . These steps (specimen bag, vaginal cuff, vaginal suture and clamp) were designed to prevent tumor
spillage during laparoscopic operation. Colpotomy was followed above the special clamp the vaginal
cuff, then the specimen is delivered vaginally. Specimens included uterus, tubes, portion of vagina,
parametrium, and pelvic lymph nodes. Statistical analysis SPSS version 17.0 (SPSS, Inc., IBM, Chicago, IL) was used to perform all statistical analyses. Data are
presented as mean ± standard deviation (SD), median (range), or absolute number (%). The difference in
means between the two groups was tested by analysis of variance (ANOVA). All P - values were two-
tailed, and a P - value < 0.05 was considered as statistically significant. Result Between January 2017 and June 2020, we recruited a total of 350 patients with cervical cancer that
underwent a radical hysterectomy for the stage Ia1 LVSI (+) to IIa1 (Table. 1). There were 5 patients
missing information on follow-up, and three patients rejected the following treatment. The baseline
clinical characteristics of the patients are summarized in Table 1. There were 83 (23.71%) patients
underwent radiotherapy and/or adjuvant chemo-radiation. Over a median follow-up of 51 (range 30–72)
months period, the disease-free survival and overall survival rate were 95.71% (335/350) and 98.86%
(346/350), respectively, the peri-operative outcomes of the study population are summarized in Table 2. The unadjusted Kaplan-Meier curves are shown in Fig. 2a-2b. In the subgroup of patients with tumor ≤ 2,
there were 3 (1.49%) relapse in 201 patients and no death. Moreover, the patients with tumor ≤ 2 that
underwent MIS had lower chance of relapse compared those with tumors greater than 2cm. (HR, 0.110;
95% CI, 0.025 to 0.488; P = 0.0004) Summary of Main Results Radical hysterectomy is the standard surgical treatment for patients diagnosed with early-stage cervical
cancer. [8] With the widespread use of LRH, data suggested comparable oncological outcomes from Page 4/11 Page 4/11 minimally invasive radical hysterectomy to ARH. [9–11] Similarly, LRH appears to provide equivalent or
better intraoperative and short-term postoperative outcomes. [12] Shah et al have shown that recurrence
rates and survival rates do not significantly differ between the two approaches. [13, 20] These benefits of
minimal invasive surgery have led to the increasing use of LRH for cervical cancer. In a meta-analysis,
Wang et al. analyzed 12 studies comparing laparoscopic radical hysterectomy with open radical
hysterectomy (754 vs 785 cases) for cervical cancer. Their results revealed no significant differences in
the 5-year rate of OS and DFS between the two approaches.[21] However, in 2018, a phase III randomized
clinical trial demonstrated that minimally invasive radical hysterectomy was associated with lower rates
of DFS (3-year rate, 91.2% vs 97.1%) and OS compared with ARH (3-year rate, 93.8% vs. 99.0%) [14]. Several recent retrospective studies also demonstrated that minimally invasive radical hysterectomy was
associated with shorter survival compared with ARH [15–17]. The reason for the shorter survival of the
minimally invasive surgery maybe the less extent of resection, the surgeon experience or the use of
uterine manipulator. However, Alexander etal [19] observed no meaningful difference in the frequency of
positive margins and in most institution only those who were trained and underwent several years of
supervised training with a long learning curve can perform laparoscopic surgery. Therefore, compared
with the first two reasons, the tumor peritoneal contamination appears to be more sensitive to survival. Chiva et al [18] used the “protective maneuvers” such as closing the vagina instead of uterine
manipulator, and found the increased overall survival in the MIS to 93% compared with 98% in the open
surgery. These conflicting data cause us to reconsider whether it is in the best interest of our patients to
abandon MIS. This resulted to the hypothesis that the increased rate of recurrence in the laparoscopic
radical hysterectomy may be driven by tumor exposure to the peritoneal cavity as a result of differences
in surgical technique between the different surgical approaches. In the time of minimal invasive approach practiced widely throughout the world, minimally invasive
radical hysterectomy with uterine manipulator to perform surgery for early-stage cervical cancer from
2013 in our department. Summary of Main Results However, we found that some cases with larger diameter tumor recurred only
about 1 year after surgery, despite the following radiotherapy or/and chemotherapy, and most of them
died as to the central recurrence. One of the patient had two central recurrent and underwent
laparoscopic surgery respectively, and has survived for 11 years. Based on the experiences of short-term
central-recurrence and poor prognosis of larger tumor, we reflected that we did not pay enough attention
to tumor exposure to the peritoneum. Since 2017, we modified the approach for the surgical treatment of
early-stage cervical cancer and designed a list of the principle of “no-tumor”, and hoped to improve
patient survival and maintain maximum quality of life. In order to promote the further promotion of the
surgical approach of abandoning the uterine manipulator in radical hysterectomy, we applied for a new
project in our hospital. Results in the Context of Published Literature The survival outcomes in all patients had comparable outcomes to the LACC trial
laparotomy group (3-year DFS and OS rate 97.1% and 99% respectively), which may be related to the the
technique of avoiding tumor containment and evolving treatment of cervical cancer. Moreover, the
subgroup of the patients with tumor ≤ 2cm that underwent MIS had significant lower chance of relapse
compared those with tumors > 2cm. (P = 0.0005). In contrast to the LACC trial, we did not find inferiority
outcomes of MIS in our trial, the strict principle of “untouched-entire tumor” may play an significant role;
in addition, lower percentage of lymph node positivity in all cases (4.29% vs 12.4%) and relative-short
following-up versus the LACC trial may have impacted the rate of OS and DFS. Moreover, some other
studies [22–23] found that MIS did not have inferior oncological outcomes; there were two cohort
studies in Denmark and Sweden, a nationwide adoption of robot-assisted MIS for cervical cancer did not
negatively impact oncological outcomes, which is consistent with our study. The following period in our
study was relatively short, however, at the time of analysis, there were 60.29% (211/350) cases had
reached the 4-year time point. From the above result, we can infer that the laparotomy might not the
most appropriate method for treating early cervical cancer, and we should reconsider the MIS
approaches in cervical cancer. Results in the Context of Published Literature There are various ways have been proposed for avoiding to contaminate the peritoneal cavity in our
study, such as vaginal cuff before surgery or a special vaginal clamp and vaginal suture before
colpotomy, in which a cervical tumor is encased vaginally. However, the clamp can only used in the non- Page 5/11 ectopic cervical cancer patients with smaller tumors (≤ 2cm) and without vaginal tumor invasion. It is
worth noting that with the the principle of “no-touch”, we need to push the clamp from the vagina to
cervix to close the vagina, or would be mobilized the tumor cells and may potentially resulted to
metastasis. As the limitations of clamp, we performed vaginal suture before colpotomy in patients with
smaller tumors (≤ 2cm). In the process of vaginal suture, it may mobilize the tumor cells, however, the
following result in our study revealed no central recurrences. The technique of vaginal cuff mainly used
for tumors that larger than 2cm. The method can effective avoid tumor spread, however, which can be
technically challenging, especially in patients who are difficult to being expose cervix such as obese or
nulliparous [19]. On the other hand, during the procedure (Type C), we incised the deep uterine vein
around the ureter and then transected the vaginal wall, however, the position of vaginal incision in
laparoscopic may not always consistent with the level of the vaginal cuff, which may lead to positioning
failure in colpotomy and resulted to difficult to continue. As the myoma drill may lead to uterine
perforation and then cause damage to pelvic vessels or ureters. Considering the possible complication,
we modified the myoma drill to uterus suture in the later period of study. In the process of our study, we applied myoma drill or uterus suture combined with protective vaginal
closure such as clamp, vaginal cuff, or vaginal suture to avoid tumor spillage before colpotomy. In
addition, we placed the specimen in a collection bag to prevent tumor spillage and extracted via the
vagina. Combined with the pathological results, 83 (23.71%) patients underwent radiotherapy or/and
adjuvant chemo-radiation. The following result revealed no central recurrences after a median follow-up
of 51 (range 30–72) months, and the favorable outcome of DFS and OS rate were 95.71% (335/350) and
98.86% (346/350). Acknowledgement Thanks are due to Lanying Jin for valuable discussion. Funding Sources This research received no specific grant from any funding agency in the public, commercial, or not-for-
profit sectors. Statement of Ethics The study was approved by the Ethics Committee of Jinhua Municipal Central Hospital (ethics approval
number: 2020 − 268). Author Contribution Min Hu conceived and designed this research study. Mingjun Shao, Xiujuan Fu and Baomei Shi acquired
data. Limei Ji and Lanying Jin analyzed and interpreted the data. Limei Ji drafted the manuscript,. Limei
Ji and Lanying Jin prepared figures 1-2 and Min Hu revized the manuscript. We declare that there are no conflicts of interest related to this research We declare that there are no conflicts of interest related to this research Strengths and Weaknesses There were some limitations associated with our current research that should be taken into
consideration. The follow-up period was short that only a median of 39 months. Our work is an Page 6/11 observational retrospective study and lack of control group. The results call for further surveillance of a
new prospective randomized trial comparing laparoscopic radical hysterectomy free from manipulator
with open radical hysterectomy to achieve optimal DFS with longer following up. Data Availability Statement All data analysed during this study are included in this article. Further enquiries can be directed to the
corresponding authorfi Page 7/11 mplications for Practice and Future Researc Implications for Practice and Future Research In conclusion, in our study we avoiding the uterine manipulator combined a protective vaginal closure
over the entire tumor to avoid tumor spillage in MIS, and we did not find inferior oncologic outcomes
especially in subgroup of the patients with tumor ≤ 2cm . synopsis of your findings: The technique of minimally invasive surgery using maneuvers to avoid peritoneal contamination during
surgery provides excellent oncologic outcome for patients with early cervical cancer. References 1. R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2020, CA Cancer J. Clin. 70 (1) (2020 Jan) 7–30. 2. Casarin J, Buda A, Bogani G, Ghezzi F etal.Predictors of recurrence following laparoscopic radical
hysterectomy for early-stage cervical cancer: A multi-institutional study. Gynecol
Oncol.2020;159:164–170. 3. L. Philp, A. Covens, D. Vicus, R. Kupets, K. Pulman, L.T. Gien, Feasibility and safety of same-day
discharge after laparoscopic radical hysterectomy for cervix cancer, Gynecol. Oncol. 147 (2017)
572–576. 4. P.T. Ramirez, M. Frumovitz, R. Pareja, A. Lopez, M. Vieira, R. Ribeiro, et al., Minimally invasive versus
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trial? J. Gynecol. Oncol. Korea (South) 29 (2018) e74. 8. Cibula D, Pötter R, Planchamp F, et al. The European Society of Gynaecological Oncology/European
Society for Radiotherapy and Oncology/European Society of Pathology guidelines for the
management of patients with cervical cancer. Int J Gynecol Cancer 2018;28:641–55. 8. Cibula D, Pötter R, Planchamp F, et al. The European Society of Gynaecological Oncology/European
Society for Radiotherapy and Oncology/European Society of Pathology guidelines for the
management of patients with cervical cancer. Int J Gynecol Cancer 2018;28:641–55. 9. Wang Y, Deng L, Xu H, Zhang Y, Liang Z. Laparoscopy versus laparotomy for the management of
early stage cervical cancer. Bmc Cancer. (2015) 15:1. doi: 10.1186/s12885-015-1818-4 9. References Wang Y, Deng L, Xu H, Zhang Y, Liang Z. Laparoscopy versus laparotomy for the management of
early stage cervical cancer. Bmc Cancer. (2015) 15:1. doi: 10.1186/s12885-015-1818-4 10. Nam JH, Park JY, Kim DY, Kim JH, Kim YM, Kim YT. Laparoscopic versus open radical hysterectomy
in early-stage cervical cancer: long-term survival outcomes in a matched cohort study. Ann Oncol. (2012) 23:903–11. doi: 10.1093/annonc/mdr360 10. Nam JH, Park JY, Kim DY, Kim JH, Kim YM, Kim YT. Laparoscopic versus open radical hysterectomy
in early-stage cervical cancer: long-term survival outcomes in a matched cohort study. Ann Oncol. (2012) 23:903–11. doi: 10.1093/annonc/mdr360 11. Chen Y, Xu H, Li Y, Wang D, Li J, Yuan J, et al. The outcome of laparoscopic radical hysterectomy and
lymphadenectomy for cervical cancer: a prospective analysis of 295 patients. Ann Surg Oncol. (2008) 15:2847–55. doi: 10.1245/s10434-008-0063-3. 11. Chen Y, Xu H, Li Y, Wang D, Li J, Yuan J, et al. The outcome of laparoscopic radical hysterectomy and
lymphadenectomy for cervical cancer: a prospective analysis of 295 patients. Ann Surg Oncol. (2008) 15:2847–55. doi: 10.1245/s10434-008-0063-3. Page 8/11 12. Gallotta V, Conte C, Federico A, Vizzielli G, Gueli Alletti S, Tortorella L, et al. Robotic versus
laparoscopic radical hysterectomy in early cervical cancer: a case matched control study. Eur J Surg
Oncol. (2018) 44:754–9. doi: 10.1016/j.ejso.2018.01.092 13. Sert BM, Boggess JF, Ahmad S, et al. Robot-assisted versus open radical hysterectomy: a multi-
institutional experience for early-stage cervical cancer. Eur J Surg Oncol 2016;42:513–22. 13. Sert BM, Boggess JF, Ahmad S, et al. Robot-assisted versus open radical hysterectomy: a multi-
institutional experience for early-stage cervical cancer. Eur J Surg Oncol 2016;42:513–22. 14. Shah CA, Beck T, Liao JB, Giannakopoulos NV, Veljovich D, Paley P. Surgical and oncologic outcomes
after robotic radical hysterectomy as compared to open radical hysterectomy in the treatment of
early cervical cancer. J Gynecol Oncol 2017; 28(6):e82. 14. Shah CA, Beck T, Liao JB, Giannakopoulos NV, Veljovich D, Paley P. Surgical and oncologic outcomes
after robotic radical hysterectomy as compared to open radical hysterectomy in the treatment of
early cervical cancer. J Gynecol Oncol 2017; 28(6):e82. 15. Wang YZ, Deng L, Xu HC, Zhang Y, Liang ZQ. Laparoscopy versus laparotomy for the management of
early stage cervical cancer. BMC Cancer 2015;15:928. 15. Wang YZ, Deng L, Xu HC, Zhang Y, Liang ZQ. Laparoscopy versus laparotomy for the management of
early stage cervical cancer. BMC Cancer 2015;15:928. 16. References Jensen PT, Schnack TH, Frøding LP, et al. Survival after a nationwide adoption of robotic minimally
invasive surgery for early-stage cervical cancer–a population-based study. Eur J Cancer. 2020;128:47–56. 23. Alfonzo E, Wallin E, Ekdahl L, et al. No survival difference between robotic and open radical
hysterectomy for women with early-stage cervical cancer: results from a nationwide
populationbased cohort study. Eur J Cancer. 2019;116:169–77. 23. Alfonzo E, Wallin E, Ekdahl L, et al. No survival difference between robotic and open radical
hysterectomy for women with early-stage cervical cancer: results from a nationwide
populationbased cohort study. Eur J Cancer. 2019;116:169–77. References Ramirez PT, Frumovitz M, Pareja R. Minimally invasive versus abdominal radical hysterectomy for
cervical cancer. N Engl J Med. (2018) 379:1895–904. doi: 10.1056/NEJMoa1806395 16. Ramirez PT, Frumovitz M, Pareja R. Minimally invasive versus abdominal radical hysterectomy for
cervical cancer. N Engl J Med. (2018) 379:1895–904. doi: 10.1056/NEJMoa1806395 17. Melamed A, Margul DJ, Chen L, Keating NL, Del Carmen MG, Yang J, et al.Survival after minimally
invasive radical hysterectomy for early-Stage cervical cancer. New Engl J Med. (2018) 379:1905–14. doi: 10.1056/NEJMoa1804923 17. Melamed A, Margul DJ, Chen L, Keating NL, Del Carmen MG, Yang J, et al.Survival after minimally
invasive radical hysterectomy for early-Stage cervical cancer. New Engl J Med. (2018) 379:1905–14. doi: 10.1056/NEJMoa1804923 18. Doo DW, Kirkland CT, Griswold LH, McGwin G, Huh WK, Leath CA, et al. Comparative outcomes
between robotic and abdominal radical hysterectomy for IB1 cervical cancer: results from a single
high volume institution. Gynecol Oncol. (2019) 153:242–7. doi: 10.1016/j.ygyno.2019. 03.001 18. Doo DW, Kirkland CT, Griswold LH, McGwin G, Huh WK, Leath CA, et al. Comparative outcomes
between robotic and abdominal radical hysterectomy for IB1 cervical cancer: results from a single
high volume institution. Gynecol Oncol. (2019) 153:242–7. doi: 10.1016/j.ygyno.2019. 03.001 19. Kim SI, Cho JH, Seol A, Kim YI, Lee M, Kim HS, et al. Comparison of survival outcomes between
minimally invasive surgery and conventional open surgery for radical hysterectomy as primary
treatment in patients with stage iB1-IIA2 cervical cancer. Gynecol Oncol. (2019) 153:3–12. doi:
10.1016/j.ygyno.2019.01.008 20. Kohler C, Hertel H, Herrmann J, et al. Laparoscopic radical hysterectomy with transvaginal closure of
vaginal cuff - a multicenter analysis. Int J Gynecol Cancer. 2019;29:845–850. 20. Kohler C, Hertel H, Herrmann J, et al. Laparoscopic radical hysterectomy with transvaginal closure of
vaginal cuff - a multicenter analysis. Int J Gynecol Cancer. 2019;29:845–850. 21. Chiva L, Zanagnolo V, Kucukmetin A, et al. SUCCOR study. An international European cohort
observational study comparing minimally invasive surgery versus open abdominal radical
hysterectomy in patients with stage IB1 cervical cancer operated in 2013–2014. 21. Chiva L, Zanagnolo V, Kucukmetin A, et al. SUCCOR study. An international European cohort
observational study comparing minimally invasive surgery versus open abdominal radical
hysterectomy in patients with stage IB1 cervical cancer operated in 2013–2014. 22. Jensen PT, Schnack TH, Frøding LP, et al. Survival after a nationwide adoption of robotic minimall
invasive surgery for early-stage cervical cancer–a population-based study. Eur J Cancer. 2020;128:47–56. 22. Tables Tables 1 and 2 are available in the Supplementary Files section. Figure 2 a The unadjusted Kaplan-Meier survival curves of disease-free survival of tumor size <= 2 cm and > 2 cm. b The unadjusted Kaplan-Meier survival curves of overall survival of tumor size <= 2 cm and > 2 cm. a The unadjusted Kaplan-Meier survival curves of disease-free survival of tumor size <= 2 cm and > 2 cm. b The unadjusted Kaplan-Meier survival curves of overall survival of tumor size <= 2 cm and > 2 cm. Page 9/11 Figures Figure 1
a We applied a myoma drill to grip the uterus to manipulate the uterine. a We applied a myoma drill to grip the uterus to manipulate the uterine. b We used the thread fixing in the uterus. c-e A special clamp was used to clamp the vaginal tube in the non-exogeneitic cervical cancer (tumor
size ≤ 2cm). c-e A special clamp was used to clamp the vaginal tube in the non-exogeneitic cervical cancer (tumor
size ≤ 2cm). f We performed vaginal suture before colpotomy in patients with smaller tumors (≤ 2cm). Page 10/11 Figure 2 Figure 2 Table1.doc
Table2.doc Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Table1.doc Table2.doc Page 11/11
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Хірургічне лікування великої ліпоми сигмоподібної кишки із розвитком товстокишкової інвагінації
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Хірургічне лікування великої ліпоми сигмоподібної кишки
із розвитком товстокишкової інвагінації У статті описано казуїстичний випадок великої ліпоми сигмоподібної кишки, що призвела до розвитку товстокишкової інвагіна
ції, з успішним хірургічним лікуванням. Ключові слова: сигмоподібна кишка; хірургічне лікування; ліпома; товстокишкова інвагінація. Якщо утворення занадто велике для ендоско
пічної резекції, можна застосувати лапароскопіч
ний метод [19, 27, 33]. Лапароскопічне видалення
використовується і коли малігнізація до втручання
не може бути виключена, а також при розмірах лі
поми більше 2 см [8]. Ліпоми шлунково-кишкового тракту – це доб
роякісні пухлини, що розвиваються зі зрілих ади
поцитів [1, 2], локалізується, як правило, на рів
ні підслизового (90 %) або субсерозного шару
(10 %) [3]. Зустрічаються рідко (в 0,2 % розтинів)
[4] та описуються в науковій літературі в рубри
ках “CaseReports”. Вперше описана Баурером у
1757 р. Може розміщуватися від стравоходу до
прямої кишки, але найчастіше – в товстій кишці
(colonic lipomas) [5], де частота ліпом є на другому
місці після аденоматозних поліпів [6, 7]. Ліпома
товстої кишки частіше локалізується в правій її
половині (в 50 % випадків) [8, 9]. Видалений препарат віддають на патогістоло
гічне дослідження для виключення онкопатології
[13, 23]. Пацієнт П., 54 років, госпіталізований у хірур
гічну клініку 25.11.2021 р. з діагнозом: стенозуюча
пухлина сигмоподібної кишки (лейоміома, лейо
міосаркома?). Попередньо 16.11.2021 р. звернувся
до гастроентеролога зі скаргами на послаблення
випорожнення після кожного приймання їжі, поя
ву слизу в калі, відчуття неповного випорожнення,
бурчання в животі, біль у лівій половині живота. На УЗД ОЧП суттєвих відхилень не знайдено. 10
років тому переніс аутогерніопластику із приводу
пупкової грижі із подальшим розвитком рецидиву
ії розмірами 3х3см та наявністю діастазу прямих
м’язів живота до 4-5 см. Більшість випадків перебігає безсимптомно,
але іноді може маніфестуватись абдомінальним
болем, кровотечею, кишковою непрохідністю, ін
вагінацією [4, 6, 9–24] або навіть перфорацією чи
інфарктом ліпоми [25]. Вважається, що симптоматика кишкових ліпом
проявляється при досягненні ними відповідних роз
мірів [12, 26, 27], найчастіше 2 см і більше [28, 29]. У попередні роки діагноз встановлювали пе
реважно рентгенологічно або під час операційно
го втручання [10, 11], або ж під час колоноскопії
[29, 30], а на даний момент методом вибору є КТ з
контрастуванням [3, 4, 9, 12, 15, 16, 18, 19, 24, 26,
27]. Диференційна діагностика включає злоякісне
новоутворення, дивертикульоз, аденоматозні полі
пи та накладені раніше анастомози [4, 24, 27]. Для
диференційної діагностики із злоякисними пухли
нами Younathan C.M. еt al. (1991) запропонували
метод МРТ з клізмою барію сульфату [31]. При відеоколоноскопії 19.11.2021 р. ©Є. М. ШЕПЕТЬКО1,2, М. В. БІЛЯЧЕНКО2, В. В.ЄФРЕМОВ2, І. М. ГО Київський медичний університет1
Інноваційний хірургічний Центр “Адоніс-лаб” СП2 Київський медичний університет1
Інноваційний хірургічний Центр “Адоніс-лаб” СП2 Київський медичний університет1
Інноваційний хірургічний Центр “Адоніс-лаб” СП2 УДК 616.349-006.326-089:616.345-007.44
DOI 10.11603/2414-4533.2022.2.13177 ©Є. М. ШЕПЕТЬКО1,2, М. В. БІЛЯЧЕНКО2, В. В.ЄФРЕМОВ2, І. М. ГОЛОВАЩУК2 ВИПАДОК З ПРАКТИКИ ВИПАДОК З ПРАКТИКИ ВИПАДОК З ПРАКТИКИ УДК 616.349-006.326-089:616.345-007.44
DOI 10.11603/2414-4533.2022.2.13177 УДК 616.349-006.326-089:616.345-007.44
DOI 10.11603/2414-4533.2022.2.13177 Хірургічне лікування великої ліпоми сигмоподібної кишки
із розвитком товстокишкової інвагінації виявлено
пухлинне утворення округлої форми, червоного
кольору, рухоме, еластичне, розміром до 10 см,
що пролабує з сигмоподібної кишки в пряму
кишку. Зайти за дане утворення не вдається, при
біопісії щипці захоплюють вкрай мало матеріа
лу. Заключення: округла пухлина сигмоподібної
кишки, що перекриває просвіт (рис. 1). Вислов
лено припущення щодо інвагінації сигмоподіб
ної кишки. Результати біопсії від 24.11.2021 р. –
фрагменти слизової оболонки товстої кишки без
ознак атипії. При ліпомах невеликого розміру можливо
спостереження, а при розмірах ліпом більше 2 см
рекомендується їх операційне видалення [2, 32]. Останнє включає ендоскопічне видалення, лапа
роскопічне видалення та традиційну відкриту хі
рургію [7, 11]. За результатами МСКТ із контрастуванням
(23.11.2021 р.) – пухлина сигмоподібної кишки
дистального відділу (гігантська ліпома?) розмі
рами 3,86х5,46х4,0 см, лімфаденопатії немає, від ISSN 1681–2778. ШПИТАЛЬНА ХІРУРГІЯ. Журнал імені Л. Я. Ковальчука. 2022. № 2 72 ВИПАДОК З ПРАКТИКИ Рис. 1. Пухлина (ліпома): а – на відеоколоноскопії; б – перекриває просвіт сигмоподібної кишки. а б б б а Рис. 1. Пухлина (ліпома): а – на відеоколоноскопії; б – перекриває просвіт сигмоподібної кишки далених метастазів немає. Дистальніше помірне
інфільтративне потовщення циркулярного харак
теру стінок ректосигмоїдного відділу ободової
кишки із залученням прямої кишки до рівня її ам
пулярного відділу включно до 0,77 см на протязі
до 9,6 см, переважно за рахунок набряку підсли
зового шару (рис. 2). далених метастазів немає. Дистальніше помірне
інфільтративне потовщення циркулярного харак
теру стінок ректосигмоїдного відділу ободової
кишки із залученням прямої кишки до рівня її ам
пулярного відділу включно до 0,77 см на протязі
до 9,6 см, переважно за рахунок набряку підсли
зового шару (рис. 2). Виконано дезінвагінацію, стінка кишки після
дезінвагінації на протязі 5-6 см набрякла, гіпере
мійована, але життєздатна. Через стінку кишки
пальпується округла пухлина 5х5 см. Виконана резекція сигмоподібної кишки ра
зом із пухлиною з накладанням апаратного тов
стокишкового анастомозу “кінець-у-бік” цирку
лярним зшивачем 28 мм із заглушуванням торця
привідної петлі сигмоподібної кишки лінійним
зшивачем GIA 60 мм. Також виконана протезуюча
пластика післяопераційної грижі сітчастим імп
лантом 10х30см sublay, ліквідація діастазу прямих
м’язів живота. Післяопераційний період перебігав
без особливостей. 26.11.2022 р. проведено операційне втручан
ня – виявлено пухлину в просвіті сигмоподібної
кишки, округлої форми з явищами товстокишкової
інвагінації, так що проксимальний відділ сигмо
видної кишки нашарувався на дистальний разом із
пухлиною, яка перемістилась та занурилась в про
світ кишки в ретроградному напрямку (рис. 3). Рис. 2. МСКТ органів черевної порожнини: а – пряма проекція; б – бокова проекція, стрілкою вказано ліпому. SSN 1681–2778. ШПИТАЛЬНА ХІРУРГІЯ. Журнал імені Л. Я. Ковальчука. 2022. № 2 Хірургічне лікування великої ліпоми сигмоподібної кишки
із розвитком товстокишкової інвагінації а б б а б а Рис. 2. МСКТ органів черевної порожнини: а – пряма проекція; б – бокова проекція, стрілкою вк органів черевної порожнини: а – пряма проекція; б – бокова проекція, стрілкою вказано ліпому. ISSN 1681–2778. ШПИТАЛЬНА ХІРУРГІЯ. Журнал імені Л. Я. Ковальчука. 2022. № 2 2778. ШПИТАЛЬНА ХІРУРГІЯ. Журнал імені Л. Я. Ковальчука. 2022. № 2 73 ВИПАДОК З ПРАКТИКИ Рис. 3. Пухлина “на ніжці”, що стала причиною
товстокишкової інвагінації (стрілкою вказано інвагінат). Патогістологічне
дослідження
препарату
(01.12.2021 р.) – субсерозна ліпома сигмоподіб
ної кишки великих розмірів (рис. 4). На 5-у добу
пацієнта виписали з клініки в задовільному стані,
оглянутий через 3 місяці, почувається добре, пра
цює за спеціальністю. Випадок стосується рідкісного спостережен
ня доброякісної пухлини – ліпоми сигмоподібної
кишки, що призвела до товстокишкової інвагінації. Можливо, поява інвагінації пов’язана з підготов
кою кишечника до колоноскопії та прийоманням
препарату “Фортранс”, що посилив перистальтич
ну активність не лише тонкої, але й товстої кишки. Це сприяло розвитку ретроградної товстокишкової
інвагінації, коли проксимальний відрізок сигмопо
дібноїної кишки нашарувався на дистальний з рет
роградним переміщенням та зануренням пухлини в
просвіт сигмоподібної кишки (рис. 5). Рис. 3. Пухлина “на ніжці”, що стала причиною
товстокишкової інвагінації (стрілкою вказано інвагінат). Рис. 4. Препарат, відправлений на патогістологічне дослідження: а – пухлина на ніжці, б – пухлина на розрізі. а б б а Рис. 4. Препарат, відправлений на патогістологічне дослідження: а – пухлина на ніжці, б – пухл Рис. 4. Препарат, відправлений на патогістологічне дослідження: а – пухлина на ніжці, б – пухлина на розрізі. Рис. 5. Ретроградна інвагінація великої ліпоми сиг
ми (відеоколоноскопія). Теоретично можна розділити товстокишкову
інвагінацію, причиною якої є пухлина на ніжці
– в даному випадку доброякісна, на 2 варіанти:
перший варіант – ретроградна інвагінація, коли
проксимальний відділ і стінка товстої кишки на
шаровується, напливає на дистальний разом з пух
линою, яка переміщується і занурюється в просвіт
кишки в ретроградному напрямку; другий варіант
– антеградна інвагінація, коли пухлиноподібне
утворення занурюється в просвіт дистального від
різку кишки таким чином, що стінка дистального
відділу навертається на проксимальний відділ ра
зом з пухлиною і вона розташовується в просвіті
кишки в зоні інвагінації. Виконання резекції киш
ки в таких ситуаціях є доцільним. Рис. 5. Ретроградна інвагінація великої ліпоми сиг
ми (відеоколоноскопія). ISSN 1681–2778. ШПИТАЛЬНА ХІРУРГІЯ. Журнал імені Л. Я. Ковальчука. 2022. № 2 ВИПАДОК З ПРАКТИКИ невеликих розмірів [10, 34, 40], разом із тим,
видалення ліпом великого розміру (більше 2 см)
ендоскопічним методом (електрокоагуляція) ста
новить високий ризик перфорації стінки кишки
[12, 28, 30, 41], тому раніше застосовувалось ви
далення методами традиційної хірургії [7, 11], а
на даний час ускладнень допомагає уникнути тех
ніка Endoloop. Цей метод є перспективним з огля
ду на профілактику післяопераційної кровотечі,
навіть при розмірах ліпоми розміром більше 2 см,
особливо у пацієнтів із захворюваннями печінки,
коагулопатією та які приймають антикоагулянти
[13]. Дана технологія (Endoloop, PolyLoop) вико
ристовується в тому числі для видалення гігант
ських ліпом [20, 29, 41, 42,43]. Для ендоскопіч
ного видалення важливо, чи є ліпома на широкій
основі, чи на ніжці [32]. При ліпомі на широкій
основі для кращої візуалізації країв застосовуєть
ся ін’єкція адреналіну з фізіологічним розчином
[13, 32]. Ліпоми товстої кишки можуть проявлятись
симптомами, схожими на злоякісні пухлини тов
стої кишки, але потребують набагато менш агре
сивного хірургічного підходу і важливо не при
йняти їх помилково за злоякісне ураження [5, 21,
24, 31, 34]. Діагноз доброякісного новоутворення
у пацієнтів із товстокишковою інвагінацією може
бути встановлений лише при патогістологічному
обстеженні, але цей стан слід лікувати як злоякіс
ну пухлину через високу частоту малігнізації [21]. Онкологічний діагноз слід особливо ретельно ви
ключити у пацієнтів похилого віку [35]. У даному випадку було прийнято рішення опе
рувати відкритим лапаротомним методом, тому що
до операції були сумніви в доброякісності пухлини. Деякі автори повідомляють про можливість
інтраопераційної експрес-біопсії пухлини, що
дозволить визначитись із обсягом операційного
втручання під час операції [36], оскільки не слід
передчасно та без гістологічного підтвердження
малігнизації робити більш обширну резекцію при
підозрі на малігнізацію [37]. При наявності великої ліпоми товстої кишки,
що не піддається лігуванню, перспективною тех
нологією є метод ендокліпування, що дає змогу
уникнути ризику перфорації та кровотечі, особли
во у пацієнтів групи високого ризику [13, 28, 32]. Застосовуються такі операційні втручання при
кишковій непрохідності, що викликана інвагіна
цією, ‒ відкрита розширена правобічна геміколек
томія з ілеотрансверзостомією “кінець-у-кінець” [1,
15], лівобічна геміколектомія [4], сегментарна ко
лектомія [14, 38], резекція інвагінованого сегмента
[15, 23, 39]. В даному випадку інтраопераційно було
прийнято рішення виконати резекцію сигмоподіб
ної кишки разом із пухлиною з накладанням апарат
ного товстокишкового анастомозу “кінець-у-бік”. Після видалення ліпоми ендоскопічно через
визначений період (як правило, 6 міс.) показана
колоноскопія з оглядом місця операції [20]. Рідкісний випадок великих розмірів доброя
кісної пухлини (ліпоми) сигмоподібної кишки, що
призвела до ретроградної товстокишкової інвагі
нації. ВИПАДОК З ПРАКТИКИ При розвитку товстокишкової інвагінації на
тлі пухлиноподібного утворення товстої кишки
доцільним є застосування резекції в межах здоро
вих тканин апаратним способом. Як альтернатива традиційній резекції кишеч
нику використовується ендоскопічна підслизо
ва дисекція (ESD) [17], насамперед при ліпомах Хірургічне лікування великої ліпоми сигмоподібної кишки
із розвитком товстокишкової інвагінації Теоретично можна розділити товстокишкову
інвагінацію, причиною якої є пухлина на ніжці
– в даному випадку доброякісна, на 2 варіанти:
перший варіант – ретроградна інвагінація, коли
проксимальний відділ і стінка товстої кишки на
шаровується, напливає на дистальний разом з пух
линою, яка переміщується і занурюється в просвіт
кишки в ретроградному напрямку; другий варіант
– антеградна інвагінація, коли пухлиноподібне
утворення занурюється в просвіт дистального від
різку кишки таким чином, що стінка дистального
відділу навертається на проксимальний відділ ра
зом з пухлиною і вона розташовується в просвіті
кишки в зоні інвагінації. Виконання резекції киш
ки в таких ситуаціях є доцільним. Рис. 5. Ретроградна інвагінація великої ліпоми сиг
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lonic intussusception 76 ISSN 1681–2778. ШПИТАЛЬНА ХІРУРГІЯ. Журнал імені Л. Я. Ковальчука. 2022. № 2 ISSN 1681–2778. ШПИТАЛЬНА ХІРУРГІЯ. Журнал імені Л. Я. Ковальчука. 2022. № 2 REFERENCES et al. (2011). Intestinal subocclusion
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36. Jiang, L., Jiang, L.-S., Li, F.Y., Ye, H., Li, N., Cheng, N.-S., & Отримано 28.04.2022 Отримано 28.04.2022 Електронна адреса для листування: shepevgen@gmail.com E. M. SHEPETKO1,2, M. V. BILYACHENKO2, V. V. IEFREMOV2, I. М. GOLOVASHCHUK2 Kyiv Medical University1,
Innovative Surgical Center “Adonis-lab” SP2 Kyiv Medical University1,
Innovative Surgical Center “Adonis-lab” SP2 Kyiv Medical University1,
Innovative Surgical Center “Adonis-lab” SP2 ISSN 1681–2778. ШПИТАЛЬНА ХІРУРГІЯ. Журнал імені Л. Я. Ковальчука. 2022. № 2 SURGICAL TREATMENT OF LARGE SIGMOID COLON LIPOMA WITH THE DEVELOPMENT
OF COLON INTUSSUSCEPTION SURGICAL TREATMENT OF LARGE SIGMOID COLON LIPOMA WITH THE DEVELOPMENT
OF COLON INTUSSUSCEPTION The aim of the work: to describe a case report of a large lipoma of the sigmoid colon, which led to the development of
intussusception, with successful surgical treatment. Key words: sigmoid colon; surgical treatment; lipoma; colonic intussusception. ISSN 1681–2778. ШПИТАЛЬНА ХІРУРГІЯ. Журнал імені Л. Я. Ковальчука. 2022. № 2 78 78
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Towards Robust Numerical Question Answering: Diagnosing Numerical Capabilities of NLP Systems
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Abstract Reasoning (Dua et al., 2019; Hu et al., 2019; Al-
Negheimish et al., 2021a), Tabular Question An-
swering (Zhong et al., 2017; Chen et al., 2020b;
Zhu et al., 2021; Chen et al., 2021). These Numer-
ical QA tasks require NLP systems to arrive at a
numerical answer from the numbers in the ques-
tion and context. By studying how existing NLP
systems perform in these Numerical QA tasks, we
could take a glimpse at what capabilities are re-
quired for building NLP systems in the future. Numerical Question Answering is the task of
answering questions that require numerical ca-
pabilities. Previous works introduce general
adversarial attacks to Numerical Question An-
swering, while not systematically exploring nu-
merical capabilities specific to the topic. In
this paper, we propose to conduct numerical
capability diagnosis on a series of Numerical
Question Answering systems and datasets. A
series of numerical capabilities are highlighted,
and corresponding dataset perturbations are de-
signed. Empirical results indicate that exist-
ing systems are severely challenged by these
perturbations. E.g., Graph2Tree experienced
a 53.83% absolute accuracy drop against the
“Extra” perturbation on ASDiv-a, and BART
experienced 13.80% accuracy drop against the
“Language” perturbation on the numerical sub-
set of DROP. As a counteracting approach, we
also investigate the effectiveness of applying
perturbations as data augmentation to relieve
systems’ lack of robust numerical capabilities. With experiment analysis and empirical studies,
it is demonstrated that Numerical Question An-
swering with robust numerical capabilities is
still to a large extent an open question. We dis-
cuss future directions of Numerical Question
Answering and summarize guidelines on future
dataset collection and system design. In an ad-hoc manner, a line of work revealed
robustness issues of handling Numerical QA in
existing NLP systems. Through adversarial at-
tacks with designed dataset perturbations, number-
related limitations were exposed: E.g., utilizing
spurious correlation in datasets (Patel et al., 2021;
Kumar et al., 2021; Al-Negheimish et al., 2021b;
Pi et al., 2022b), incorrectly representing numbers
(Nogueira et al., 2021; Kim et al., 2021) and fail-
ing to extrapolate (Kim et al., 2021; Pal and Baral,
2021). This line of work inspires us to ask follow-
ing questions: 1) What is the overall landscape of
robustness issues of numerical capabilities in exist-
ing NLP systems? Can we find a more systematic
way to investigate the number-related limitations? ∗The contributions by Jialiang Xu and Xinyi He have
been conducted and completed during their internships at
Microsoft Research Asia, Beijing, China. 1Our code and datasets used are available in the link:
https://github.com/microsoft/NumberDiagnosis Jialiang Xu1∗, Mengyu Zhou2†, Xinyi He3*, Shi Han2, Dongmei Zhang2
1 University of Illinois at Urbana-Champaign
2 Microsoft Research 3 Xi’an Jiaotong University
jx17@illinois.edu, hxyhxy@stu.xjtu.edu.cn,
{mezho, shihan, dongmeiz}@microsoft.com Jialiang Xu1∗, Mengyu Zhou2†, Xinyi He3*, Shi Han2, Dongmei Zhang2
1 University of Illinois at Urbana-Champaign
2 Microsoft Research 3 Xi’an Jiaotong University
jx17@illinois.edu, hxyhxy@stu.xjtu.edu.cn,
{mezho, shihan, dongmeiz}@microsoft.com Jialiang Xu1∗, Mengyu Zhou2†, Xinyi He3*, Shi Han2, Dongmei Zhang2
1 University of Illinois at Urbana-Champaign
2 Microsoft Research 3 Xi’an Jiaotong University
jx17@illinois.edu, hxyhxy@stu.xjtu.edu.cn,
{mezho, shihan, dongmeiz}@microsoft.com Abstract 2) How to diagnose each numerical capability and
evaluate the severity of it not being captured in a
system? Can we further develop new adversarial
perturbation methods on Numerical QA for diag-
nosis and evaluation? 3) How to address the numer-
ical robustness issues? How do existing solutions
work and what are possible future directions? Proceedings of the 2022 Conference on Empirical Methods in Natural Language Processing, pages 7950–7966
December 7-11, 2022 ©2022 Association for Computational Linguistics † Corresponding author. 1
Introduction Numeracy is an essential part for real-world NLP
applications (Sundaram et al., 2022; Thawani et al.,
2021b; Sundararaman et al., 2020; Spithourakis
and Riedel, 2018). Numerical QA (Question
Answering) is one representative group of such
number-dependent NLP tasks. E.g., Math Word
Problem Solving (Zhang et al., 2020a; Miao et al.,
2020; Koncel-Kedziorski et al., 2016), Discrete To answer the above questions, in this pa-
per we propose the DNC (Diagnosing Numerical
Capbilities) framework1 as shown in Figure 1. Most existing Numerical QA systems (see §2.1)
take a two-stage approach to extract and manipu-
late numbers. As shown in the QA Stages part of
Figure 1, systems usually first recognize numbers 7950 7950 7950 Systems
T5
BART
TagOps
GPT2
Tokenization
Graph2Tree
GPT2pre
Replacement
Compare
Math Word
Problem
Discrete
Reasoning
Tabular
Understanding
ASDiv-a
DROP-num
TATQA-a
Datasets
Stages
Capabilities
Numerical
Parsing
Semantic
Parsing
Perturbs
Language
Type
Noise
Distribution
Verbosity
Extra
Logic
Order
Noise
Test
Noise
Train
Noise
Val
Apply
Split Dataset
Compare
Attack Setting
Diagnose
Defense Setting
QA Stages
E.g., A room is 12 feet long, 8 feet wide and 14 feet high. How much carpet does one need to cover the whole floor? Num1 (12)
Num2 (8)
Num3 (14)
Stage 1: recognizing numbers from context & question
Num1 (12)
Num2 (8)
×
96
Stage 2: translating problem to logical form & executing
DNC Framework
Number
Detection
Number Value
Understanding
Operand
Selection
Operation
Reasoning
Noise
Test
Train
Val
To evaluate the relief by training on perturbed data. To reveal systems’ lack of numerical capabilities. Figure 1: Overview of DNC Framework. The process of Numerical QA solving is divided into two logical stages. Four capabilities are required to complete the stages, each maps to two perturbations. Perturbations can be applied to
appropriate train / validation / test splits of Numerical QA datasets under Attack or Defense Setting. Models of the
NLP systems are trained and then evaluated on the perturbed datasets as a diagnosis of their numerical capabilities. Num1 (12)
Num2 (8)
×
96
Stage 2: translating problem to logical form & executing Tabular
Understanding Figure 1: Overview of DNC Framework. The process of Numerical QA solving is divided into two logical stages. Four capabilities are required to complete the stages, each maps to two perturbations. Perturbations can be applied to
appropriate train / validation / test splits of Numerical QA datasets under Attack or Defense Setting. Models of the
NLP systems are trained and then evaluated on the perturbed datasets as a diagnosis of their numerical capabilities. verifies their lack of robust numerical capabilities. E.g., Graph2Tree experienced a 53.83% absolute
accuracy drop against the “Extra” perturbation on
ASDiv-a, and BART experienced 13.80% accuracy
drop against the “Language” perturbation on the
numerical subset of DROP. in the context and question and treat them as can-
didate operands. Then, with the understanding of
the question semantics, they select corresponding
operands, and explicitly generate logical forms or
implicitly execute operations to get the final result. 7950 The above two stages correspond to the two
groups of numerical capabilities (see §4.1) covered
by our DNC Framework (as shown in Figure 1). In
Stage 1, we focus on a system’s capabilities to
recognize different forms of numbers (“Number
Detection”), and to parse and represent number val-
ues correctly (“Number Value Understanding”). In
Stage 2, we focus on the capabilities to correctly
choose operands (“Operand Selection”) and oper-
ations (“Operation Reasoning”) by understanding
context and question. For each of these four capa-
bilities, two perturbations (see §4.2) are proposed
by us to diagnose the capability. Each perturbation
is designed to be trivial to humans and thus cannot
easily fool humans, but it could bring down exist-
ing NLP systems (under the “Attack” setting), and
therefore expose the robustness issue of lacking its
corresponding capability. From another point of view, the perturbations are
also applicable for data augmentation. Under the
“Defense” setting (see §4.3), perturbations are ap-
plied to all splits of the dataset. A system’s perfor-
mance of the same perturbation under both “Attack”
and “Defense” settings are compared (in §5.2) to
show if the corresponding robustness issue could
be relieved by augmenting the training data. Em-
pirical results indicate that despite the recovery in
most cases, the performance still fall lower than the
original level. Finally, based on the “Attack” and “Defense” re-
sults in §5 and additional experiments, in §6 we
compare some existing design choices in Numer-
ical QA, such as: Is it better to generate logical
forms (and then execute the program/expression)
or predict answers directly in an end-to-end way? Shall we break numbers into subword tokens or
substitute them with a placeholder that can be later
re-substituted? We also discuss the open questions
and future directions on the robust numerical capa- By applying the above diagnosis to various NLP
Systems and Numerical QA Datasets (as shown
in Figure 1), in §5 we find that existing systems
experience significant performance drops, which 7951 bilities of NLP systems, including recent relevant
development such as neural program execution and
numerical data synthesizing. in the domain of tabular data, which often involves
a large amount of numbers and requires arithmetic
aggregations to arrive at the final answer. 2.2
Numeracy Limitations in NLP Systems Efforts have been dedicated to reveal numeracy
limitations in NLP systems. (Patel et al., 2021;
Kumar et al., 2021; Al-Negheimish et al., 2021b;
Pi et al., 2022b; Nogueira et al., 2021; Kim et al.,
2021; Pal and Baral, 2021). However, previous
work mainly focused on borrowing adversarial at-
tack methods from general QA such as re-ordering
sentences (Patel et al., 2021; Al-Negheimish et al.,
2021b; Kumar et al., 2021), substituting synonyms
(Kumar et al., 2021; Pi et al., 2022b), or adding
irrelevant information (Patel et al., 2021; Pi et al.,
2022b), while having limited exploration into ca-
pabilities specific to Numerical QA problems such
as understanding different number values, recog-
nizing different number surface forms or selecting
related numbers. • Based on experiments and previous work, we
provide guidelines for existing numerically-
robust NLP system designs and discussions
for future directions on robust Numerical QA. 2.1
Numerical Question Answering Previous work has proposed Numerical QA
datasets and systems. In this paper we consider
as examples the domains of Math Word Problem,
Discrete Reasoning and Tabular QA. 7950 Tabular
QA systems (Dong et al., 2022; Liu et al., 2022;
Iida et al., 2021; Herzig et al., 2020; Yin et al.,
2020) are mainly based on Pretrained Language
Models with Transformer backbones. Tabular QA
systems mainly aim at converting natural language
utterance into executable expressions such as com-
mands in SQL language. In summary, our major contributions are: • The DNC framework is proposed by us to sys-
tematically diagnose the robustness of NLP
systems on numerical capabilities. A series
of number-related perturbation methods are
designed for the capabilities. • Comprehensive diagnosing experiments on
adversarial attacks and data augmentations are
conducted by us on five systems over three
Numerical QA tasks. We show the overall
picture of numerical robustness issues of the
systems, and the partial effectiveness of our
simple defense mechanism. 3
Preliminaries Math Word Problem (Kushman et al., 2014;
Upadhyay and Chang, 2017; Miao et al., 2020;
Qin et al., 2020; Lan et al., 2022) concerns arith-
metic questions collected from lower-grade el-
ementary school coursework. Neural network
are employed with different architectures such as
Seq2Seq (Wang et al., 2017; Chiang and Chen,
2019), Seq2Tree (Xie and Sun, 2019; Liang et al.,
2021) and Graph2Tree (Zhang et al., 2020b; Shen
and Jin, 2020). Recently, large end-to-end pre-
trained language models (Chowdhery et al., 2022;
Pi et al., 2022a) have also been showing impressive
results in Math Word Problem. A Numerical Question Answering problem is de-
fined to consist of a problem prompt (question) P
and a problem body (context) B. Depending on
the task type, the problem body takes the form of
either a paragraph or a mixture of free-form text
paragraphs and structured data such as tables. Let
V be the vocabulary of the textual words, Q be
the set of the numerical values in P ∪B, and Q+
be the numerical values that can be arithmetically
computed with Q, then the problem prompt and
body can be formulated as P = L
i pi, pi ∈V ∪Q and B =
(L
j bj
L
i,j τi,j ⊕L
k bk
, τi,j, bk ∈V ∪Q. Discrete Reasoning (Dua et al., 2019; Al-
Negheimish et al., 2021a; Hu et al., 2019) concerns
questions requiring logistic and arithmetic opera-
tions on real-world paragraphs. Discrete Reason-
ing Systems are mainly based on Graph Attention
Networks (Chen et al., 2020a) or the Transformer
architecture (Ran et al., 2019). , τi,j, bk ∈V ∪Q. (
,j
Here ⊕denotes the concatenation operation, p· and
b· are prompt and body textual words, and τ·,· are
the body tabular cells. (
,j
Here ⊕denotes the concatenation operation, p· and
b· are prompt and body textual words, and τ·,· are
the body tabular cells. The target output T of the problem is either
a numerical value Tans that is an element in Q+
or a mathematical expression Teq that consists of
elements in the concerned numerical values Q
and the simple operators O = {+, −, ×, ÷}. I.e. 2https://github.com/savoirfairelinux/num2words 4
DNC Framework Perturbations are designed according to each nu-
merical capabilities. In Table 1, an example prob-
lem is provided for each of the perturbations. The
formal definition of the perturbations is provided
in Appendix A. Our approach aims at diagnosing the numerical
weakness of existing Numerical Question Answer-
ing models. We list out and explain a series of
numerical capabilities that are critical to solving
Numerical Question Answering problems in §4.1. We then design numerical perturbations targeting
these capabilities in §4.2. With the designed per-
turbations, we examine the weaknesses under two
different perturbations settings in §4.3. Language Perturbation targets the Number De-
tection capability and diagnoses how accurate can
systems detect numbers in different surface forms. To perturb a number string ns, we replace it with
its English form of the number with Num2Words.2
This perturbation changes number surface forms
but not their values. These three sections are represented in Figure 1. as the “Capabilities” stripe, the “Perturbs” stripe,
and the “Attack Setting” and “ Defense Setting”. Type Perturbation targets the Number Detec-
tion capability and challenges systems to detect
numbers in their float-point forms. To perturb a
number string ns, we concatenate it with the string
“.0”. Similar to Language Perturbation, only the
number detection capability is diagnosed with this
perturbation. Contrary to the Noise perturbation in
the next paragraph, the Type perturbation does not
propose additional calculation difficulty by chang-
ing number values. (1) Note that this expression not only describes the
Numerical QA tasks, but also generalizes to other
numeracy-related NLP tasks such as Tabular En-
tailment (Chen et al., 2020b) and Timeseries-based
Fraudulent Detection (Padhi et al., 2021). In this paper, we design and apply perturba-
tions to the samples in the dataset to form per-
turbed prompt P⋆, perturbed body B⋆and per-
turbed ground truth target T ⋆. We show that ex-
isting systems are fragile against numerical pertu-
bation by showing that on a large portion of the
dataset, the previous mapping fails to generate cor-
rect perturbed target, i.e.: Operation Reasoning is the capability of infer-
ring operations from the logic pattern described in
the text. In an arithmetic process, the operation is
independent from the operands, therefore different
operations can be applied to the same set of selected
related numbers in different questions. Systems are
expected to decouple operation from operands and
select the operation in an operand-agnostic way. f : (P⋆, B⋆) ̸→T ⋆
(2) (2) 3
Preliminaries Tabular QA and Semantic Parsing (Zhu et al.,
2021; Chen et al., 2021; Zhong et al., 2017; Pasu-
pat and Liang, 2015) concerns question answering Tabular QA and Semantic Parsing (Zhu et al.,
2021; Chen et al., 2021; Zhong et al., 2017; Pasu-
pat and Liang, 2015) concerns question answering 7952 T =
(
Tans : q ∈Q+
Teq : L
i ti, ti ∈Q ∪O
. With P and B T =
(
Tans : q ∈Q+ as included in the BERT tokenizer vocabulary). Robust Numerical QA systems are also expected
to handle values such as float-point numbers and
numbers larger than 500. T =
(
Teq : L
i ti, ti ∈Q ∪O
. (
q
i
as input and T as output, a trained Numerical QA
system can be regarded as a mapping f such that (
q
i
as input and T as output, a trained Numerical QA
system can be regarded as a mapping f such that Operand Selection is the capability of decid-
ing which numbers to select as the operands in the
arithmetic process. One important aspect of select-
ing related values is to exclude numbers that are 1)
irrelevant to the Numerical QA problem scenario,
or 2) relevant to the problem scenario but not essen-
tial to the question solving. Systems are expected
to select as operands the important values from the
unimportant values. f : (P, B) →T
(1) 4.1
Numerical Capabilities We classify numerical capabilities into three major
categories, concerning different aspects of numeri-
cal understanding, as below: Number Detection is the capability of recog-
nizing numbers of different surface forms. For
instance, the English word "Forty-two" and the
Arabic number "42.0" are regarded the same num-
ber in Numerical QA and should not affect the final
arithmetic answer of a question. Noise Perturbation targets the Number Value
Understanding capability and challenges systems
to not only understand arithmetic operations of
not only integers but also float-point numbers. To
perturb a number n, we randomly attach a one- Number Value Understanding is the capabil-
ity of understanding numbers of different value
distributions. Systems are expected to not only
apply arithmetic calculation on a specific set of
numbers (e.g., integers of values smaller than 500 7953 Capability
Perturbation Example Problem Pair Capability
Perturbation
Example Problem Pair
T5 Prediction
Number
Detection
Language
Original: A mailman has to give out 192 pieces of junk mail. If he goes to 4 blocks, how
many pieces of junk mail should he give each block? Perturbed: A mailman has to give out one hundred and ninety-two pieces of junk mail. If
he goes to four blocks, how many pieces of junk mail should he give each block? Original: 192 / 4 ✓
Perturbed: 92 / 4 ×
Expected: 192 / 4
Type
Original: There were 105 parents in the program and 698 pupils, too. How many people
were present in the program? Perturbed: There were 105.0 parents in the program and 698.0 pupils, too. How many
people were present in the program? Original: 105 + 698 ✓
Perturbed: 105 + 688 ×
Expected: 105 + 698
Number Value
Understanding
Noise
Original: Tony had $20. He paid $8 for a ticket to a baseball game. At the game, he
bought a hot dog for $3. What amount of money did Tony have then? Perturbed: Tony had $20.2. He paid $8.5 for a ticket to a baseball game. At the game, he
bought a hot dog for $3.5. What amount of money did Tony have then? Original: 20 - 8 - 3 ✓
Perturbed: 208.52 - 3.5 ×
Expected: 20.2 - 8.5 - 3.5
Distribution
Original: Frank had $16. After buying some new toys he had $8 left.How much did he
spend on toys? Perturbed: Frank had $1281. 3https://spacy.io/ 4.1
Numerical Capabilities After buying some new toys he had $478 left.How much did
he spend on toys? Original: 16 - 8 ✓
Perturbed: 1215 - 878 ×
Expected: 1281 - 478
Operand
Selection
Extra
Original: John has twelve shirts. Later he bought four more shirts. How many shirts does
John have in total? Perturbed: John has twelve shirts. Later he bought four more shirts. Frank had $16. How
many shirts does John have in total? Original: 12 + 4 ✓
Perturbed: 16 + 12 ×
Expected: 12 + 4
Verbosity
Original: The roller coaster at the state fair costs 6 tickets per ride. If 8 friends were going
to ride the roller coaster, how many tickets would they need? Perturbed: The roller coaster at the state fair costs 6 (not 30) tickets per ride. If 8 (not 119)
friends were going to ride the roller coaster, how many tickets would they need? Original: 6 * 8 ✓
Perturbed: 8 * 119 ×
Expected: 6 * 8
Operation
Reasoning
Logic
Original: Jack received 8 emails in the morning and 2 emails in the afternoon. How many
emails did Jack receive in the day? Perturbed: Jack received 8 emails in the morning and 2 emails in the afternoon. How many
more emails did Jack receive in the morning than in the afternoon? Original: 8 + 2 ✓
Perturbed: 8 + 2 ×
Expected: 8 - 2
Order
Original: A DVD book holds 126 DVDs. There are 81 DVDs already in the book. How
many more DVDs can be put in the book? Perturbed: There are 81 DVDs already in the book. A DVD book holds 126 DVDs. How
many more DVDs can be put in the book? Original: 126 - 81 ✓
Perturbed: 81 - 126 ×
Expected: 126 - 81
Table 1: Examples of DNC Perturbations and Corresponding Predictions by T5. For each perturbation an example
original and perturbed problem pair is shown. The rightmost column shows some error cases where T5 generates
correct equation on the original problem but fails on the perturbed. The ground truth equation of the perturbed
problem is also provided after “Expected”. Table 1: Examples of DNC Perturbations and Corresponding Predictions by T5. For each perturbation an example
original and perturbed problem pair is shown. The rightmost column shows some error cases where T5 generates
correct equation on the original problem but fails on the perturbed. 4.1
Numerical Capabilities The ground truth equation of the perturbed
problem is also provided after “Expected”. 4.3
Perturbing Settings Systems. We selected representative systems
on each dataset and test their performance against
perturbations. For the ASDiv-a dataset, we use
Graph2Tree (Patel et al., 2021). For the DROP
dataset, we use BART-base and T5-base from Hug-
gingface.4
For the TATQA dataset, we utilize
TagOps with the RoBERTa backbone as described
in the original paper. With the aforementioned perturbations, we con-
struct perturbed datasets under different settings
to investigate systems’ numerical capabilities and
the effectiveness of the perturbations from different
perspectives. For a specific dataset with a training
/ validation / testing split, different splits are per-
turbed under different settings. In this paper we
consider the following two settings of Attack and
Defense, as compared in Table 2: Compute Environment. All experiments are
done on a Linux machine equipped with 4 NVIDIA
Tesla V100 16GB GPUs. The average runtime of
our experiments ranges from one to three hours. Compute Environment. All experiments are
done on a Linux machine equipped with 4 NVIDIA
Tesla V100 16GB GPUs. The average runtime of
our experiments ranges from one to three hours. Attack. By applying the perturbations to the
testing split of the dataset, we construct a challenge
set to evaluate the corresponding numerical capa-
bility of existing systems. Systems are trained on
the original datasets and evaluated on the perturbed
challenge set. Hyperparameters. In our experiments, we
adopt a general setting of hyperparameters of epoch
number = 40, learning rate = 1e −5 and batch size
= 32. It is observed in our exploratory experiments
that while the hyperparameters such as learning rate
and batchsize do affect the absolute performance
of the models, they have a modest effect on the gen-
eral trend of the models’ strengths and weaknesses
against the numerical perturbations. The details
and analysis are provided in Appendix C. Hyperparameters. In our experiments, we
adopt a general setting of hyperparameters of epoch
number = 40, learning rate = 1e −5 and batch size
= 32. It is observed in our exploratory experiments
that while the hyperparameters such as learning rate
and batchsize do affect the absolute performance
of the models, they have a modest effect on the gen-
eral trend of the models’ strengths and weaknesses
against the numerical perturbations. The details
and analysis are provided in Appendix C. Defense. Under the defense setting, perturba-
tions are applied to all of training, validation, and
testing split of the dataset. 5
Experiments Order Perturbation targets the Operation Rea-
soning capability and challenges systems to choose
correct operations for the same set of numbers. On
ASDiv-a, the order of sentences in the problem
body is manually altered in a manner that changes
the order of number occurrence but not the prob-
lem logic. This perturbation does not break the
operation distribution within the dataset. 4.3
Perturbing Settings By comparing sys-
tems’ performance under the Defense with Attack
settings, we investigate to what extent the perfor-
mance drop can be alleviated by using the pertur-
bations as a data augmentation approach. 4https://github.com/huggingface/transformers 5.1
Experiment Setup Datasets. In this paper, we used ASDiv-a (Miao
et al., 2020), DROP (Dua et al., 2019), and TATQA
(Zhu et al., 2021) as our Numerical Question An-
swering datasets. For DROP and TATQA, we fil-
tered out DROP-num and TATQA-a, the numerical
subsets of them. The statistics of these datasets are
shown in Table 4. of relevant numbers in the dataset. of relevant numbers in the dataset. digit fractional part with uniform distribution. This
perturbation introduces new float-point numbers
and breaks the original number value distribution
in the dataset by adding an random variable. Extra Perturbation targets the Operand Selec-
tion capability and challenges systems to exclude ir-
relevant numbers. To perturb a problem (B, P), An
irrelvant sentence containing numbers randomly
sampled from the corpus is added to the body B. This perturbation breaks the number distribution
by introducing extra instances of different numbers
for the same problem. Distribution Perturbation targets the Number
Value Understanding capability and challenges sys-
tems to conduct arithmetic with larger integers. To
perturb a number n, we randomly offset the value
with a normal distribution. Based on the observa-
tions in Wallace et al. (2019), we choose to perturb
the majority of the numbers to larger than 500. This
perturbation introduces large numbers and breaks
original number value distribution in the dataset. Logic Perturbation targets the Operation Rea-
soning capability and challenges systems to choose
correct operations for the same set of numbers. In this paper, for two datasets described in §5.1,
TATQA and ASDiv-a, the Operation perturbation
demands additional attention. On TATQA it is
based on template matching via SpaCy3 and auto-
matic conversions, while on ASDiv-a it is based
on manual annotation due to the diversity of pat-
terns in the ASDiv-a dataset. This perturbation Verbosity Perturbation targets the Operand Se-
lection capability and challenges systems to select
the correct quantity in the problem by adding ex-
plicitly irrelevant numbers into the problem. To
perturb a number string ns, we concatenate it with
an irrelevant number in parentheses, the irrelevant
number is preceded by “not”. This perturbation in-
troduces numbers without breaking the distribution 7954 introduces extra problems of different operations. introduces extra problems of different operations. 5.2
Experiment Results and Analysis The experiment results are provided in Table 3. The metric we report is 1) the metric on original
datasets (Original), and 2) the absolute change of
the metric on perturbed datasets, denoted by “∆”. We additionally provide the raw metric and relative
drop in Table 9 and Table 10 in the Appendix. The
calculation details of the observation can be found
in Appendix D.2. Setting
Attack
Defense
Train on
train
train⋆
Validate on
val
val⋆
Test on
test⋆
test⋆
Table 2: The Comparison between Two Settings in DNC. Perturbations (denoted by “⋆”) are applied to different
dataset splits (train / val / test) under each setting. Table 2: The Comparison between Two Settings in DNC. Perturbations (denoted by “⋆”) are applied to different
dataset splits (train / val / test) under each setting. Attack. As can be observed in Table 3 and Ta-
ble 10, most systems were severely challenged un-
der the Attack setting and experienced significant
performance drop, ranging from 5% to 50% abso-
lute drop and 5% to 80% relative drop in answer
denotation accuracy. Between the two DNC goals,
Semantic Parsing causes a more severe challenge,
averaging 19.66% absolute drop and 31.79% rela- To perturb under Attack or Defense setting, suit-
able samples are first filtered according to a series
of conditions. The perturbations are applied only
to these filtered samples. The filtered samples in
the dataset split(s) are replaced with their perturbed
version to form the perturbed dataset. The filter-
ing conditions and the formalized algorithm are
provided in Appendix B. 5.2
Experiment Results and Analysis The symbol “∆” stands for the absolute metric difference between the current
setting and the original setting. The color scale represents the distance from the original setting, deeper means
further from the original setting. For ASDiv-a, Acceq and Accans refer to the prediction accuracy of ground truth
equations and denotation accuracy of answers, respectively. For DROP-num and TATQA-a, Acc refers to the
denotation accuracy of the answers. We provide the raw performance and relative change of the metrics w.r.t. the
original setting in Appendix D.1. “-” denotes that automatic perturbation and automatic data augmentation as
described by §4.3 is not applicable here. We provide detailed explanation of the reason why they are not applicable
in Appendix E. Table 3: The Results of DNC Framework. Five NLP systems are evaluated with three Numerical QA tasks under
both Attack and Defense settings. The symbol “∆” stands for the absolute metric difference between the current
setting and the original setting. The color scale represents the distance from the original setting, deeper means
further from the original setting. For ASDiv-a, Acceq and Accans refer to the prediction accuracy of ground truth
equations and denotation accuracy of answers, respectively. For DROP-num and TATQA-a, Acc refers to the
denotation accuracy of the answers. We provide the raw performance and relative change of the metrics w.r.t. the
original setting in Appendix D.1. “-” denotes that automatic perturbation and automatic data augmentation as
described by §4.3 is not applicable here. We provide detailed explanation of the reason why they are not applicable
in Appendix E. Dataset
# Training
# Validation
# Testing
ASDiv-a
974
122
122
DROP-num
42258
5282
5283
TATQA-a
1971
245
247
Table 4: The Statistics of the Datasets Used. solute improvement and 26.95% relative improve-
ment vs. 6.52% absolute improvement and 11.42%
relative improvement). Among the considered systems, Transformer-
based Seq2Seq systems benefits more from De-
fense than the Graph2Tree system (12.53% abso-
lute improvement and 20.52% relative improve-
ment vs. 11.58% absolute improvement and
16.88% relative improvement). Table 4: The Statistics of the Datasets Used. Table 4: The Statistics of the Datasets Used. tive drop, as compared to the 13.15% absolute drop
and 19.66% relative drop by Numerical Parsing. Despite the recovery from Defense, the chal-
lenge is still not solved. As the majority of the
defense performance is still more than 10% below
the original performance. 5.2
Experiment Results and Analysis 7955 ASDiv-a
DROP-num
TATQA-a
Configuration
T5
BART
GPT2
Graph2Tree
T5
BART
TagOps
Setting
Perturbation
Acceq
Accans
Acceq
Accans
Acceq
Accans
Acceq
Accans
Acc
Acc
Acc
Language
-18.85%
-18.85%
-23.77%
-27.05%
-12.30%
-12.30%
-7.65%
-7.38%
-10.62%
-14.73%
-18.62%
Type
-37.70%
-11.48%
-32.79%
-15.57%
-17.21%
-10.66%
0.27%
1.09%
-7.70%
-11.06%
-5.34%
Noise
-36.89%
-36.89%
-18.85%
-21.31%
-9.84%
-9.02%
0.27%
0.55%
-
-
-
Distribution
-16.39%
-14.75%
-29.51%
-18.03%
-13.11%
-13.11%
-6.56%
-6.56%
-
-
-
Verbosity
-41.80%
-44.26%
-25.41%
-29.51%
-10.66%
-11.48%
-33.33%
-33.88%
-9.58%
-13.31%
-1.90%
Extra
-25.41%
-27.87%
-41.80%
-45.90%
-28.69%
-28.69%
-53.83%
-54.64%
-11.79%
-11.67%
-1.21%
Logic
-29.51%
-27.87%
-36.89%
-35.25%
-25.41%
-23.77%
-28.42%
-21.86%
-
-
-14.29%
Attack (∆)
Order
-34.43%
-5.74%
-33.61%
-4.10%
-27.87%
-7.38%
-33.33%
-7.10%
-
-
1.12%
Language
-12.30%
-13.93%
-19.67%
-24.59%
2.46%
2.46%
-7.65%
-7.38%
0.07%
-1.84%
-7.59%
Type
-11.48%
-12.30%
-4.92%
-6.56%
3.28%
4.10%
1.64%
1.91%
0.46%
-0.95%
2.93%
Noise
-14.75%
-14.75%
-3.28%
-4.92%
3.28%
4.10%
0.55%
0.27%
-
-
-
Distribution
-20.49%
-20.49%
-8.20%
-9.84%
-8.20%
-9.02%
-6.83%
-6.01%
-
-
-
Verbosity
-15.57%
-16.39%
-5.74%
-7.38%
-0.82%
0.00%
-0.27%
1.09%
-5.13%
-1.84%
2.25%
Extra
0.00%
1.64%
-2.46%
-4.10%
-17.21%
-18.03%
-20.22%
-17.76%
-11.32%
-10.44%
-9.14%
Logic
-
-
-
-
-
-
-
-
-
-
13.64%
Defense (∆)
Order
-25.41%
-4.10%
-27.87%
-7.38%
-1.64%
23.77%
-29.23%
-7.92%
-
-
19.47%
Original
None
68.03%
72.95%
67.21%
72.95%
44.26%
45.08%
66.94%
68.58%
49.42%
50.36%
42.41%
Table 3: The Results of DNC Framework. Five NLP systems are evaluated with three Numerical QA tasks under
both Attack and Defense settings. The symbol “∆” stands for the absolute metric difference between the current
setting and the original setting. The color scale represents the distance from the original setting, deeper means
further from the original setting. For ASDiv-a, Acceq and Accans refer to the prediction accuracy of ground truth
equations and denotation accuracy of answers, respectively. For DROP-num and TATQA-a, Acc refers to the
denotation accuracy of the answers. We provide the raw performance and relative change of the metrics w.r.t. the
original setting in Appendix D.1. “-” denotes that automatic perturbation and automatic data augmentation as
described by §4.3 is not applicable here. We provide detailed explanation of the reason why they are not applicable
in Appendix E. Table 3: The Results of DNC Framework. Five NLP systems are evaluated with three Numerical QA tasks under
both Attack and Defense settings. 6
Guidelines and Open Directions In this section, phenomena observed on different
systems and datasets were summarized to provide
comparison for existing methods. Also, recent re-
lated efforts corresponding to these phenomena
were discussed to point open directions in the do-
main of Numerical QA. Table 5: Comparing Models with Different Prediction
Targets on ASDiv-a. For a model M, Meq / Mans
predicts equation / equation and answer, respectively. Acceq and Accans stand for the denotation accuracy of
the generated equation and the accuracy of the directly
predicted answer, respectively. Table 5: Comparing Models with Different Prediction
Targets on ASDiv-a. For a model M, Meq / Mans
predicts equation / equation and answer, respectively. Acceq and Accans stand for the denotation accuracy of
the generated equation and the accuracy of the directly
predicted answer, respectively. p
g
Targets on ASDiv-a. For a model M, Meq / Mans
predicts equation / equation and answer, respectively. Acceq and Accans stand for the denotation accuracy of
the generated equation and the accuracy of the directly
predicted answer, respectively. 5.2
Experiment Results and Analysis This observation indi-
cates that the lack of Numerical Capabilities is still
an open question. Among the considered systems, Transformer-
based Seq2Seq systems (T5, BART, GPT2) are
more sensitive than the tasks-specific Graph2Tree
system against the perturbations stemming from
the Numerical Parsing goal. The former resulted
in 17.42% absolute drop and 27.06% relative drop,
while Graph2Tree only experienced 3.07% abso-
lute drop and 4.48% relative drop. The masking of
numbers used by Graph2Tree allows it to remain
unaffected against a portion of the perturbations
targeting the Numerical Parsing goal. Summary. Our DNC framework provides in-
sights on two major aspects of the diagnosis to
Numerical QA systems: 1) It is demonstrated that severe numerical weak-
nesses exist in current Numerical QA systems (“At-
tack”), and they can not be trivially eliminated via,
although benefiting from, an automatic data aug-
mentation process (“Defense”). 1) It is demonstrated that severe numerical weak-
nesses exist in current Numerical QA systems (“At-
tack”), and they can not be trivially eliminated via,
although benefiting from, an automatic data aug-
mentation process (“Defense”). Defense. As a counteracting approach, the de-
fense mechanism helps alleviate systems’ lack of
corresponding numerical capabilities by applying
automatic perturbations to the training and valida-
tion set. Via Defense, the lack according to the Se-
mantic Parsing gets more recovery of (17.96% ab- Defense. As a counteracting approach, the de-
fense mechanism helps alleviate systems’ lack of
corresponding numerical capabilities by applying
automatic perturbations to the training and valida-
tion set. Via Defense, the lack according to the Se-
mantic Parsing gets more recovery of (17.96% ab- 2) The systems’ weaknesses are explicitly pro-
filed in a quantitative and interpretable manner
through the models’ susceptibility difference to 7956 Model
Accans
Acceq
GPT2ans
6.56%
-
GPT2eq
45.08%
44.26%
BARTans
9.02%
-
BARTeq
72.95%
67.21%
T5ans
2.46%
-
T5eq
72.95%
68.03%
Table 5: Comparing Models with Different Prediction
Targets on ASDiv-a. For a model M, Meq / Mans
predicts equation / equation and answer, respectively. Acceq and Accans stand for the denotation accuracy of
the generated equation and the accuracy of the directly
predicted answer, respectively. Model
Accans
Acceq
GPT2ans
6.56%
-
GPT2eq
45.08%
44.26%
BARTans
9.02%
-
BARTeq
72.95%
67.21%
T5ans
2.46%
-
T5eq
72.95%
68.03% a diversity of perturbations. 6.1
Target: Logical Form Generation vs.
Answer Predicting One attribute specific to Numerical QA is the rea-
soning processes leading to the numerical answers,
which is usually described by logical forms. On
datasets where the ground truth logical forms are
provided as an additional supervision (e.g., ASDiv-
a and TATQA), the systems have two options for
the target: 1) Logical Form Generation, where
systems generate the logical form which is later in-
put to external symbolic executing systems such as
Python scripts or SQL engines, and 2) Answer Pre-
dicting, where systems directly predict the output
answer in an end-to-end manner. On datasets where
ground truth logical forms are not provided (e.g.,
DROP), the latter is the most frequently adopted
approach. Logical Form Generation and Answer
Predicting differ in the actual object to conduct the
executing step of the logical form insinuated by
the question (external symbolic systems vs. neural
systems). With Answer Predicting, systems are
expected to possess the capability of executing the
logical forms internally. been leveraging data synthesizing and intermedi-
ate pretraining to learn neural program executors
and achieved state-of-the-art results over systems
leveraging Logical Form Generation. This recent
development shows the potential of neural systems
with enhanced execution capability on the Numeri-
cal QA task. been leveraging data synthesizing and intermedi-
ate pretraining to learn neural program executors
and achieved state-of-the-art results over systems
leveraging Logical Form Generation. This recent
development shows the potential of neural systems
with enhanced execution capability on the Numeri-
cal QA task. 6.2
Numbers: Tokenization vs. Replacement We also investigate the impact of different ways of
manipulating numbers. There are two mainstream
existing methods to process and represent num-
bers, herein referred to as the Tokenization and
Replacement methods. Tokenization methods such as WordPiece (Wu
et al., 2016) and BPE (Sennrich et al., 2016)
adopted by existing Numerical QA systems divides
numbers into potentially multiple sub-word level
tokens. E.g., The number 768 will be divided into
tokens 7 and 68 by T5’s tokenizer. This approach
stems from the fundamental fact that existing sys-
tems’ vocabularies are finite while the occurrences
of numbers in a Numerical QA dataset can be too
diverse to include in a finite vocabulary. Tokeniza-
tion causes extra representation cost and erases the
digit integrity by potentially introducing multiple
tokens for a single number. We investigate to what extent do existing sys-
tems possess this execution capability, by compar-
ing the impact of the problem target T in Numer-
ical QA on ASDiv-a. The systems are trained to
predict two different targets: 1) the logical form
(i.e., the MWP equation), and 2) the logical form
and the execution result. Since most MWP-specific
systems are incapable of predicting answers di-
rectly, we choose the Transformer-based systems
GPT2, BART and T5. Results in Table 5 indicate
that: 1) on existing systems, Logical Form Genera-
tion is beneficial for higher accuracy, and 2) even
though models managed to compose equations with
high accuracy, they struggle to faithfully execute
an equation to get the correct answer. Replacement substitutes numbers with special
tokens in the input ([NUM1], [NUM2], etc.), which
are later re-substituted with the original number
in the output logical forms. This approach avoids
multiple tokens by providing exactly one represen-
tation for each number, but has its own limitations
handling number diversity since the recognition
of numbers are usually performed with rule-based
matching, which is often non-exhaustive. Recent work also pays increasing attention to
the execution capability. 7
Conclusion In this paper we aim at diagnosing numerical ca-
pabilities in existing NLP systems. We list out a
series of numerical capabilities and design corre-
sponding dataset perturbations. Empirical results
show that existing systems still lack numerical ca-
pabilities to a large extent, and this lack cannot
be eliminated in a trivial manner. Analysis into
the empirical results, discussion of the the existing
practices, and insights for future directions of Nu-
merical QA dataset collection and system design
are also provided. Limitations Table 6: The Results of Tokenization and Replacement
on GPT2. GPT2token adopts the Tokenization method
and GPT2replace adopts the Replacement method. Table 6: The Results of Tokenization and Replacement
on GPT2. GPT2token adopts the Tokenization method
and GPT2replace adopts the Replacement method. Our pipeline has limitations in the following two
aspects that we plan to address in the future: Dependency on ground truth equation. Cur-
rently, three of the eight DNC perturbations have
strong dependency on the ground truth solving
equation, which is missing in datasets such as
DROP. We hope to utilize semi-supervised ap-
proaches in the future to enlarge the coverage of
the DNC perturbations. placement. We implement two variations of GPT2:
GPT2token and GPT2replace to compare their ro-
bustness against different perturbations on the
ASDiv-a dataset.Results in Table 6 indicate that Re-
placement has an advantage when no perturbation
is present or when the perturbation only involves
changes in number value. However, when the per-
turbation changes number values, the Replacement-
based system is more severely challenged. Perturbing scalability. Currently our filters
cover only a portion of the whole dataset due to
DNC filtering and perturbing questions based on
manual rules and templates. we hope to develop
more automatic filtering and perturbing in the fu-
ture. Also, DNC can only apply perturbations to
numbers provided by the problem, which limits its
diagnosing power in questions where an unspeci-
fied number is used, e.g., when numerical common-
sense knowledge is involved. We hypothesize that the Replacement method re-
moves all numerical information such as the format
and value of numbers in the problem and lost nu-
meracy capabilities, therefore the system receives
only textual signals such as number order or word
frequency, which further encouraged systems to
learn from spurious correlations as stated in Patel
et al. (2021). This hypothesis is consistent with
the observations of a recent study (Thawani et al.,
2021a) that investigates of the mutual-enhancement
between numeracy and literacy. 6.2
Numbers: Tokenization vs. Replacement Systems such as TAPEX
(Liu et al., 2022) and POET (Pi et al., 2022a) have In this paper, T5, BART, GPT2 and TagOps
adopts Tokenization, while Graph2Tree adopts Re- 7957 Model
Perturbation
Acceq
Accans
∆Acceq
∆Accans
Language
31.97%
32.79%
-12.30%
-12.30%
Type
27.05%
34.43%
-17.21%
-10.66%
Noise
34.43%
36.07%
-9.84%
-9.02%
Distribution
31.15%
31.97%
-13.11%
-13.11%
Verbosity
33.61%
33.61%
-10.66%
-11.48%
Extra
15.57%
16.39%
-28.69%
-28.69%
Logic
18.85%
21.31%
-25.41%
-23.77%
Order
16.39%
37.70%
-27.87%
-7.38%
GPT2token
Original
44.26%
45.08%
0.00%
0.00%
Language
46.72%
47.54%
-9.84%
-9.84%
Type
56.56%
57.38%
0.00%
0.00%
Noise
56.56%
57.38%
0.00%
0.00%
Distribution
56.56%
57.38%
0.00%
0.00%
Verbosity
22.13%
22.13%
-34.43%
-35.25%
Extra
10.66%
11.48%
-45.90%
-45.90%
Logic
32.79%
40.16%
-23.77%
-17.21%
Order
18.03%
36.07%
-38.52%
-21.31%
GPT2replace
Original
56.56%
57.38%
0.00%
0.00%
Table 6: The Results of Tokenization and Replacement
on GPT2. GPT2token adopts the Tokenization method
and GPT2replace adopts the Replacement method. Model
Perturbation
Acceq
Accans
∆Acceq
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Copenhagen, Denmark. Association for Computa-
tional Linguistics. 7961 containing numbers from the corpus is added to the
body B, i.e., B
Details of the Perturbing Process Language Perturbation. The concerned num-
ber string ns is replaced by the English word de-
scribing the same quantity, i.e., A
Formal Definition of Perturbations We provide the formalized definition of the pertur-
bations as follows. In all definitions, “⋆” denotes
perturbed version. P∆= SampleOtherQs()
P⋆= P ⊕P∆
B⋆= B Noise Perturbation. To apply noise perturba-
tion to an number n, an variable X is uniformly
sampled on the interval (1, 10). Then a fractional
part corresponding to X is added the concerned
number n, i.e., Logic Perturbation To apply the Logic Pertur-
bation to a problem (B, P), the prompt is altered
to convert the problem logic used in the problem,
i.e., X ∼U(1, 10)
n⋆= n + 0.1 × ⌊X⌋ X ∼U(1, 10) P⋆= ConvertLogic(P)
B⋆= B n⋆= n + 0.1 × ⌊X⌋ Distribution Perturbation. The Distribution
Perturbation changes the number distribution in the
dataset by adding an normally distributed random
variable X to the concerned number n. I.e., Order Perturbation For the Order Perturbation,
the sentence order in the problem body is manu-
ally altered in a manner that changes the order of
number occurrence but not the problem logic, i.e., X ∼N(µd, δ2
d)
n⋆= n + ⌊X⌋ P⋆= ChangeOrder(P) P⋆= ChangeOrder(P)
B⋆= B In this paper we adopt µd = 1000 and δd = 300. B.1
The Filtering Conditions The filtering conditions for Perturbing Algorithms
is different across perturbations. The perturbations
can be divided into two major categories: 1) pertur-
bations that do not change the solving equation or
final results (Language, Type, Verbosity, Extra, Or-
der), and 2) perturbations that changes the solving
equation or final results (Noise, Distri, Operation). n⋆
s = Num2Words(ns) Type Perturbation. To apply the Type Pertur-
bation, the concerned number is expected to be an
integral number. The number string ns is concate-
nated with an extra “.0” string to change the type of
the concerned number from integer to float-point,
i.e., For perturbations in category 1), there is no limi-
tation on the perturbing process, thus all questions
naturally pass the the filtering condition. For perturbations in category 2), the filtering
conditions follow the principles of Unambiguity,
Suitability and Visibility. B.2
The Formalized Perturbing Process B.2
The Formalized Perturbing Process B.2
The Formalized Perturbing Process
Algorithm 1: The Perturbing Process
Data: D = {Dtrain, Dval, Dtest}
AllPert = {Noise, ...} as in §4.2
AllSet ∈{Attack, ...} as in §4.3
Filter = {Integral, ...} as in §B
Result: D⋆= {D⋆
train, D⋆
val, D⋆
test}
/* Decide perturbations to use
*/
Perturbs ←SelectBy(AllPert, D);
/* Create perturbed dataset for
each perturbation and setting */
for setting ∈AllSet do
/* Decide split to perturb
*/
D∆, Dremain ←
SelectBy(D, setting);
for perturb ∈Perturbs do
D⋆
∆←{};
for d ∈D∆do
if Filter(d) then
D⋆
∆←D⋆
∆+ perturb(d);
end
else
D⋆
∆←D⋆
∆+ d;
end
end
D⋆
perturb = {D⋆
∆, Dremain}
end
end
D⋆= {D⋆
perturb}| perturb ∈Perturbs B.2
The Formalized Perturbing Process
Algorithm 1: The Perturbing Process
Data: D = {Dtrain, Dval, Dtest}
AllPert = {Noise, ...} as in §4.2
AllSet ∈{Attack, ...} as in §4.3
Filter = {Integral, ...} as in §B
Result: D⋆= {D⋆
train, D⋆
val, D⋆
test}
/* Decide perturbations to use
*/
Perturbs ←SelectBy(AllPert, D);
/* Create perturbed dataset for
each perturbation and setting */
for setting ∈AllSet do
/* Decide split to perturb
*/
D∆, Dremain ←
SelectBy(D, setting);
for perturb ∈Perturbs do
D⋆
∆←{};
for d ∈D∆do
if Filter(d) then
D⋆
∆←D⋆
∆+ perturb(d);
end
else
D⋆
∆←D⋆
∆+ d;
end
end
D⋆
perturb = {D⋆
∆, Dremain}
end
end
D⋆= {D⋆
perturb}| perturb ∈Perturbs B.2
The Formalized Perturbing Process
Algorithm 1: The Perturbing Process
Data: D = {Dtrain, Dval, Dtest}
AllPert = {Noise, ...} as in §4.2
AllSet ∈{Attack, ...} as in §4.3
Filter = {Integral, ...} as in §B
Result: D⋆= {D⋆
train, D⋆
val, D⋆
test}
/* Decide perturbations to use
*/
Perturbs ←SelectBy(AllPert, D);
/* Create perturbed dataset for
each perturbation and setting */
for setting ∈AllSet do
/* Decide split to perturb
*/
D∆, Dremain ←
SelectBy(D, setting);
for perturb ∈Perturbs do
D⋆
∆←{};
for d ∈D∆do
if Filter(d) then
D⋆
∆←D⋆
∆+ perturb(d);
end
else
D⋆
∆←D⋆
∆+ d;
end
end
D⋆
perturb = {D⋆
∆, Dremain}
end
end
D⋆= {D⋆
perturb}| perturb ∈Perturbs Perturbation
Acceq
lr=1e-2
Acceq
batchsize=128
Default
Language
-4.10%
-9.84%
-7.65%
Type
3.28%
3.28%
0.27%
Noise
4.10%
1.64%
0.27%
Distribution
-4.92%
-10.66%
-6.56%
Verbosity
-33.61%
-38.52%
-33.33%
Extra
-44.26%
-57.38%
-53.83%
Logic
-18.03%
-27.87%
-28.42%
Order
-33.61%
-13.11%
-33.33%
Original
62.30%
71.31%
66.94%
Table 7: The results of the attack Acceq for Graph2Tree
on ASDiv-a Table 7: The results of the attack Acceq for Graph2Tree
on ASDiv-a Similar behavior can also be observed on large
transformer-based model such as T5, as shown in
Table 8: Perturbation
Acceq
lr=1e-4
Acceq
batchsize=16
Default
Language
-17.21%
-17.21%
-18.85%
Type
-20.49%
-32.79%
-37.70%
Noise
-23.77%
-32.79%
-36.89%
Distribution
-18.03%
-13.11%
-16.39%
Verbosity
-39.34%
-38.52%
-41.80%
Extra
-40.16%
-21.31%
-25.41%
Logic
-27.05%
-28.69%
-29.51%
Order
-31.15%
-33.61%
-34.43%
Original
61.48%
63.11%
68.03%
Table 8: The results of the attack Acceq for T5 on
ASDiv-a Table 8: The results of the attack Acceq for T5 on
ASDiv-a n⋆
s = Concat(ns, Stringfy(.0)) Verbosity Perturbation. The Verbosity Pertur-
bation aims to introduce irrelevant numbers without
changing the semantics of the problem. To perturb
a number string ns, we concatenate it with an irrel-
evant number in parentheses, the irrelevant number
is preceded by "not", i.e., Unambiguity. The filtered question should have
an unambiguous mapping between the number to
be perturbed and the their location in the context. One example that violates this principle is when
there are duplicated numbers in the problem body,
then it cannot be determined which occurrence of
the number affects the final result. X ∼N(µv, δ2
v) i
Suitability. The number to be perturbed should
be suitable for the perturbation to be conducted. E.g. A float-point number should not be used as the
target of the Noise perturbation which adds frac-
tional part to integral numbers. In DNC, the Noise
and Type perturbations requires the concerned num-
ber to be integral, and the Operation perturbation n⋆
s = Concat(ns, (not, Stringfy(X))) n⋆
s = Concat(ns, (not, Stringfy(X))) In this paper we adopt µv = 100 and δv = 30. Extra Perturbation To apply the Extra Pertur-
bation to a problem (B, P), an irrelevant sentence 7962 requires the question to match a manually created
template. 32 for batch size), the following results are ob-
served: On Graph2Tree, the results of changing the learn-
ing rate and batch size are shown in Table 7, the
trend of results with the varied hyperparameters
align with the default result as shown in Table 3. Visibility. The concerned number should be
occur in the the problem since the perturbations
can only be applied to known input numbers. E
DNC Experiments that Are Not
Applicable The following types of experiments are not appli-
cable in current DNC framework: E.1
The Defense of the Logic perturbation on
ASDiv-a The Logic perturbation requires the problem to be
perturbed in a way that the logic is changed while
the semantics of the problem is still cohesive. This
requirement proposes challenge on the scalability
of the perturbation. For the Attack setting, we
utilized manually annotated labels. However, under
the Defense setting the perturbations are expected
to automatically augment the dataset. Thus, the
Defense setting results of Logic perturbation on
ASDiv-a is not applicable. D.2
Observation Calculation Details We denote the experiment results table in Table 11. The values, observation explanation, and the for-
mula used are provided in Table 12. D.1
Raw Performance And Relative
Performance Drop We provide the original result in Table 9 and the
relative performance drop in Table 10. E.2
Noise and Type perturbations on
DROP-num and TATQA-a DROP-num and TATQA-a do not provide supervi-
sion of the operand origins, therefore a mapping
from the operands in equation to the context quan-
tities cannot be built, which results in the Noise
and Distribution perturbation not applicable on the
DROP-num and TATQA-a datasets. E.3
Logic Perturbation on DROP-num DROP-num does not provide ground truth reason-
ing steps or logical forms, thus Logic perturbations
that has dependency on the provided supervision is
not applicable on DROP-num. C
Hyperparameters In our exploratory experiment, it it observed that
while the hyperparameters such as learning rate
and batchsize do affect the absolute performance
of the models, they have a modest effect on the gen-
eral trend of the models’ strengths and weaknesses
against the numerical perturbations. We hypothe-
size that this is due to the numerical capabilities
of a model being contributed mostly by the model
architecture instead of hyperparameters. Considering this observation, and the fact that
the number of our experiment is large due to the
combination of different models, datasets, DNC
settings, and DNC perturbations, we chose one
general setting to reduce search space. We chose
the setting as close as possible to the reported set-
ting in the original papers of Graph2Tree and T5. We verified that this setting provides sufficiently
good performance to demonstrate the performance
gap corresponding to the perturbations, since our
experiment focused more on the performance of a For example, when the hyperparameters are vary-
ing from the default setting (1e-5 for learning rate, 7963 the semantic of the paragraph and will also confuse
humans. Thus Order perturbation is not valid on
DROP-num and the results are not applicable. the semantic of the paragraph and will also confuse
humans. Thus Order perturbation is not valid on
DROP-num and the results are not applicable. same model checkpoint against the datasets before
and after the perturbations. E.4
Order Perturbation on DROP-num DROP-num is a reasonding dataset based on real-
world paragraphs that usually have logical or tem-
poral order information. Order perturbation breaks 7964 Configuration
ASDiv-a
DROP-num
TATQA-a
T5
BART
GPT2
Graph2Tree
T5
BART
TagOps
Setting
Perturbation
Acceq
Accans
Acceq
Accans
Acceq
Accans
Acceq
Accans
Acc
Acc
Acc
Attack
Language
49.18%
54.10%
43.44%
45.90%
31.97%
32.79%
59.29%
61.20%
38.80%
35.62%
23.79%
Type
30.33%
61.48%
34.43%
57.38%
27.05%
34.43%
67.21%
69.67%
41.72%
39.30%
37.07%
Noise
31.15%
36.07%
48.36%
51.64%
34.43%
36.07%
67.21%
69.13%
-
-
-
Distribution
51.64%
58.20%
37.70%
54.92%
31.15%
31.97%
60.38%
62.02%
-
-
-
Verbosity
26.23%
28.69%
41.80%
43.44%
33.61%
33.61%
33.61%
34.70%
39.84%
37.04%
40.52%
Extra
42.62%
45.08%
25.41%
27.05%
15.57%
16.39%
13.11%
13.93%
37.63%
38.69%
41.21%
Logic
38.52%
45.08%
30.33%
37.70%
18.85%
21.31%
38.52%
46.72%
-
-
28.12%
Order
33.61%
67.21%
33.61%
68.85%
16.39%
37.70%
33.61%
61.48%
-
-
43.53%
Defense
Language
55.74%
59.02%
47.54%
48.36%
46.72%
47.54%
59.29%
61.20%
49.49%
48.52%
34.83%
Type
56.56%
60.66%
62.30%
66.39%
47.54%
49.18%
68.58%
70.49%
49.88%
49.41%
45.34%
Noise
53.28%
58.20%
63.93%
68.03%
47.54%
49.18%
67.49%
68.85%
-
-
-
Distribution
47.54%
52.46%
59.02%
63.11%
36.07%
36.07%
60.11%
62.57%
-
-
-
Verbosity
52.46%
56.56%
61.48%
65.57%
43.44%
45.08%
66.67%
69.67%
44.29%
48.52%
44.67%
Extra
68.03%
74.59%
64.75%
68.85%
27.05%
27.05%
46.72%
50.82%
38.10%
39.92%
33.28%
Logic
-
-
-
-
-
-
-
-
-
-
56.05%
Order
42.62%
68.85%
39.34%
65.57%
42.62%
68.85%
37.70%
60.66%
-
-
61.89%
Original
None
68.03%
72.95%
67.21%
72.95%
44.26%
45.08%
66.94%
68.58%
49.42%
50.36%
42.41%
Table 9: The raw results of the DNC Framework. E.4
Order Perturbation on DROP-num Notations here follow the ones in the main experiment results ASDiv-a
DROP-num
TATQA-a
Configuration
T5
BART
GPT2
Graph2Tree
T5
BART
TagOps
Setting
Perturbation
Acceq
Accans
Acceq
Accans
Acceq
Accans
Acceq
Accans
Acc
Acc
Acc
Language
-27.71%
-25.84%
-35.37%
-37.08%
-27.78%
-27.27%
-11.43%
-10.76%
-21.49%
-29.26%
-43.90%
Type
-55.42%
-15.73%
-48.78%
-21.35%
-38.89%
-23.64%
0.41%
1.59%
-15.59%
-21.96%
-12.60%
Noise
-54.22%
-50.56%
-28.05%
-29.21%
-22.22%
-20.00%
0.41%
0.80%
-
-
-
Distribution
-24.10%
-20.22%
-43.90%
-24.72%
-29.63%
-29.09%
-9.80%
-9.56%
-
-
-
Verbosity
-61.45%
-60.67%
-37.80%
-40.45%
-24.07%
-25.45%
-49.80%
-49.40%
-19.38%
-26.44%
-4.47%
Extra
-37.35%
-38.20%
-62.20%
-62.92%
-64.81%
-63.64%
-80.41%
-79.68%
-23.86%
-23.17%
-2.85%
Logic
-43.37%
-38.20%
-54.88%
-48.31%
-57.41%
-52.73%
-42.45%
-31.87%
-
-
-33.69%
Attack (∆%)
Order
-50.60%
-7.87%
-50.00%
-5.62%
-62.96%
-16.36%
-49.80%
-10.36%
-
-
2.64%
Language
-18.07%
-19.10%
-29.27%
-33.71%
5.56%
5.45%
-11.43%
-10.76%
0.14%
-3.65%
-17.89%
Type
-16.87%
-16.85%
-7.32%
-8.99%
7.41%
9.09%
2.45%
2.79%
0.93%
-1.88%
6.91%
Noise
-21.69%
-20.22%
-4.88%
-6.74%
7.41%
9.09%
0.82%
0.40%
-
-
-
Distribution
-30.12%
-28.09%
-12.20%
-13.48%
-18.52%
-20.00%
-10.20%
-8.76%
-
-
-
Verbosity
-22.89%
-22.47%
-8.54%
-10.11%
-1.85%
0.00%
-0.41%
1.59%
-10.38%
-3.65%
5.31%
Extra
0.00%
2.25%
-3.66%
-5.62%
-38.89%
-40.00%
-30.20%
-25.90%
-22.90%
-20.72%
-21.54%
Logic
-
-
-
-
-
-
-
-
-
-
32.16%
Defense (∆%)
Order
-37.35%
-5.62%
-41.46%
-10.11%
-3.70%
52.73%
-43.67%
-11.55%
-
-
45.92%
Original
None
68.03%
72.95%
67.21%
72.95%
44.26%
45.08%
66.94%
68.58%
49.42%
50.36%
42.41%
Table 10: The relative drop results of the DNC Framework. Notations here follow the ones in the main experiment
results Table 10: The relative drop results of the DNC Framework. E.4
Order Perturbation on DROP-num Notations here follow the ones in the main experiment
results 7965 A
B
C
D
E
F
G
H
I
J
K
ASDiv-a
DROP-num
TATQA-a
Configuration
T5
BART
GPT2
Graph2Tree
T5
BART
TagOps
Setting
Perturbation
Acceq
Accans
Acceq
Accans
Acceq
Accans
Acceq
Accans
Acc
Acc
Acc
1
Language
-18.85%
-18.85%
-23.77%
-27.05%
-12.30%
-12.30%
-7.65%
-7.38%
-10.62%
-14.73%
-18.62%
2
Type
-37.70%
-11.48%
-32.79%
-15.57%
-17.21%
-10.66%
0.27%
1.09%
-7.70%
-11.06%
-5.34%
3
Noise
-36.89%
-36.89%
-18.85%
-21.31%
-9.84%
-9.02%
0.27%
0.55%
-
-
-
4
Distribution
-16.39%
-14.75%
-29.51%
-18.03%
-13.11%
-13.11%
-6.56%
-6.56%
-
-
-
5
Verbosity
-41.80%
-44.26%
-25.41%
-29.51%
-10.66%
-11.48%
-33.33%
-33.88%
-9.58%
-13.31%
-1.90%
6
Extra
-25.41%
-27.87%
-41.80%
-45.90%
-28.69%
-28.69%
-53.83%
-54.64%
-11.79%
-11.67%
-1.21%
7
Logic
-29.51%
-27.87%
-36.89%
-35.25%
-25.41%
-23.77%
-28.42%
-21.86%
-
-
-14.29%
8
Attack (∆)
Order
-34.43%
-5.74%
-33.61%
-4.10%
-27.87%
-7.38%
-33.33%
-7.10%
-
-
1.12%
9
Language
-12.30%
-13.93%
-19.67%
-24.59%
2.46%
2.46%
-7.65%
-7.38%
0.07%
-1.84%
-7.59%
10
Type
-11.48%
-12.30%
-4.92%
-6.56%
3.28%
4.10%
1.64%
1.91%
0.46%
-0.95%
2.93%
11
Noise
-14.75%
-14.75%
-3.28%
-4.92%
3.28%
4.10%
0.55%
0.27%
-
-
-
12
Distribution
-20.49%
-20.49%
-8.20%
-9.84%
-8.20%
-9.02%
-6.83%
-6.01%
-
-
-
13
Verbosity
-15.57%
-16.39%
-5.74%
-7.38%
-0.82%
0.00%
-0.27%
1.09%
-5.13%
-1.84%
2.25%
14
Extra
0.00%
1.64%
-2.46%
-4.10%
-17.21%
-18.03%
-20.22%
-17.76%
-11.32%
-10.44%
-9.14%
15
Logic
-
-
-
-
-
-
-
-
-
-
13.64%
16
Defense (∆)
Order
-25.41%
-4.10%
-27.87%
-7.38%
-1.64%
23.77%
-29.23%
-7.92%
-
-
19.47%
Original
None
68.03%
72.95%
67.21%
72.95%
44.26%
45.08%
66.94%
68.58%
49.42%
50.36%
42.41%
Table 11: The main experiment result table with cell coordinates Value
Explanation
Formula
19.66%
Average performance drop of all models
caused by perturbations according to the
Semantic Parsing stage
AVERAGE(B5:B8,D5:D8,F5:F8,H5:H8,I5:I8,J5:J8,K5:K8)
13.15%
Average performance drop of all models
caused by perturbations according to the
Numerical Parsing stage
AVERAGE(B1:B4,D1:D4,F1:F4,H1:H4,I1:I4,J1:J4,K1:K4)
17.42%
Average performance drop of all Transformer-
based models caused by perturbations according
to the Semantic Parsing stage
AVERAGE(B5:B8,D5:D8,F5:F8,I5:I8,J5:J8,K5:K8)
3.07%
Average performance drop of the Graph2Tree
system caused by perturbations according to
the Semantic Parsing stage
AVERAGE(B1:B4,D1:D4,F1:F4,I1:I4,J1:J4,K1:K4)
17.96%
Average performance recovery of all models
caused by perturbations according to the
Semantic Parsing stage
AVERAGE(B13:B16,D13:D16,F13:F16,H13:H16,I13:I16,J13:J16,K13:K16)
6.52%
Average performance recovery of all models
caused by perturbations according to the
Numerical Parsing stage
AVERAGE(B9:B12,D9:D12,F9:F12,H9:H12,I9:I12,J9:J12,K9:K12)
12.53%
Average performance recovery of all Transformer-
based models caused by perturbations according
to the Semantic Parsing stage
AVERAGE(B13:B16,D13:D16,F13:F16,I13:I16,J13:J16,K13:K16)
11.58%
Average performance recovery of the Graph2Tree
system caused by perturbations according to the
Semantic Parsing stage
AVERAGE(B9:B12,D9:D12,F9:F12,I9:I12,J9:J12,K9:K12)
Table 12: The Value, Explanation, And Formula Used of The Experiment Observations. E.4
Order Perturbation on DROP-num Formula 7966
|
https://openalex.org/W4392471793
|
https://www.nature.com/articles/s43018-024-00740-1.pdf
|
English
| null |
A deep learning model of tumor cell architecture elucidates response and resistance to CDK4/6 inhibitors
|
Nature cancer
| 2,024
|
cc-by
| 22,009
|
A deep learning model of tumor cell
architecture elucidates response and
resistance to CDK4/6 inhibitors Sungjoon Park
1,6, Erica Silva2,6, Akshat Singhal
3,6, Marcus R. Kelly1,4,
Kate Licon1, Isabella Panagiotou1, Catalina Fogg1, Samson Fong5, John J. Y. Lee1,
Xiaoyu Zhao1, Robin Bachelder
1, Barbara A. Parker1,4, Kay T. Yeung1,4 &
Trey Ideker
1,3,4,5 Cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6is) have revolutionized
breast cancer therapy. However, <50% of patients have an objective
response, and nearly all patients develop resistance during therapy. To elucidate the underlying mechanisms, we constructed an interpretable
deep learning model of the response to palbociclib, a CDK4/6i, based on a
reference map of multiprotein assemblies in cancer. The model identifies
eight core assemblies that integrate rare and common alterations across
90 genes to stratify palbociclib-sensitive versus palbociclib-resistant cell
lines. Predictions translate to patients and patient-derived xenografts,
whereas single-gene biomarkers do not. Most predictive assemblies can
be shown by CRISPR–Cas9 genetic disruption to regulate the CDK4/6i
response. Validated assemblies relate to cell-cycle control, growth factor
signaling and a histone regulatory complex that we show promotes S-phase
entry through the activation of the histone modifiers KAT6A and TBL1XR1
and the transcription factor RUNX1. This study enables an integrated
assessment of how a tumor’s genetic profile modulates CDK4/6i resistance. treatment outcomes, increasing progression-free and overall sur-
vival with manageable adverse effects4,5. However, objective tumor
response is observed in <50% of patients who receive CDK4/6 inhibitors
as first-line therapy, and nearly all initially responsive patients develop
drug resistance with subsequent mortality6,7. Cell-cycle activation and sustained proliferation are hallmarks of can-
cer1. Cyclin-dependent kinases 4 and 6 (CDK4/6) trigger cells to pass
the G1/S cell-cycle restriction point by phosphorylating the retinoblas-
toma (RB) transcriptional repressor and its paralogs. Inhibiting these
kinases has been of high interest in cancer drug development2,3. Thus
far, three CDK4/6 inhibitors (CDK4/6is) (palbociclib, ribociclib and abe-
maciclib) have been approved in combination with endocrine therapy
for the treatment of hormone receptor-positive, human epidermal
growth factor (EGF) receptor 2-negative (HR+, HER2−) breast cancer,
and clinical trials are underway in a spectrum of other tissue types4. In metastatic breast cancer, these agents have appreciably improved treatment outcomes, increasing progression-free and overall sur-
vival with manageable adverse effects4,5. However, objective tumor
response is observed in <50% of patients who receive CDK4/6 inhibitors
as first-line therapy, and nearly all initially responsive patients develop
drug resistance with subsequent mortality6,7. Article https://doi.org/10.1038/s43018-024-00740-1 1Department of Medicine, University of California, San Diego, La Jolla, CA, USA. 2Program in Biomedical Sciences, University of California, San Diego,
La Jolla, CA, USA. 3Department of Computer Science and Engineering, University of California, San Diego, La Jolla, CA, USA. 4Moores Cancer Center,
University of California, San Diego, San Diego, CA, USA. 5Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA.
6These authors contributed equally: Sungjoon Park, Erica Silva, Akshat Singhal.
e-mail: tideker@health.ucsd.edu nature cancer nature cancer Evaluation of prediction performance We constructed NeST-VNN drug response models for palbociclib and
separately for each of the 50 benchmark drugs, using standard neural
network learning procedures based on backpropagation (Methods). Each model was trained to use the gene alteration profile of a cell line to
predict the corresponding area under the dose–response curve (AUC). Training and performance assessment was conducted using nested five-
fold cross-validation (Methods), with each fold setting aside 64% of cell
lines for training, 16% for validation (used for tuning hyperparameters)
and 20% for testing. Although nested cross-validation is computation-
ally intensive, it fully insulates model testing from parameter tuning
while maximizing the amount of testing that can be performed. We com-
pared the NeST-VNN approach to three state-of-the-art alternatives:
ElasticNet, random forest (RF) and a conventional black-box artificial
neural network (ANN) (Extended Data Fig. 2a,b and Supplementary
Table 1). The overall performance of NeST-VNN was generally compa-
rable to that of the state-of-art models and often better, with NeST-VNN
achieving the best performance for more than half of the tested drugs
(62.7%; Extended Data Fig. 2a,b). NeST-VNN trained for palbociclib
was one of the top-performing models, significantly outperforming
the ElasticNet and ANN models and slightly, but not significantly, out-
performing RF (Extended Data Fig. 2c and Supplementary Table 1). Thus far, VNN models have been structured using Gene Ontol-
ogy17 or Reactome18, two general human expert-curated databases of
known cellular components and functions that have not been explicitly
designed to capture the molecular pathways of cancer. To define and
discover cancer mechanisms systematically, we recently developed
a hierarchical map of multiprotein assemblies called NeST (Nested
Systems in Tumors)19. To build this map, we used affinity purification–
mass spectrometry (AP–MS) to interrogate the physical interactions
of a broad set of frequently altered cancer proteins. These data were
integrated with other systematic omics datasets to create a large can-
cer protein–protein association network. Structural analysis of this
network revealed a hierarchy of protein assemblies in which small,
specific complexes nest within larger communities corresponding to
broad processes and organelles. NeST was defined as the final hierarchy
of 395 assemblies found to be under significant selection pressure for
somatic mutations in one or more adult tumor types (Fig. 1a)19. Beyond
the identification of the mutated protein assemblies, NeST has not yet
been used to inform drug response models. Article bank of artificial neurons, reflecting its biological state or ‘in silico
activity’ (Extended Data Fig. 1a). Connections were also established
between the neurons of an assembly and those of larger assemblies
that contain it (Extended Data Fig. 1b), allowing for the flow of genetic
information from small focal assemblies (for example, ‘CDK holoen-
zyme complex’) to larger-scale assemblies and superassemblies (for
example, ‘cell cycle’). The final protein assembly at the root of the hier-
archy represented the model output—the predicted drug response
of a tumor sample given the input set of genetic alterations (Fig. 1b). assessments obtained primarily through retrospective analyses4,5. RB1
mutation bears the strongest burden of evidence, as it has been exten-
sively associated with CDK4/6 drug resistance in cell lines and patient
cohorts8. However, it and other markers have met with inconsistent
results in prospective clinical trials8, suggesting that our understand-
ing of this drug response is still incomplete. assessments obtained primarily through retrospective analyses4,5. RB1
mutation bears the strongest burden of evidence, as it has been exten-
sively associated with CDK4/6 drug resistance in cell lines and patient
cohorts8. However, it and other markers have met with inconsistent
results in prospective clinical trials8, suggesting that our understand-
ing of this drug response is still incomplete. Deep learning is a powerful general methodology in precision
medicine, including the use of molecular profiles to predict drug
responses9. Such models are typically trained to maximize the accuracy
of outcome prediction (for example, whether a patient will respond to
a drug) without attempting to reveal the internal cellular and molecular
mechanisms by which that outcome is achieved. In this regard, it is
notoriously difficult to interpret which molecular features are relevant
and even more difficult to describe how these features integrate with
one another in the logic of molecular pathways10. To create models that
are both predictive and interpretable11,12, we and others have advanced
a series of ‘visible’ neural network (VNN) architectures13–16 that are
guided by knowledge maps of cellular components and functions. For
example, using such a model, Elmarakeby et al. found that metastatic
outcomes in prostate cancer were well predicted by convergent genetic
alterations within a mouse double minute (MDM)–tumor protein 53
(TP53) inhibition pathway, implicating MDM4 in resistance to antian-
drogen therapy15. Evaluation of prediction performance To translate predictions to discrete tumor response outcomes,
we thresholded the AUC such that predictions below a value tlow were
labeled ‘sensitive’, those above a value thigh were labeled ‘resistant’ and
those between these two thresholds were labeled ‘undefined’ (Fig. 2a). At the most inclusive setting, tlow = thigh = median(AUC), NeST-VNN
could accurately discriminate between actual sensitive and resistant
cell lines in heldout test data, with a diagnostic odds ratio (OR) of 6.0. Discriminative power increased substantially with more stringent
thresholds. For instance, setting the thresholds 1 s.d. from the median
(tlow, thigh = median(AUC) ± s.d.(AUC)) yielded a very high OR of 40.1,
indicating that samples predicted as resistant were approximately 40
times more likely to test as resistant than samples predicted as sensi-
tive (Fig. 2b). The trade-off for higher accuracy was that samples were
left undefined (66%), increasing specificity but decreasing sensitivity
(Extended Data Fig. 2d). Here, we use this experimentally derived NeST map as the founda-
tion for a visible deep learning approach to understand how patterns
of genetic alterations govern the tumor response to CDK4/6 inhibi-
tion. This model is functionally predictive of palbociclib treatment
outcomes and can be structurally interpreted, revealing a focal set
of protein assemblies on which common and rare cancer mutations
converge to affect drug resistance or sensitivity (Fig. 1b). Article To train NeST-VNN, we leveraged drug response data for 1,244
genomically characterized tumor cell lines23, obtained by harmonizing
the Cancer Therapeutics Response Portal (CTRP)24,25 and Genomics of
Drug Sensitivity in Cancer (GDSC)26,27 databases (Methods). These data
included the response to the CDK4/6i palbociclib, which had been well
characterized in 947 cell lines. For comparative benchmarking, we also
examined 50 non-CDK-related drugs investigated in at least 200 cell
lines, for which the cellular responses displayed sufficient variability,
with many examples of sensitivity and resistance (Methods). Implementation of a cancer-oriented VNN We defined a set of 718 genes assessed by one or more clinical cancer
gene panels and studies, including the FoundationOne CDx panel20,
Tempus xT21 and Project GENIE (Genomics Evidence Neoplasia Infor-
mation Exchange)22. We then queried NeST to extract a hierarchy of
131 protein assemblies containing proteins encoded by the clinically
assessed genes. This hierarchy was used to guide the architecture of
a VNN following a previously described approach13 (Fig. 1b, Extended
Data Fig. 1a and Methods). This model of cancer cell structure and
response, which we call NeST-VNN, allowed for three binary input fea-
tures per gene, describing the presence or absence of point mutation/
insertion/deletion, copy number amplification (CNA) or copy number
deletion (CND) (Fig. 1b and Methods). These gene-level input features
were integrated within their respective protein assemblies in subse-
quent layers of the NeST-VNN, with each assembly represented by a A deep learning model of tumor cell
architecture elucidates response and
resistance to CDK4/6 inhibitors Studies of resistance to these drugs have largely defined two
groups of molecular biomarkers: loss-of-function alterations to anti-
proliferative CDK pathway genes (for example, CDKN2A/B/C or RB1) or
gain-of-function alterations to progrowth genes (for example, CDK2,
CDK4/6, CCND1, CCNE1, E2F or PIK3CA). These markers have been char-
acterized predominantly in preclinical in vitro studies, with clinical 1Department of Medicine, University of California, San Diego, La Jolla, CA, USA. 2Program in Biomedical Sciences, University of California, San Diego,
La Jolla, CA, USA. 3Department of Computer Science and Engineering, University of California, San Diego, La Jolla, CA, USA. 4Moores Cancer Center,
University of California, San Diego, San Diego, CA, USA. 5Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA. 6These authors contributed equally: Sungjoon Park, Erica Silva, Akshat Singhal. e-mail: tideker@health.ucsd.edu Nature Cancer | Volume 5 | July 2024 | 996–1009 996 https://doi.org/10.1038/s43018-024-00740-1 Article Nature Cancer | Volume 5 | July 2024 | 996–1009 Interpreting the model to identify important protein
assemblies tumor volume) than those predicted to be resistant, suggesting
that predicted sensitivity was associated with impaired tumor
growth (log-rank P = 0.04, hazard ratio 0.53, 95% confidence interval
0.30–0.97; Fig. 2c and Methods). Having seen that the NeST-VNN model was predictive of drug response
in tumor cell lines, PDX samples and patients, we sought to interpret
which protein assemblies were important to this process. Following a
previous method13, we computed a quantitative importance score for
each assembly according to how well its in silico activity was associated
with the final drug response prediction (Fig. 3a, Supplementary Table 2
and Methods). Assemblies containing the primary CDK4 and CDK6
drug targets were of significantly higher importance than expected
by chance, serving as positive controls (P = 5 × 10–5; Fig. 3a and Sup-
plementary Table 2). For example, one of the important CDK assem-
blies was NeST:110 (CDK holoenzyme complex I; Fig. 3b,c), comprising
the cyclin D–CDK4–CDK6 complex along with upstream inhibitors
(CDKN1/2 protein families) and downstream targets (RB1). Positive
control assemblies were also observed for other top-performing drug
models; the model for the drug nutlin-3a, which targets TP53 activity
through MDM2, placed high importance on assemblies containing
these proteins (P = 6.8 × 10–10; Extended Data Figs. 2a and 3a). After this analysis in xenografts, we evaluated model performance
in predicting treatment outcomes for 226 patients with breast cancer
from the GENIE metastatic breast cancer cohort22. These patients
had been treated with baseline endocrine therapy with (n = 67) or
without (n = 159) a CDK4/6i. Each patient was classified as ‘sensitive’
or ‘resistant’ using the threshold tlow = thigh = median(AUC) (no ‘unde-
fined’ category was used because the number of treated samples was
less than that for the earlier PDX or cell line analysis). The resistant
category was further equally split to denote ‘partially resistant’ and
‘strongly resistant’ subgroups. For patients treated with a CDK4/6i,
those predicted to be sensitive had significantly longer survival than
those predicted to be strongly resistant (log-rank P = 0.02, hazard
ratio 0.21, 95% confidence interval 0.05–0.91; Fig. 2d). Moreover, for
the strongly resistant subgroup, the addition of the CDK4/6i failed to
produce a significant increase in overall survival compared to base-
line therapy (P = 0.37). https://doi.org/10.1038/s43018-024-00740-1 Predicted drug response
Sensitive
Resistant
Tumor cell lines
Patient tumors
PDX tumors
or
or
Mutations
CNAs
CNDs
Deep neural network
in NeST architecture
Assembly
activity
Neuron active
Neuron inactive
Model
explanation
a
Afinity purification
of cancer proteins
Other published
interactions
3× FLAG
Bait
Cancer mutations (13 tumor types)
Multiscale analysis
of protein communities
Systematic proteomics datasets
b
NeST
Hierarchy of 395 commonly mutated assemblies
Fig. 1 | Architecture and features of the visible deep learning model. a, Workflow depicting the construction of the NeST hierarchy of cancer
protein assemblies by Zheng et al.19. AP–MS data for 61 cancer protein baits
were combined with a compendium of other systematic proteomics and omics
datasets to produce an integrated protein network. This network was analyzed
by multiscale community detection to identify a hierarchy of nested protein
assemblies. Those assemblies under mutational selection pressure in different
tumor types were then identified, yielding the NeST map. b, VNN architecture
for translating tumor genetic alterations (top) to drug responses (bottom)
by genetic flow through the NeST map (middle). NeST is reduced to the 131
assemblies that involve genes measured by clinical gene panels (see text). Predicted drug response
Sensitive
Resistant
Tumor cell lines
Patient tumors
PDX tumors
or
or
Mutations
CNAs
CNDs
Deep neural network
in NeST architecture
Assembly
activity
Neuron active
Neuron inactive
Model
explanation
b
d l
bli
Th
bli
d
t ti
l
l
ti
i diff
t a
Afinity purification
of cancer proteins
Other published
interactions
3× FLAG
Bait
Cancer mutations (13 tumor types)
Multiscale analysis
of protein communities
Systematic proteomics datasets
NeST
Hierarchy of 395 commonly mutated assemblies Predicted drug response
Sensitive
Resistant
Tumor cell lines
Patient tumors
PDX tumors
or
or
utations
CNAs
CNDs
Deep neural network
in NeST architecture
ly
Model
explanation
assemblies. Those assemblies under mutational selection pressure in different
tumor types were then identified, yielding the NeST map. b, VNN architecture
for translating tumor genetic alterations (top) to drug responses (bottom)
by genetic flow through the NeST map (middle). NeST is reduced to the 131
assemblies that involve genes measured by clinical gene panels (see text). https://doi.org/10.1038/s43018-024-00740-1 b a PDX tumors Patient tumors Systematic proteomics datasets Tumor cell lines or Multiscale analysis
of protein communities Assembly
activity Deep neural network
in NeST architecture NeST
Hierarchy of 395 commonly mutated assemblies Predicted drug response
Sensitive
Resistant
Model
explanation Sensitive NeST NeST
hy of 395 commonly muta NeST
Hierarchy of 395 commonly mutated assemblies NeST
Hierarchy of 395 commonly mutated assemblies Model
explanation NeST
Hierarchy of 395 commonly mutated assemblies Fig. 1 | Architecture and features of the visible deep learning model. assemblies. Those assemblies under mutational selection pressure in different
tumor types were then identified, yielding the NeST map. b, VNN architecture
for translating tumor genetic alterations (top) to drug responses (bottom)
by genetic flow through the NeST map (middle). NeST is reduced to the 131
assemblies that involve genes measured by clinical gene panels (see text). itecture and features of the visible deep learning model. a, Workflow depicting the construction of the NeST hierarchy of cancer
protein assemblies by Zheng et al.19. AP–MS data for 61 cancer protein baits
were combined with a compendium of other systematic proteomics and omics
datasets to produce an integrated protein network. This network was analyzed
by multiscale community detection to identify a hierarchy of nested protein a, Workflow depicting the construction of the NeST hierarchy of cancer
protein assemblies by Zheng et al.19. AP–MS data for 61 cancer protein baits
were combined with a compendium of other systematic proteomics and omics
datasets to produce an integrated protein network. This network was analyzed
by multiscale community detection to identify a hierarchy of nested protein Translation to patient-derived xenografts and patients Next, we examined the performance of NeST-VNN in a study of
patient-derived xenografts (PDXs)28, including n = 172 samples
treated with a CDK4/6i (ribociclib). Each PDX sample was classified
as sensitive, resistant or undefined using thresholds at 1 s.d. from
the median (tlow, thigh = median(AUC) ± s.d.(AUC)). PDX samples pre-
dicted to be sensitive exhibited significantly longer progression-free
survival (duration from the start of treatment to the doubling of Nature Cancer | Volume 5 | July 2024 | 996–1009 997 https://doi.org/10.1038/s43018-024-00740-1 Article Interpreting the model to identify important protein
assemblies These predictions outperformed single-gene
markers of palbociclib resistance29 (RB1 mutation) or sensitivity30,31
(CCND1 CNA) that had been previously suggested, consistent with
the mixed results of these markers in clinical trials8 (Extended Data
Fig. 2e,f). In patients who did not receive a CDK4/6i, no significant
survival differences were observed among the predicted sensitive/
resistant/strongly resistant class labels (all comparisons with log-rank
P > 0.1; Fig. 2d). These results indicate that the NeST-VNN palbociclib
model translates to the population of patients with breast cancer and
is specifically predictive of response rather than generally prognostic
of patient survival. For all drug models, assembly importance tended to increase with
size and depth in the hierarchy, reflecting the progressive integration
of genetic information. Assembly importance was similar between cell
lines and patient tumors (Fig. 3d) or PDX samples (Fig. 3e). In contrast,
little correlation was observed between cell lines and clinical samples
when examining the importance of individual gene mutations (Fig. 3f)
or copy number aberrations (Extended Data Fig. 3b,c). These results
are consistent with the premise that most individual genetic altera-
tions are rare, with variable incidence across contexts32, and suggest
that the effects of genetic alterations on protein assemblies can be
substantially more stable. Nature Cancer | Volume 5 | July 2024 | 996–1009 998 https://doi.org/10.1038/s43018-024-00740-1 Article 0
20
40
60
80
100
120
Overall survival (months)
0
20
40
60
80
100
120
Overall survival (months)
0
0.2
0.4
0.6
0.8
1.0
Surviving fraction
0
0.2
0.4
0.6
0.8
1.0
Surviving fraction
Hazard
ratio 0.21*
Sensitive (n = 22)
Resistant (n = 33)
Hazard
ratio 0.53*
Sensitive
Partially
resistant
n = 34
n = 16
n = 17
Prediction
Strongly
resistant
n = 80
n = 39
n = 40
Endocrine therapy
without CDK4/6i
Endocrine therapy
+ CDK4/6i
Prediction
0.2
0.4
0.6
0.8
1.0
0
Fraction of progression-free tumors
0
20
40
60
80
100
120
Time after treatment (days)
a
b
Survival
AUC
Dose
Sensitivity
Resistance
Predicted AUC
Undefined
Frequency
tlow
thigh
c
d
Cell lines (n = 161)
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
True AUC
OR 40.1
Predicted sensitive
Predicted resistant
True sensitive
cell lines
True resistant
cell lines
Fig. 2 | Predictive performance of the palbociclib model. a, Scanning cell
survival to measure a continuous AUC that is thresholded to assign class labels
(sensitive, undefined or resistant). Interpreting the model to identify important protein
assemblies Colors denote class labels for predicted
CDK4/6i-sensitive (blue) and CDK4/6i-resistant (yellow/orange) patients, with
additional stratification of a strongly resistant category (orange). Patients not
treated with CDK4/6i therapy are shown in gray. The hazard ratio was 0.21 for
strongly resistant versus sensitive predicted subgroups. *P < 0.05 by log-
rank test. survival to measure a continuous AUC that is thresholded to assign class labels
(sensitive, undefined or resistant). b, Waterfall plot showing the true dose
responses of tumor cell lines, with colors indicating the predicted class of
each. Predicted AUC is thresholded to produce class labels (sensitive, resistant;
see text). c, Survival curve analysis for predicting the sensitive or resistant
status of PDX samples. *P < 0.05 by log-rank test. d, Survival curve analysis for Of 33 assemblies that were of high importance for palbociclib
response prediction in cell lines (importance ≥ 0.5), we focused on eight
distinct minimally overlapping assemblies whose importance scores
remained significant under multiple-hypothesis correction (hereafter
referred to as ‘core assemblies’; Methods). Beyond regulation of CDK
activity, core assemblies spanned histone and chromatin regulation,
DNA damage response and growth factor signaling (Fig. 3a), integrat-
ing rare and common genetic alterations across 90 genes (Extended
Data Fig. 3d). Most core assemblies were also important for predicting
outcomes in clinical and PDX samples (Fig. 3d,e). KOs in a diversity of genes, including roughly a dozen with extreme
loss-of-fitness phenotypes (Fig. 4c; for example, BCL6, CCND3, CDK4,
CDK5, RAD51C, TOP2A, BARD, AURKA, AURKB) and several causing gain
of fitness (for example, BRCA2, CTNNB1, CDKN2B, MSH6, MLH3). Enrich-
ment was not observed for a genome-wide KO screen without palbo-
ciclib treatment34, indicating that at least some of the effect was due
to gene–drug interactions rather than independent gene essentiality
(Methods and Fig. 4d). We then moved on to our de novo dual CRISPR KO
screen and noted that this screen and the earlier chemogenetic screen
were reasonably consistent with respect to gene KO fitness effects
(Pearson ρ = 0.48; Fig. 4e). Disruptions in all six of the core assem-
blies with sufficient coverage in our gene KO panel displayed a trend
toward increased cell fitness (Fig. 4f,g and Supplementary Table 3). Taken together, these results indicate that engineered genetic disrup-
tions in protein assemblies identified by NeST-VNN can influence tumor
cell growth in the setting of CDK4/6 inhibition, whether such inhibition
is induced by a drug (Fig. Systematic validation of core assemblies by loss-of-function
screens We next sought to validate the palbociclib core assemblies using two
CRISPR (clustered regularly interspaced short palindromic repeat)
loss-of-function screens (Fig. 4a): a published chemogenetic screen
involving genome-wide knockout (KO) of single genes in combination
with palbociclib treatment33 and a de novo dual CRISPR screen in which
we paired gene KOs in selected NeST-VNN assemblies with a second gene
KO targeting CDK4 or CDK6 (Fig. 4a and Methods). For the chemoge-
netic screen, we assessed each assembly in NeST for the enrichment
of genes whose KOs modulate cell fitness in the context of palbociclib
treatment (Methods). The enrichments of the eight core assemblies
tended to be significantly higher than those of nonimportant controls
(P = 0.005, Mann–Whitney U test), with four of these assemblies in
particular (regulation of CDK activity, histone-mediated transcription
regulation, DNA damage response, promyelocytic leukemia (PML)
body) showing stronger effects than any assembly in the control set
(Fig. 4b and Supplementary Table 3). Such enrichment was due to Interpreting the model to identify important protein
assemblies 4b) or CDK4/6 KO (Fig. 4f,g). Interpreting the model to identify important protein
assemblies b, Waterfall plot showing the true dose
responses of tumor cell lines, with colors indicating the predicted class of
each. Predicted AUC is thresholded to produce class labels (sensitive, resistant;
see text). c, Survival curve analysis for predicting the sensitive or resistant
status of PDX samples. *P < 0.05 by log-rank test. d, Survival curve analysis for
GENIE clinical trial patients treated with CDK4/6i plus endocrine therapy (left)
or endocrine therapy alone (right). Colors denote class labels for predicted
CDK4/6i-sensitive (blue) and CDK4/6i-resistant (yellow/orange) patients, with
additional stratification of a strongly resistant category (orange). Patients not
treated with CDK4/6i therapy are shown in gray. The hazard ratio was 0.21 for
strongly resistant versus sensitive predicted subgroups. *P < 0.05 by log-
rank test. a
b
Survival
AUC
Dose
Sensitivity
Resistance
Predicted AUC
Undefined
Frequency
tlow
thigh
Cell lines (n = 161)
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
True AUC
OR 40.1
Predicted sensitive
Predicted resistant
True sensitive
cell lines
True resistant
cell lines b a Sensitive (n = 22)
Resistant (n = 33)
Hazard
ratio 0.53*
Prediction
0.2
0.4
0.6
0.8
1.0
0
Fraction of progression-free tumors
0
20
40
60
80
100
120
Time after treatment (days)
c d c Time after treatment (days) Overall survival (months) Fig. 2 | Predictive performance of the palbociclib model. a, Scanning cell
survival to measure a continuous AUC that is thresholded to assign class labels
(sensitive, undefined or resistant). b, Waterfall plot showing the true dose
responses of tumor cell lines, with colors indicating the predicted class of
each. Predicted AUC is thresholded to produce class labels (sensitive, resistant;
see text). c, Survival curve analysis for predicting the sensitive or resistant
status of PDX samples. *P < 0.05 by log-rank test. d, Survival curve analysis for Fig. 2 | Predictive performance of the palbociclib model. a, Scanning cell GENIE clinical trial patients treated with CDK4/6i plus endocrine therapy (left)
or endocrine therapy alone (right). Colors denote class labels for predicted
CDK4/6i-sensitive (blue) and CDK4/6i-resistant (yellow/orange) patients, with
additional stratification of a strongly resistant category (orange). Patients not
treated with CDK4/6i therapy are shown in gray. The hazard ratio was 0.21 for
strongly resistant versus sensitive predicted subgroups. *P < 0.05 by log-
rank test. GENIE clinical trial patients treated with CDK4/6i plus endocrine therapy (left)
or endocrine therapy alone (right). https://doi.org/10.1038/s43018-024-00740-1 c, Diagram of known functional associations among NeST:110 proteins in the context of cell-cycle progr associations among NeST:110 proteins in the context of cell-cycle progression. The cyclin D–CDK complex inhibits RB1 by phosphorylation, such that it
no longer transcriptionally represses genes required for S-phase entry and
subsequent DNA replication. d, Scatterplot of assembly importance in the
clinical versus cell line contexts (x axis versus y axis). e, Scatterplot of assembly
importance in the PDX versus cell line contexts (x axis versus y axis). f, Scatterplot
of gene mutation importance in the clinical versus cell line contexts (x axis
versus y axis). DREAM, dimerization partner, RB-like, E2F and multivulval class
B; MAPK, mitogen-activated protein kinase; RTK, receptor tyrosine kinase; Reg.,
regulation; tx, transcription; med., mediated; stim., stimulation. associations among NeST:110 proteins in the context of cell-cycle progression. The cyclin D–CDK complex inhibits RB1 by phosphorylation, such that it
no longer transcriptionally represses genes required for S-phase entry and
subsequent DNA replication. d, Scatterplot of assembly importance in the
clinical versus cell line contexts (x axis versus y axis). e, Scatterplot of assembly
importance in the PDX versus cell line contexts (x axis versus y axis). f, Scatterplot
of gene mutation importance in the clinical versus cell line contexts (x axis
versus y axis). DREAM, dimerization partner, RB-like, E2F and multivulval class
B; MAPK, mitogen-activated protein kinase; RTK, receptor tyrosine kinase; Reg.,
regulation; tx, transcription; med., mediated; stim., stimulation. Table 3). However, most of the frequent genetic alterations affecting
this assembly in tumor cell lines or patients were not loss-of-function
events but gene CNAs (Fig. 5b), which were especially prevalent in
lung, oropharyngeal and gynecologic tumors (frequencies 15–35%;
Extended Data Fig. 4a). CNAs also accounted for the top five genetic
alterations in this assembly that were most predictive of palbociclib
resistance, in particular those of MYC, TERT, KAT6A, TBL1XR1 and RUNX1
(Fig. 5c and Methods). Each of these amplifications had a resistance
OR of approximately 2.0, indicating that cells harboring CNAs are twice as likely to exhibit resistance to palbociclib than cells without
CNAs (Fig. 5c). Motivated by these findings, we turned to the technique of CRISPR
activation (CRISPRa), which uses the dCas9 (dead Cas9 endonuclease)–
VPR (VP64–p65–Rta) transcriptional activator to increase expression
from gene promoters targeted with CRISPR single guide RNAs (Fig. 5d). Exploration of gain-of-function alterations in a histone
transcriptional assembly An open question is how CDK4/6 and the G1/S transcriptional program
interact with other cell functions, including upstream modulators and
downstream effectors. A notable assembly in this regard was NeST:85
(histone-mediated transcription regulation), a densely connected
complex of 15 proteins with roles in histone acetylation, deacetylation
and transcriptional activation (Fig. 5a). This assembly was important
for the CDK4/6i response in cell lines (Fig. 3a), PDX samples (Fig. 3e
and Extended Data Fig. 4b) and patients (Fig. 3d). It had also been vali-
dated by CRISPR loss-of-function analysis (Fig. 4b and Supplementary Nature Cancer | Volume 5 | July 2024 | 996–1009 999 icle
https://doi.org/10.1038/s43018-024-0
CDKN1A
CDKN2A
CDKN1A
CDKN2A
−0.02
0
0.02
0.04
0.06
Importance of gene feature
(mutations, clinical samples)
−0.02
0
0.02
0.04
0.06
Importance of gene feature
(mutations, cell lines)
−0.04
−0.04
ρ = 0.09
d
e
f
Assembly part
of additional
assembly
Assembly part
of larger
assembly
Importance
Importance > 0.5
Core
Importance > 0.5
FDR < 0.1
Distinct assembly
Nonimportant
5 proteins
60
20
700
Assembly size
Contains
CDK4 or CDK6
a
Histone-med. tx regulation
Cell
Nucleus
Signaling pathways
Chromosome
organization
Tx of cell-
cycle genes
Cyclin D–CDK complex
Cyclin E–CDK
complex
DNA replication
Cell-cycle progression
CDKN2C
CKS1B
CCND1
CCND2
CCND3
CCNE1
CCNE2
CDK4
CDK6
CDKN1B
CDKN2B
RB1
TP53
Phosphorylates
Inhibits activity of
Promotes activity of
CDKN2C
CKS1B
CCND1
CCND2
CCND3
CCNE1
CCNE2
CDK4
CDK6
CDKN1B
CDKN2B
RB1
TP53
Cyclin D holoenzyme complex I
b
c
Weak
Strong
Protein–protein association
Reg. of E2F tx
by DREAM
Cytoskeleton
CDK
holoenzyme
complex II
CDK
holoenzyme
complex I
mRNA
processing
Cell-cycle
arrest
Cell motility
Cyclin D-
associated
events in G1
Cytoplasm and
extracellular space
Regulation
of CDK
activity
Reg. of
transcription
AKT/activin
signaling
Adherens
junction
Transmembrane
RTK signaling
MAPK
signaling
EGF/FGF
stim. of cell
proliferation
Immune system
Cell cycle
PML
body
Ribo-
nucleoprotein
complexes
MDM2–p53
pathway
DNA damage
response
AR
signaling
Core assemblies (8)
Other assemblies (123)
ρ = 0.79
Importance of assembly
(PDX)
Cyclin D-
associated
events in G1
Regulation of
CDK activity
DNA damage
response
Cell-cycle
arrest
EGF/FGF stim. of cell proliferation
Histone-med. Exploration of gain-of-function alterations in a histone
transcriptional assembly tx regulation
AR
signaling
PML
body
Importance of assembly
(clinical samples)
0
0.2
0.4
0.6
0.8
1.0
0
0.2
0.4
0.6
0.8
1.0
Importance of assembly
(cell lines)
Importance of assembly
(cell lines)
ρ = 0.68
0
0.2
0.4
0.6
0.8
1.0
0
0.2
0.4
0.6
0.8
1.0
Regulation of
CDK activity
DNA damage
response
Cell-cycle
arrest
EGF/FGF stim. of cell proliferation
Histone-med. tx regulation
AR
signaling
PML
body
Cyclin D-
associated
events in G1
Genes (718)
3 | Interpretation of palbociclib response mechanisms. a, NeST-VNN
rpretation of the palbociclib response. Nodes indicate assemblies, whereas
e sizes indicate assembly sizes in numbers of proteins. Colors indicate
degree of importance for response prediction: yellow, assemblies with
ortance > 0.5; red, ‘core’ assemblies, which bring the additional requirement
DR ≤ 0.1 and exclude redundant assemblies of lesser importance (Jaccard
larity > 0.5). Assemblies containing CDK4 or CDK6 are marked with small
k squares. b, Protein interaction network defining the CDK holoenzyme
plex I (NeST:110), which contains CDK4 and CDK6. Edges represent
hysical protein–protein associations, with the edge thickness reflecting
trength of the evidence for association. c, Diagram of known functional
associations among NeST:110 proteins in the context of cell-cycle progres
The cyclin D–CDK complex inhibits RB1 by phosphorylation, such that it
no longer transcriptionally represses genes required for S-phase entry an
subsequent DNA replication. d, Scatterplot of assembly importance in th
clinical versus cell line contexts (x axis versus y axis). e, Scatterplot of asse
importance in the PDX versus cell line contexts (x axis versus y axis). f, Sca
of gene mutation importance in the clinical versus cell line contexts (x axi
versus y axis). DREAM, dimerization partner, RB-like, E2F and multivulval
B; MAPK, mitogen-activated protein kinase; RTK, receptor tyrosine kinas
regulation; tx, transcription; med., mediated; stim., stimulation. https://doi.org/10.1038/s43018-024-00740-1 https://doi.org/10.1038/s43018-024-00740-1 tx regulation
AR
signaling
PML
body
0
0.2
0.4
0.6
0.8
1.0
Importance of assembly
(cell lines)
0
0.2
0.4
0.6
0.8
1.0 f e 0.02
0
0.02
0.04
0.0
Importance of gene feature
(mutations, clinical samples)
0.04 Importance of assembly
(clinical samples) p
(clinical samples) Fig. 3 | Interpretation of palbociclib response mechanisms. a, NeST-VNN
interpretation of the palbociclib response. Nodes indicate assemblies, whereas
node sizes indicate assembly sizes in numbers of proteins. Colors indicate
the degree of importance for response prediction: yellow, assemblies with
importance > 0.5; red, ‘core’ assemblies, which bring the additional requirement
of FDR ≤ 0.1 and exclude redundant assemblies of lesser importance (Jaccard
similarity > 0.5). Assemblies containing CDK4 or CDK6 are marked with small
black squares. b, Protein interaction network defining the CDK holoenzyme
complex I (NeST:110), which contains CDK4 and CDK6. Edges represent
biophysical protein–protein associations, with the edge thickness reflecting
the strength of the evidence for association. c, Diagram of known functional Fig. 3 | Interpretation of palbociclib response mechanisms. a, NeST-VNN Fig. 3 | Interpretation of palbociclib response mechanisms. a, NeST-VNN
interpretation of the palbociclib response. Nodes indicate assemblies, whereas
node sizes indicate assembly sizes in numbers of proteins. Colors indicate
the degree of importance for response prediction: yellow, assemblies with
importance > 0.5; red, ‘core’ assemblies, which bring the additional requirement
of FDR ≤ 0.1 and exclude redundant assemblies of lesser importance (Jaccard
similarity > 0.5). Assemblies containing CDK4 or CDK6 are marked with small
black squares. b, Protein interaction network defining the CDK holoenzyme
complex I (NeST:110), which contains CDK4 and CDK6. Edges represent
biophysical protein–protein associations, with the edge thickness reflecting
the strength of the evidence for association. c, Diagram of known functional Fig. 3 | Interpretation of palbociclib response mechanisms. a, NeST-VNN
interpretation of the palbociclib response. Nodes indicate assemblies, whereas
node sizes indicate assembly sizes in numbers of proteins. Colors indicate
the degree of importance for response prediction: yellow, assemblies with
importance > 0.5; red, ‘core’ assemblies, which bring the additional requirement
of FDR ≤ 0.1 and exclude redundant assemblies of lesser importance (Jaccard
similarity > 0.5). Assemblies containing CDK4 or CDK6 are marked with small
black squares. b, Protein interaction network defining the CDK holoenzyme
complex I (NeST:110), which contains CDK4 and CDK6. Edges represent
biophysical protein–protein associations, with the edge thickness reflecting
the strength of the evidence for association. https://doi.org/10.1038/s43018-024-00740-1 Article Assembly part
of additional
assembly
Assembly part
of larger
assembly
Importance
Importance > 0.5
Core
Importance > 0.5
FDR < 0.1
Distinct assembly
Nonimportant
5 proteins
60
20
700
Assembly size
Contains
CDK4 or CDK6
a
Histone-med. tx regulation
Cell
Nucleus
Signaling pathways
Chromosome
organization
Tx of cell-
cycle genes
Reg. of E2F tx
by DREAM
Cytoskeleton
CDK
holoenzyme
complex II
CDK
holoenzyme
complex I
mRNA
processing
Cell-cycle
arrest
Cell motility
Cyclin D-
associated
events in G1
Cytoplasm and
extracellular space
Regulation
of CDK
activity
Reg. of
transcription
AKT/activin
signaling
Adherens
junction
Transmembrane
RTK signaling
MAPK
signaling
EGF/FGF
stim. of cell
proliferation
Immune system
Cell cycle
PML
body
Ribo-
nucleoprotein
complexes
MDM2–p53
pathway
DNA damage
response
AR
signaling a CDKN1A
CDKN2A
CDKN1A
CDKN2A
Cyclin D–CDK complex
Cyclin E–CDK
complex
DNA replication
Cell-cycle progression
CDKN2C
CKS1B
CCND1
CCND2
CCND3
CCNE1
CCNE2
CDK4
CDK6
CDKN1B
CDKN2B
RB1
TP53
Phosphorylates
Inhibits activity of
Promotes activity of
CDKN2C
CKS1B
CCND1
CCND2
CCND3
CCNE1
CCNE2
CDK4
CDK6
CDKN1B
CDKN2B
RB1
TP53
Cyclin D holoenzyme complex I
b
c
Weak
Strong
Protein–protein association b CDKN1A
CDKN2A
CDKN2C
CKS1B
CCND1
CCND2
CCND3
CCNE1
CCNE2
CDK4
CDK6
CDKN1B
CDKN2B
RB1
TP53
Cyclin D holoenzyme complex I
b
Weak
Strong
Protein–protein association CDKN1A
CDKN2A
Cyclin D–CDK complex
Cyclin E–CDK
complex
DNA replication
Cell-cycle progression
CDKN2C
CKS1B
CCND1
CCND2
CCND3
CCNE1
CCNE2
CDK4
CDK6
CDKN1B
CDKN2B
RB1
TP53
Phosphorylates
Inhibits activity of
Promotes activity of
c c d
Core assemblies (8)
Importance of assembly
(clinical samples)
0
0.2
0.4
0.6
0.8
1.0
Importance of assembly
(cell lines)
ρ = 0.68
0
0.2
0.4
0.6
0.8
1.0
Regulation of
CDK activity
DNA damage
response
Cell-cycle
arrest
EGF/FGF stim. of cell proliferation
Histone-med. tx regulation
AR
signaling
PML
body
Cyclin D-
associated
events in G1 d −0.02
0
0.02
0.04
0.06
Importance of gene feature
(mutations, clinical samples)
−0.02
0
0.02
0.04
0.06
Importance of gene feature
(mutations, cell lines)
−0.04
−0.04
ρ = 0.09
f
Genes (718) e
Other assemblies (123)
ρ = 0.79
Importance of assembly
(PDX)
Cyclin D-
associated
events in G1
Regulation of
CDK activity
DNA damage
response
Cell-cycle
arrest
EGF/FGF stim. of cell proliferation
Histone-med. https://doi.org/10.1038/s43018-024-00740-1 For these experiments, we selected A549 lung carcinoma epithelial
cells, which harbor few genetic alterations in the NeST:85 assembly
compared to many other common tumor cell models for which multiple Nature Cancer | Volume 5 | July 2024 | 996–1009 1000 Article https://doi.org/10.1038/s43018-024-00740-1 Perturbation 1:
gene KO in assembly
Lentiviral particles
Cas9 nuclease-expressing
MCF7 breast tumor cell lines
Cell line propagation
Fitness
a
Perturbation 2:
CDK4/6i (palbociclib) or
CDK4 KO or
CDK6 KO
Genes in
assembly 1
Genes in
assembly 2
Genes in
assembly 3
Palbociclib
(genome-wide)
Genes
CDK4 KO
(69-gene panel)
CDK6 KO
(69-gene panel)
c
f
d
–4
–2
0
2
4
Cell fitness of gene KO × palbociclib
e
Reg. of CDK activity
Histone-med. tx reg. PML body
DNA damage response
Several top assemblies
0
0.5
1.0
1.5
2.0
Core
assemblies
(n = 8)
Nonimportant
assemblies
(n = 8)
b
0
0.5
1.0
1.5
2.0
Core
assemblies
(n = 8)
Nonimportant
assemblies
(n = 8)
NS
Mean cell fitness of
gene KO × CDK4 KO
*
**
Cyclin D-
associated
events in G1
PML
body
Histone-med. transcription
regulation
Cell-
cycle
arrest
EGF/FGF stim. of
cell proliferation
Regulation of
CDK activity
*
Cyclin D-
associated
events in G1
EGF/FGF stim. of
cell proliferation
Histone-med. transcription
regulation
Regulation of
CDK activity
Mean cell fitness of
gene KO × CDK6 KO
g
–1
1
–2
–1
1
2
TP53
CDKN2B
BRCA1
BRCA2
CDKN2A
RB1
CREBBP
PARP1
MLH1
MSH6
POLE
NPM1
BRIP1
MSH2
MYC
RUNX1
CHEK1
FANCD2
ρ = 0.48
Cell fitness of
gene KO × palbociclib
Cell fitness of
gene KO × CDK6 KO
**
Histone-med. transcription
regulation
Regulation of
CDK activity
DNA
damage
response
PML body
Gene KOs in
top four enriched
assemblies
(n = 65)
Gene KOs in
nonimportant
assemblies
(n = 349)
DAXX
BCL6
CCND3
HLA-A
TOP2A
RAD51C
AURKB
CDK4
PMS1
RBL2
CDK5
BRCA2
CTNNB1
CDKN2B
MLH3
MSH6
YEATS4
TP53
AURKA
BARD1
Enrichment of gene KOs modulating
cell fitness under palbociclib treatment
Enrichment of gene KOs modulating
cell fitness without palbociclib treatment
–0.5
0
0.5
1.0
1.5
–0.5
0
0.5
1.0
1.5
Core
assemblies
(n = 6)
Nonimportant
assemblies
(n = 6)
Core
assemblies
(n = 6)
Nonimportant
assemblies
(n = 6)
Fig. 4 | Systematic validation of palbociclib response mechanisms. a, Schematic overview of CRISPR screens in MCF7 breast tumor cells. https://doi.org/10.1038/s43018-024-00740-1 Cell fitness is z score
normalized across all tested gene KOs, with z > 0 indicating increased fitness
relative to average and z < 0 indicating decreased fitness. *P < 0.05 by two-tailed
Mann–Whitney U test. d, Violin plots illustrating the enrichment of assemblies
for gene KOs modulating cell fitness without palbociclib treatment, comparing
the core assemblies versus the same number of nonimportant assemblies. NS,
not significant by one-tailed Mann–Whitney U test. e, Scatterplot of cell fitness
of gene KOs in the context of CDK4/6i (x axis) versus CDK6 KO (y axis). Genes
shown are from the top four assemblies in b (n = 18). f, Violin plots illustrating the
mean fitness across gene KOs in core assemblies versus the same number of gene
KOs from nonimportant assemblies in combination with CDK4 KO. **P < 0.01 by
two-tailed Mann–Whitney U test. g, Same as f, except gene KOs are combined
with CDK6 KO. *P < 0.05 by two-tailed Mann–Whitney U test. In f and g, two core
assemblies did not have sufficient coverage in the gene panel; thus, six of the
eight core assemblies were tested. gene KOs in nonimportant assemblies (negative control). Cell fitness is z score
normalized across all tested gene KOs, with z > 0 indicating increased fitness
relative to average and z < 0 indicating decreased fitness. *P < 0.05 by two-tailed
Mann–Whitney U test. d, Violin plots illustrating the enrichment of assemblies
for gene KOs modulating cell fitness without palbociclib treatment, comparing
the core assemblies versus the same number of nonimportant assemblies. NS,
not significant by one-tailed Mann–Whitney U test. e, Scatterplot of cell fitness
of gene KOs in the context of CDK4/6i (x axis) versus CDK6 KO (y axis). Genes
shown are from the top four assemblies in b (n = 18). f, Violin plots illustrating the
mean fitness across gene KOs in core assemblies versus the same number of gene
KOs from nonimportant assemblies in combination with CDK4 KO. **P < 0.01 by
two-tailed Mann–Whitney U test. g, Same as f, except gene KOs are combined
with CDK6 KO. *P < 0.05 by two-tailed Mann–Whitney U test. In f and g, two core
assemblies did not have sufficient coverage in the gene panel; thus, six of the
eight core assemblies were tested. Fig. 4 | Systematic validation of palbociclib response mechanisms. a, Schematic overview of CRISPR screens in MCF7 breast tumor cells. https://doi.org/10.1038/s43018-024-00740-1 **P < 0.01 by
two-tailed Mann–Whitney U test. g, Same as f, except gene KOs are combined
with CDK6 KO. *P < 0.05 by two-tailed Mann–Whitney U test. In f and g, two core
assemblies did not have sufficient coverage in the gene panel; thus, six of the 0
0.5
1.0
1.5
2.0
Core
assemblies
(n = 8)
Nonimportant
assemblies
(n = 8)
b
**
Histone-med. transcription
regulation
Regulation of
CDK activity
DNA
damage
response
PML body
Enrichment of gene KOs modulating
cell fitness under palbociclib treatment c
–4
–2
0
2
4
Cell fitness of gene KO × palbociclib
Reg. of CDK activity
Histone-med. tx reg. PML body
DNA damage response
Several top assemblies
*
Gene KOs in
top four enriched
assemblies
(n = 65)
Gene KOs in
nonimportant
assemblies
(n = 349)
DAXX
BCL6
CCND3
HLA-A
TOP2A
RAD51C
AURKB
CDK4
PMS1
RBL2
CDK5
BRCA2
CTNNB1
CDKN2B
MLH3
MSH6
YEATS4
TP53
AURKA
BARD1 d
0
0.5
1.0
1.5
2.0
Core
assemblies
(n = 8)
Nonimportant
assemblies
(n = 8)
NS
Enrichment of gene KOs modulating
cell fitness without palbociclib treatment b d c Cell fitness of gene KO × palbociclib 0
Core
assemblies
(n = 8)
Nonimportant
assemblies
(n = 8)
*
Cyclin D-
associated
events in G1
EGF/FGF stim. of
cell proliferation
Histone-med. transcription
regulation
Regulation of
CDK activity
Mean cell fitness of
gene KO × CDK6 KO
g
–0.5
0
0.5
1.0
1.5
Core
assemblies
(n = 6)
Nonimportant
assemblies
(n = 6) f
Mean cell fitness of
gene KO × CDK4 KO
**
Cyclin D-
associated
events in G1
PML
body
Histone-med. transcription
regulation
Cell-
cycle
arrest
EGF/FGF stim. of
cell proliferation
Regulation of
CDK activity
(n = 65)
(n = 349)
–0.5
0
0.5
1.0
1.5
Core
assemblies
(n = 6)
Nonimportant
assemblies
(n = 6) f e
–1
1
–2
–1
1
2
TP53
CDKN2B
BRCA1
BRCA2
CDKN2A
RB1
CREBBP
PARP1
MLH1
MSH6
POLE
NPM1
BRIP1
MSH2
MYC
RUNX1
CHEK1
FANCD2
ρ = 0.48
Cell fitness of
gene KO × palbociclib
Cell fitness of
gene KO × CDK6 KO e g EGF/FGF stim. of
cell proliferation Nonimportant
assemblies
(n = 6) Core
assemblies
(n = 6) Fig. 4 | Systematic validation of palbociclib response mechanisms. gene KOs in nonimportant assemblies (negative control). https://doi.org/10.1038/s43018-024-00740-1 Individual
sgRNAs targeting genes in protein assemblies were combined with palbociclib
(CDK4/6i) or a second sgRNA targeting CDK4 or CDK6. Cells harboring the Cas9
nuclease were infected with lentiviral-packaged sgRNAs and propagated under
33
gene KOs in nonimportant assemblies (negative control). Cell fitness is z score
normalized across all tested gene KOs, with z > 0 indicating increased fitness
relative to average and z < 0 indicating decreased fitness. *P < 0.05 by two-tailed
Mann–Whitney U test. d, Violin plots illustrating the enrichment of assemblies
for gene KOs modulating cell fitness without palbociclib treatment, comparing Perturbation 1:
gene KO in assembly
Lentiviral particles
Cas9 nuclease-expressing
MCF7 breast tumor cell lines
Cell line propagation
Fitness
a
Perturbation 2:
CDK4/6i (palbociclib) or
CDK4 KO or
CDK6 KO
Genes in
assembly 1
Genes in
assembly 2
Genes in
assembly 3
Palbociclib
(genome-wide)
Genes
CDK4 KO
(69-gene panel)
CDK6 KO
(69-gene panel)
Histone-med. transcription
Regulation of Perturbation 1:
gene KO in assembly
Lentiviral particles
Cas9 nuclease-expressing
MCF7 breast tumor cell lines
Cell line propagation
a
Perturbation 2:
CDK4/6i (palbociclib) or
CDK4 KO or
CDK6 KO a Genes Pa
(genome
(69-gene p
(69-gene p
c
f
d
–4
–2
0
2
4
Cell fitness of gene KO × palbociclib
e
Reg. of CDK activity
Histone-med. tx reg. PML body
DNA damage response
Several top assemblies
0
0.5
1.0
1.5
2.0
Core
assemblies
(n = 8)
Nonimportant
assemblies
(n = 8)
b
0
0.5
1.0
1.5
2.0
Core
assemblies
(n = 8)
Nonimportant
assemblies
(n = 8)
NS
Mean cell fitness of
gene KO × CDK4 KO
*
**
Cyclin D-
associated
events in G1
PML
body
Histone-med. transcription
regulation
Cell-
cycle
arrest
EGF/FGF stim. of
cell proliferation
Regulation of
CDK activity
*
Cyclin D-
associated
events in G1
EGF/FGF stim. of
cell proliferation
Histone-med. transcription
regulation
Regulation of
CDK activity
Mean cell fitness of
gene KO × CDK6 KO
g
–1
1
–2
–1
1
2
TP53
CDKN2B
BRCA1
BRCA2
CDKN2A
RB1
CREBBP
PARP1
MLH1
MSH6
POLE
NPM1
BRIP1
MSH2
MYC
RUNX1
CHEK1
FANCD2
ρ = 0.48
Cell fitness of
gene KO × palbociclib
Cell fitness of
gene KO × CDK6 KO
**
Histone-med. https://doi.org/10.1038/s43018-024-00740-1 transcription
regulation
Regulation of
CDK activity
DNA
damage
response
PML body
Gene KOs in
top four enriched
assemblies
(n = 65)
Gene KOs in
nonimportant
assemblies
(n = 349)
DAXX
BCL6
CCND3
HLA-A
TOP2A
RAD51C
AURKB
CDK4
PMS1
RBL2
CDK5
BRCA2
CTNNB1
CDKN2B
MLH3
MSH6
YEATS4
TP53
AURKA
BARD1
Enrichment of gene KOs modulating
cell fitness under palbociclib treatment
Enrichment of gene KOs modulating
cell fitness without palbociclib treatment
–0.5
0
0.5
1.0
1.5
–0.5
0
0.5
1.0
1.5
Core
assemblies
(n = 6)
Nonimportant
assemblies
(n = 6)
Core
assemblies
(n = 6)
Nonimportant
assemblies
(n = 6)
Fig. 4 | Systematic validation of palbociclib response mechanisms. a, Schematic overview of CRISPR screens in MCF7 breast tumor cells. Individual
sgRNAs targeting genes in protein assemblies were combined with palbociclib
CDK4/6i) or a second sgRNA targeting CDK4 or CDK6. Cells harboring the Cas9
nuclease were infected with lentiviral-packaged sgRNAs and propagated under
selection. The palbociclib screen was from Carpintero-Fernández et al.33;
CDK4 and CDK6 KO screens were newly generated in the present study. b, Violin plots illustrating the enrichment of assemblies for gene KOs modulating
cell fitness in the context of palbociclib treatment, comparing core assemblies
defined by NeST-VNN versus the same number of nonimportant assemblies
randomly selected among those with importance < 0.5). **P < 0.01 by one-tailed
Mann–Whitney U test. GSEA76 was conducted to calculate enrichment scores. c, Left, violin plot illustrating the effects on cell fitness due to CRISPR KO of
each gene in the top four enriched assemblies shown in b. Point color indicates
gene KOs in nonimportant assemblies (negative control). Cell fitness is z score
normalized across all tested gene KOs, with z > 0 indicating increased fitness
relative to average and z < 0 indicating decreased fitness. *P < 0.05 by two-tailed
Mann–Whitney U test. d, Violin plots illustrating the enrichment of assemblies
for gene KOs modulating cell fitness without palbociclib treatment, comparing
the core assemblies versus the same number of nonimportant assemblies. NS,
not significant by one-tailed Mann–Whitney U test. e, Scatterplot of cell fitness
of gene KOs in the context of CDK4/6i (x axis) versus CDK6 KO (y axis). Genes
shown are from the top four assemblies in b (n = 18). f, Violin plots illustrating the
mean fitness across gene KOs in core assemblies versus the same number of gene
KOs from nonimportant assemblies in combination with CDK4 KO. https://doi.org/10.1038/s43018-024-00740-1 Individual
sgRNAs targeting genes in protein assemblies were combined with palbociclib
(CDK4/6i) or a second sgRNA targeting CDK4 or CDK6. Cells harboring the Cas9
nuclease were infected with lentiviral-packaged sgRNAs and propagated under
selection. The palbociclib screen was from Carpintero-Fernández et al.33;
CDK4 and CDK6 KO screens were newly generated in the present study. b, Violin plots illustrating the enrichment of assemblies for gene KOs modulating
cell fitness in the context of palbociclib treatment, comparing core assemblies
defined by NeST-VNN versus the same number of nonimportant assemblies
(randomly selected among those with importance < 0.5). **P < 0.01 by one-tailed
Mann–Whitney U test. GSEA76 was conducted to calculate enrichment scores. c, Left, violin plot illustrating the effects on cell fitness due to CRISPR KO of
each gene in the top four enriched assemblies shown in b. Point color indicates
the assembly relevant to each gene. Right, similar plot showing the effects for Fig. 4 | Systematic validation of palbociclib response mechanisms. a, Schematic overview of CRISPR screens in MCF7 breast tumor cells. Individual
sgRNAs targeting genes in protein assemblies were combined with palbociclib
(CDK4/6i) or a second sgRNA targeting CDK4 or CDK6. Cells harboring the Cas9
nuclease were infected with lentiviral-packaged sgRNAs and propagated under
selection. The palbociclib screen was from Carpintero-Fernández et al.33;
CDK4 and CDK6 KO screens were newly generated in the present study. b, Violin plots illustrating the enrichment of assemblies for gene KOs modulating
cell fitness in the context of palbociclib treatment, comparing core assemblies
defined by NeST-VNN versus the same number of nonimportant assemblies
(randomly selected among those with importance < 0.5). **P < 0.01 by one-tailed
Mann–Whitney U test. GSEA76 was conducted to calculate enrichment scores. c, Left, violin plot illustrating the effects on cell fitness due to CRISPR KO of
each gene in the top four enriched assemblies shown in b. Point color indicates
the assembly relevant to each gene. Right, similar plot showing the effects for p
,
not significant by one-tailed Mann–Whitney U test. e, Scatterplot of cell fitness
of gene KOs in the context of CDK4/6i (x axis) versus CDK6 KO (y axis). Genes
shown are from the top four assemblies in b (n = 18). f, Violin plots illustrating the
mean fitness across gene KOs in core assemblies versus the same number of gene
KOs from nonimportant assemblies in combination with CDK4 KO. https://doi.org/10.1038/s43018-024-00740-1 **P < 0.01 by
two-tailed Mann–Whitney U test. g, Same as f, except gene KOs are combined
with CDK6 KO. *P < 0.05 by two-tailed Mann–Whitney U test. In f and g, two core
assemblies did not have sufficient coverage in the gene panel; thus, six of the
eight core assemblies were tested. Nature Cancer | Volume 5 | July 2024 | 996–1009 1001 https://doi.org/10.1038/s43018-024-00740-1 Article genes are already amplified (Fig. 5b). We transfected short guide RNAs
(sgRNAs) targeting KAT6A, TBL1XR1, RUNX1, TERT or MYC into A549
cells expressing dCas9–VPR and confirmed by qPCR that constructs
exhibited substantial overexpression of the target gene compared to
nontargeting control (NTC) sgRNAs (all except for MYC; Extended Data
Fig. 4c). sgRNAs targeting MYC did not have a significant effect, consist-
ent with prior reports that this gene is already highly expressed in A549
cells35. We used the thymidine analog 5-ethynyl-2′-deoxyuridine (EdU)
to count the fraction of cells undergoing active DNA replication in the
S phase. Overexpression of the histone modifiers KAT6A and TBL1XR1
produced significant increases in the proportion of cells entering
the S phase under palbociclib treatment compared to the untreated
group (Fig. 5e,f; approximately 2.5-fold; P < 0.05); the transcription
factor RUNX1 also led to significant increases, albeit to a lesser degree
(1.5-fold). We also examined the effects of KAT6A or TBL1XR1 overex-
pression on the phosphorylation status of RB1, the direct target of
the CDK4–CDK6–cyclin D complex. Capillary western blot analysis
demonstrated that overexpression of these factors is indeed associ-
ated with a more than twofold increase in phospho-RB levels (Fig. 5g,h,
Extended Data Fig. 4d and Methods). Together, these results indicate
several NeST:85 genes whose overexpression serves to promote the cell
cycle, supporting our earlier observation (Fig. 5c) that CNAs in these
genes are predictive of palbociclib resistance. candidate biomarkers in downstream precision medicine applications. Alternatively, a model can be used in its entirety to produce a single
resistance score integrating the mutational status of all proteins and
assemblies. NeST-VNN is based on NeST, a whole-cell map of cancer protein
complexes derived from systematic proteomics data (see the ‘Struc-
tural architecture of the NeST-VNN model’ section in Methods). Previ-
ous drug response models have generally not incorporated outside
knowledge of cell structure (many approaches, reviewed here9,36)
or have modeled structure using databases of cellular components
or pathways drawn from literature curation9,14,15. https://doi.org/10.1038/s43018-024-00740-1 Biological insights
informed by NeST-VNN are uniquely dependent on the composition
of NeST, generating both strengths and limitations. One strength
is that the model can incorporate information from numerous rare
mutations in predicting a drug response insofar as these rare altera-
tions aggregate to affect the activity of commonly altered protein
assemblies with documented cancer relevance. A limitation is that
NeST almost certainly does not include all relevant protein assem-
blies (false negatives), and some assemblies that are included may be
imperfect or irrelevant to a given tumor population (false positives). Regardless, the NeST knowledgebase positions the precision medicine
model as a dynamic entity, which can be updated either functionally
with new incoming drug response data or structurally as NeST (or
another future map) is improved by additional data. These new data
need not be limited to AP–MS experiments (the primary source inform-
ing NeST thus far37) but, in the future, might incorporate information
from complementary proteomics technologies such as proximity liga-
tion38, size-exclusion chromatography39,40 or spatial imaging41. While
pathway databases are sometimes treated as gold standards (especially
literature-curated databases such as Gene Ontology and Reactome),
knowledge of molecular pathways remains incomplete, particularly
as it relates to specific tumor states and subtypes. Discussion CDK4/6 inhibitors are a well-studied class of drugs for which numer-
ous candidate biomarkers have been identified8. Why has the predic-
tion of CDK4/6i responses remained challenging? One reason is that
markers with promise in cell lines (for example, CCND1 amplification)
do not consistently translate to patient populations30,31. Another is
that individual genetic alterations that are clinically predictive may
occur too rarely to have broad utility (for example, RB1 deletion or
mutation). A wider, more integrative analysis is needed to understand
CDKi resistance fully5,8. Using this platform, we identified a set of eight core assemblies for
which genetic alterations are associated with anti-CDK4/6 response,
seven of which were validated by one or more CRISPR screens (Figs. 4
and 5). These assemblies are not focused solely on cyclin-dependent
control of the cell cycle (Fig. 3a). Nonetheless, ample literature support
can be found for the involvement of many of these other assemblies
in anti-CDK responses, such as those related to androgen receptor
(AR) signaling42, EGF/fibroblast growth factor (FGF) signaling43, DNA
damage response44 and the MDM2–p53 pathway45. Regarding the iden-
tification of an EGF/FGF signaling assembly, recent studies have found
that the EGF receptors EGFR and ERBB2 are associated with palbociclib
response46 and that the genetic alteration status of FGFR1/2 and their
associated FGF ligands has promise as a marker of acquired resistance43. Toward this goal, NeST-VNN synthesizes both rare and common
genetic events across a repertoire of drug response pathways, with
the aim of facilitating a quantitative, integrated assessment of drug
response. The modeling process begins with a map of tumor cell com-
ponents, which is used to guide the topology of deep neural network
models as they learn to translate genetic alterations to drug responses
(Extended Data Fig. 1). The key subcellular assemblies of models that
accurately capture drug responses in vitro and that translate to in vivo
(for example, PDX) and clinical settings (Fig. 2) can be validated through
directed CRISPR loss-of-function and/or activation screens (Figs. 4
and 5). Assemblies that pass this validation pipeline are a source of e, Cell microscopy images from an EdU incorporation assay for NTC (left),
TBL1XR1 overexpression (middle) or KAT6A overexpression (right). EdU-positive
cells indicating active DNA synthesis are stained in green versus nuclei stained
in blue with DAPI. Images are shown for palbociclib-untreated (top) versus
palbociclib-treated (bottom) cells. Discussion f, Bar plot depicting the fold increase in cells
undergoing active DNA synthesis (S phase) due to overexpression of specific
target genes (x axis) relative to NTC. *P < 0.05 by two-tailed Welch’s t test. Bars
indicate mean; error bars indicate ±standard error; individual replicates are
shown. Circle points indicate biological replicate 1 (n technical replicates = 3),
and square points indicate biological replicate 2 (n technical replicates = 3). g, Capillary western blot analysis of phospho-RB levels for NTC, TBL1XR1
overexpression or KAT6A overexpression in palbociclib-treated or palbociclib-
untreated (DMSO) conditions. A representative image from two independent
experiments is shown. h, Bar plot depicting the fold increase in relative phospho-
RB level (phospho-RB/actin) for the overexpression of specific target genes
(x axis) relative to NTC. *P < 0.05 by two-tailed Welch’s t test. Bars indicate mean;
error bars indicate ±standard error; individual replicates are shown. Circle points
indicate biological replicate 1 (n technical replicates = 3), and square points
indicate biological replicate 2 (n technical replicates = 4). Fig. 5 | Exploring the NeST:85 histone-related assembly in the palbociclib
response. a, Network diagram of NeST:85 depicting the histone-mediated
transcription regulation assembly. Edges show protein–protein biophysical
associations, with the edge thickness corresponding to the strength of the
evidence for association. Three subgroups of protein functions are indicated in
boxes. b, OncoPrint illustrating the genetic alteration patterns of NeST:85 genes
(rows) in patient tumors from the TCGA/ICGC (International Cancer Genome
Consortium) pan-cancer cohort (columns) along with representative cell lines
(far right columns). Genes are sorted based on relative importance for drug
resistance and then by alteration frequency (Freq) within each important or
nonimportant group. c, ORs of important gene amplifications (amp) in NeST:85
with respect to palbociclib resistance in the TCGA/ICGC pan-cancer cohort. Error
bars indicate the 95% confidence interval. d, Schematic overview of the CRISPRa
gene overexpression screen. sgRNAs targeting the promoter regions of target
genes were transfected into cells expressing the dCas9–VPR transcriptional
activator. Effects were characterized by an EdU assay, which quantifies the
number of cells undergoing active DNA synthesis, and by the phosphorylation
status of RB, the molecular target of CDK4/6. Both palbociclib-treated and
palbociclib-untreated conditions were examined. Created with BioRender.com. e, Cell microscopy images from an EdU incorporation assay for NTC (left),
TBL1XR1 overexpression (middle) or KAT6A overexpression (right). EdU-positive
cells indicating active DNA synthesis are stained in green versus nuclei stained
in blue with DAPI. Article https://doi.org/10.1038/s43018-024-00740-1 Article Accordingly, genetic alterations affecting proteins of the NeST:85
assembly, including the histone acetylases CREBBP and EP300 (ref. 47),
the HDACs HDAC1 and HDAC2 (ref. 48), and transcription factors such
as TP53 (ref. 49) and MYC50, have been previously documented to modu-
late the anti-CDK4/6 drug response. Using CRISPRa to model the effects
of CNAs, we observed that increased expression of KAT6A and TBL1XR1,
which are also components of the NeST:85 assembly, leads to increased
S-phase entry (Fig. 5). KAT6A, also known as MYST3/MOZ, encodes
a histone lysine acetyltransferase that is amplified in many cancer
types51 (Fig. 5b). Relevant to the NeST:85 assembly, KAT6A has been
previously documented to regulate cell-cycle arrest and differentia-
tion through the transcription factors p53 (ref. 52) and RUNX1 (ref. 53);
it is a frequent translocation partner of other assembly members such Accordingly, genetic alterations affecting proteins of the NeST:85
assembly, including the histone acetylases CREBBP and EP300 (ref. 47),
the HDACs HDAC1 and HDAC2 (ref. 48), and transcription factors such
as TP53 (ref. 49) and MYC50, have been previously documented to modu-
late the anti-CDK4/6 drug response. Using CRISPRa to model the effects
of CNAs, we observed that increased expression of KAT6A and TBL1XR1,
which are also components of the NeST:85 assembly, leads to increased
S-phase entry (Fig. 5). KAT6A, also known as MYST3/MOZ, encodes
a histone lysine acetyltransferase that is amplified in many cancer
types51 (Fig. 5b). Relevant to the NeST:85 assembly, KAT6A has been
previously documented to regulate cell-cycle arrest and differentia-
tion through the transcription factors p53 (ref. 52) and RUNX1 (ref. 53);
it is a frequent translocation partner of other assembly members such Furthermore, ongoing clinical trials are assessing the combination of
anti-CDK4/6 treatments with insulin-like growth factor inhibition (trial
no. NCT03099174) or with EGFR inhibition (trial no. NCT03065387)
in various tumor types. In NeST-VNN, the EGF/FGF complex combines
each of these alterations, which have largely been reported separately,
into a single integrated effect including alterations in yet additional
receptor tyrosine kinases (for example, ERBB3/4). The model also highlights a notable role for NeST:85 (histone-
mediated transcription regulation), which integrates both well-known
and understudied factors. Treatment with CDK4/6 inhibitors
induces chromatin structure remodeling mediated by histone
acetyltransferases and histone deacetylases (HDACs), leading to
the expression signatures of senescence and cell differentiation3. Discussion Images are shown for palbociclib-untreated (top) versus
palbociclib-treated (bottom) cells. f, Bar plot depicting the fold increase in cells
undergoing active DNA synthesis (S phase) due to overexpression of specific
target genes (x axis) relative to NTC. *P < 0.05 by two-tailed Welch’s t test. Bars
indicate mean; error bars indicate ±standard error; individual replicates are
shown. Circle points indicate biological replicate 1 (n technical replicates = 3),
and square points indicate biological replicate 2 (n technical replicates = 3). g, Capillary western blot analysis of phospho-RB levels for NTC, TBL1XR1
overexpression or KAT6A overexpression in palbociclib-treated or palbociclib-
untreated (DMSO) conditions. A representative image from two independent
experiments is shown. h, Bar plot depicting the fold increase in relative phospho-
RB level (phospho-RB/actin) for the overexpression of specific target genes
(x axis) relative to NTC. *P < 0.05 by two-tailed Welch’s t test. Bars indicate mean;
error bars indicate ±standard error; individual replicates are shown. Circle points
indicate biological replicate 1 (n technical replicates = 3), and square points
indicate biological replicate 2 (n technical replicates = 4). Nature Cancer | Volume 5 | July 2024 | 996–1009 1002 Ig = BatchNorm(tanh(Linear(I))) ‘BatchNorm’ indicates batch normalization63; ‘tanh’ indicates a
hyperbolic tangent function; and ‘Linear’ indicates a linear transfor-
mation. Here, the linear transformation is applied for each row in I so
that the three gene alteration values for each gene are converted into
a single value. The remaining seven layers of NeST-VNN follow the
structure of the NeST protein assembly hierarchy, where each assem-
bly is represented by some number of neurons N, a hyperparameter. A dropout64 of 0.3 (selected through hyperparameter optimization)
was added to the last four layers. Assembly state is defined as a function
of the states of its K child assemblies and M additional genes (genes
for which the protein products are not present in any descendant
assemblies). Denoting an assembly input vector as Is and an output
vector as Os, we have Drug response data for model training Drug response data were retrieved from the GDSC and CTRP data-
bases24–27, covering a total of 692,859 cell line–drug pairs comprising
1,244 cell lines and 888 drugs. The data from the two datasets were
harmonized as follows. Drug information: each molecule’s published
name, synonym or SMILES (Simplified Molecular Input Line Entry Sys-
tem) string was queried using PubChemPy. The associated InChIKey
was extracted and used to identify duplicate drugs (within or between
datasets). Cell viability data: for CTRP, the vehicle control-normalized
average percent viability files were used. For GDSC1 and GDSC2, data
were normalized to ‘cells-only’ and ‘dimethyl sulfoxide (DMSO) control’
wells, respectively, on a per-plate basis. Data were averaged across rep-
licates within each dataset. For drug response measurement, we used
AUC, in which AUC = 0 corresponds to complete cell killing and AUC = 1
corresponds to no cell killing; AUC > 1 represents a growth advantage
conferred by the drug. AUCs calculated in this study agreed with AUCs
reported by the original consortia (Pearson correlations of 0.92, 0.83,
0.91 and 0.91 for CTRP1, CTRP2, GDSC1 and GDSC2, respectively). For
multiple AUCs for the same drug across different consortia, we used
each replicate sample as a separate training instance. Genetic altera-
tion data: a panel of 718 clinical genes was assembled from the union of
genes assessed by FoundationOne CDx20, Tempus xT21, PALOMA-3 trial61
or Project GENIE22, each of which assesses mutations and/or copy num-
ber aberrations. To compile genotypes for all cell lines, we extracted
nonsynonymous coding mutations and copy number alterations for the
718 clinical panel genes from the Cancer Cell Line Encyclopedia (CCLE,
release 22Q1)23. Genes were marked as either mutated (‘1’) or unmu-
tated (‘0’), with mutations filtered for the following types: missense/
nonsense/nonstop mutations, frameshift insertions/deletions, splice
site/region variations and in-frame insertions/deletions. Similarly,
genes were marked as amplified (‘1’) or unamplified (‘0’) and deleted
(‘1’) or undeleted (‘0’). Together, mutations, CNAs and CNDs served as Structural architecture of the NeST-VNN model Construction of the NeST hierarchy of cancer protein assemblies
has been thoroughly detailed elsewhere19. Briefly, AP–MS protein
interaction data for 61 known cancer proteins were integrated with a
compendium of other systematically generated datasets informing
protein–protein associations, including protein–protein interaction,
mRNA coexpression, protein coexpression, genetic codependency
and sequence similarity. Such integration resulted in a large network
of approximately 1.8 × 108 protein–protein interactions among 19,035
proteins. Multiscale community detection was performed to detect
approximately 2,300 densely connected sets of proteins, herein called
protein assemblies. Assemblies were nested (that is, organized hierar-
chically), with larger assemblies containing smaller ones, forming ‘par-
ent–child’ assembly relations. This hierarchy has been used earlier19 to
perform a comprehensive analysis of somatic coding mutations in The
Cancer Genome Atlas (TCGA)62, identifying significant convergence of
mutations on a set of 395 protein assemblies, named NeST19. Here, we
filtered the NeST hierarchy to identify the subset of assemblies encoded
by at least five genes represented on the 718-gene clinical panel, pro-
ducing a final hierarchy of 131 assemblies distributed over seven layers. In summary, the predictive models presented in this study build
from and substantially develop the concept of an integrated response
to therapy. In such an integrated response, diverse effects converge on
biological machinery at multiple levels to produce an overall treatment
outcome. This concept may explain the difficulty in identifying indi-
vidual genetic biomarkers of palbociclib drug response. It also speaks
to the challenge of patient-to-patient heterogeneity and illustrates one
means by which knowledge of cellular machinery can be used to score
a diverse population of cancer patients presenting unique patterns
of mutational aberrations. Such an integrated model may provide a
worthwhile asset in achieving improved outcomes for patients and in
efforts to evaluate novel therapeutics to overcome resistance. Model training The filtered NeST hierarchy was used to embed a deep neural network
for drug response prediction, which we refer to as NeST-VNN (Extended
Data Fig. 1a). We define an m × 3 input matrix as I, where Ii,j ∈ {0,1}, with m
denoting the number of genes and 3 the number of gene alteration types
(mutation, CNA and CND). For any input sample (tumor cell line, PDX or
patient tumor), somatic genetic alterations for each gene and type are
marked by 1 and otherwise 0. The first layer in NeST-VNN converts these
input features to gene-level representations, Ig ∈ℝm, as follows: Article TBL1XR1, also known as TBLR1, is an F-box-like
protein involved in the recruitment of the ubiquitin conjugation sys-
tem to histone modifier and transcriptional repression complexes55,56. Subsequent proteasomal degradation of these complexes is essen-
tial for transcriptional activation by AR, as captured by the NeST:85
assembly, as well as other transcription factors such as the estrogen
receptor (ER)57. Notably, increases in KAT6A and TBL1XR1 expression
were associated with higher phosphorylation levels of RB, the central
transcriptional repressor targeted by CDK cell-cycle control (Fig. 5g,h),
suggesting that they may promote drug resistance by increasing the
transcription, abundance or activity of the upstream CDK4–CDK6–cyc-
lin D regulatory complex. The possible combination of HDAC inhibitor
therapies with cell-cycle inhibitors has been previously proposed48;
this study further underscores this potential and delineates alternative
targets. Indeed, KAT6A inhibitors are under development and have
demonstrated promising effectiveness for inducing cellular senes-
cence58–60 (clinical trial NCT04606446). In the tumor cells character-
ized here (A549; Fig. 5), TBL1XR1 has a T290A missense mutation of
unknown significance whose impact will require further investigation. In summary, the predictive models presented in this study build
from and substantially develop the concept of an integrated response
to therapy. In such an integrated response, diverse effects converge on
biological machinery at multiple levels to produce an overall treatment
outcome. This concept may explain the difficulty in identifying indi-
vidual genetic biomarkers of palbociclib drug response. It also speaks
to the challenge of patient-to-patient heterogeneity and illustrates one
means by which knowledge of cellular machinery can be used to score
a diverse population of cancer patients presenting unique patterns
of mutational aberrations. Such an integrated model may provide a
worthwhile asset in achieving improved outcomes for patients and in
efforts to evaluate novel therapeutics to overcome resistance. as EP300 and CREBBP54. TBL1XR1, also known as TBLR1, is an F-box-like
protein involved in the recruitment of the ubiquitin conjugation sys-
tem to histone modifier and transcriptional repression complexes55,56. Subsequent proteasomal degradation of these complexes is essen-
tial for transcriptional activation by AR, as captured by the NeST:85
assembly, as well as other transcription factors such as the estrogen
receptor (ER)57. Notably, increases in KAT6A and TBL1XR1 expression
were associated with higher phosphorylation levels of RB, the central
transcriptional repressor targeted by CDK cell-cycle control (Fig. Article 5g,h),
suggesting that they may promote drug resistance by increasing the
transcription, abundance or activity of the upstream CDK4–CDK6–cyc-
lin D regulatory complex. The possible combination of HDAC inhibitor
therapies with cell-cycle inhibitors has been previously proposed48;
this study further underscores this potential and delineates alternative
targets. Indeed, KAT6A inhibitors are under development and have
demonstrated promising effectiveness for inducing cellular senes-
cence58–60 (clinical trial NCT04606446). In the tumor cells character-
ized here (A549; Fig. 5), TBL1XR1 has a T290A missense mutation of
unknown significance whose impact will require further investigation. features for each of the clinical panel genes. Of the 888 drugs available
from the CCLE and/or GDSC, we selected the 51 drugs (palbociclib and
50 others) with the highest variation in the observed drug responses
across cell lines (corresponding to s.d. ≥ 0.3). Article Nature Cancer | Volume 5 | July 2024 | 996–1009
1003
BCL6
TNF
TP53
HDAC2
KAT6A
HDAC1
EP300
TERT
MYC
YEATS4
AR
CREBBP
PML
RUNX1
TBL1XR1
Protein–protein
association
Weak
Strong
Histone-mediated transcription regulation (NeST:85)
Histone
deacetylation
associated
Histone
acetylation
Transcription
factor activity
e
h
Palbociclib
(–)
Palbociclib
(+)
Control
(NTC)
TBL1XR1
(+)
KAT6A
(+)
–
–
–
a
c
f
b
d
+/– palbociclib treatment
Selection with puromycin
sgRNA transfection
dCas9–VPR-
expressing A549 cells
sgRNAs
KAT6A, RUNX1, TBL1XR1, TERT, MYC
Gene-overexpressed
cell population
RB phosphorylation
(western)
Target gene:
OR for
palbociclib resistance
Entry into S phase
(EdU assay)
MYC
TERT
KAT6A
TBL1XR1
AR
RUNX1
TP53
BCL6
TNF
CREBBP
EP300
YEATS4
PML
HDAC2
HDAC1
19%
11%
9%
9%
8%
2.5%
37%
8%
6%
6%
5%
5%
2.9%
2.7%
1.5%
Mutation
Amplification
Deep deletion
No alterations
Important
gene
selection
Important
Nonimportant
Freq
Gene
Patients
Cell lines
MCF7
A549
T47D
ZR751
All
KAT6A amp
RUNX1 amp
TBL1XR1 amp
TERT amp
MYC amp
1
2
3
4
5
TCGA/ICGC pan-cancer
+
+
+
TBL1XR1
NTC
KAT6A
NTC
TBL1XR1
KAT6A
g
Phospho-
RB
Actin
TBL1XR1
KAT6A
RUNX1
TERT
MYC
0
1
2
3
4
5
*
*
*
Fold change in cells in
S phase compared to NTC
Overexpressed gene
Palbociclib (–)
Palbociclib
Palbociclib (+)
0
1
2
3
4
5
Fold change in phospho-RB level
compared to NTC
TBL1XR1
KAT6A
Overexpressed gene
Palbociclib (–)
Palbociclib (+)
*
*
EdU-positive cells
Nuclei
50 µm c
b
OR for
palbociclib resi
MYC
TERT
KAT6A
TBL1XR1
AR
RUNX1
TP53
BCL6
TNF
CREBBP
EP300
YEATS4
PML
HDAC2
HDAC1
19%
11%
9%
9%
8%
2.5%
37%
8%
6%
6%
5%
5%
2.9%
2.7%
1.5%
Mutation
Amplification
Deep deletion
No alterations
Important
gene
selection
Important
Nonimportant
Freq
Gene
Patients
Cell lines
MCF7
A549
T47D
ZR751
All
KAT6A amp
RUNX1 amp
TBL1XR1 amp
TERT amp
MYC amp
1
2
3
TCGA/ICGC pan-cancer b
MYC
TERT
KAT6A
TBL1XR1
AR
RUNX1
TP53
BCL6
TNF
CREBBP
EP300
YEATS4
PML
HDAC2
HDAC1
19%
11%
9%
9%
8%
2.5%
37%
8%
6%
6%
5%
5%
2.9%
2.7%
1.5%
Mutation
Amplification
Deep deletion
No alterations
Important
gene
selection
Important
Nonimportant
Freq
Gene
Patients
Cell lines
MCF7
A549
T47D
ZR751
TCGA/ICGC pan-cancer c
OR for
palbociclib resistance
ns
All
KAT6A amp
RUNX1 amp
TBL1XR1 amp
TERT amp
MYC amp
1
2
3
4
5 b BCL6
TNF
TP53
HDAC2
KAT6A
HDAC1
EP300
TERT
MYC
YEATS4
AR
CREBBP
PML
RUNX1
TBL1XR1
Protein–protein
association
Weak
Strong
Histone-mediated transcription regulation (NeST:85)
Histone
deacetylation
associated
Histone
acetylation
Transcription
factor activity
a c a OR for
palbociclib resistance palbociclib resistance e
Palbociclib
(–)
Palbociclib
(+)
Control
(NTC)
TBL1XR1
(+)
KAT6A
(+)
d
+/– palbociclib treatment
Selection with puromycin
sgRNA transfection
dCas9–VPR-
expressing A549 cells
sgRNAs
KAT6A, RUNX1, TBL1XR1, TERT, MYC
Gene-overexpressed
cell population
RB phosphorylation
(western)
Target gene:
Entry into S phase
(EdU assay)
EdU-positive cells
Nuclei
50 µm e
Palbociclib
(–)
Palbociclib
(+)
Control
(NTC)
TBL1XR1
(+)
KAT6A
(+)
ment
ycin
on
EdU-positive cells
Nuclei
50 µm d
+/– palbociclib treatment
Selection with puromycin
sgRNA transfection
dCas9–VPR-
expressing A549 cells
sgRNAs
KAT6A, RUNX1, TBL1XR1, TERT, MYC
Gene-overexpressed
cell population
RB phosphorylation
(western)
Target gene:
Entry into S phase
(EdU assay) e
Palbociclib
(–)
Control
(NTC)
TBL1XR1
(+)
n
50 µm KAT6A
(+) d e h
Pa f
TBL1XR1
KAT6A
RUNX1
TERT
MYC
0
1
2
3
4
5
*
*
*
Fold change in cells in
S phase compared to NTC
Overexpressed gene
Palbociclib (–)
Palbociclib (+) 0
1
2
3
4
5
Fold change in phospho-RB level
compared to NTC
TBL1XR1
KAT6A
Overexpressed gene
Palbociclib (–)
Palbociclib (+)
*
* h
–
–
–
+
+
+
TBL1XR1
NTC
KAT6A
NTC
TBL1XR1
KAT6A
g
Phospho-
RB
Actin
Palbociclib h f g g Nature Cancer | Volume 5 | July 2024 | 996–1009 1003 https://doi.org/10.1038/s43018-024-00740-1 Article as EP300 and CREBBP54. Os = BatchNorm(tanh(Linear(Dropout(Is)))) Here, Is has dimension N × (N × K + M) and Os has dimension N. We
define ‘in silico activity’, a representative singular value for assembly
state, as the first principal component65. The NeST-VNN objective
function (Loss) aggregates the mean squared error (MSE) across every
assembly in the hierarchy: Translation to cancer patients Data from the American Association for Cancer Research Project GENIE
metastatic breast cohort22 were used to validate the performance of the
NeST-VNN model in retrospective clinical application. We extracted
nonsynonymous coding mutations, CNAs and CNDs across 360 genes
for 226 patients with ER+, HER2– metastatic breast cancer along with
their overall survival (months) and censorship information. We did
not consider gender or sex. Of these patients, 67 had been treated
with CDK4/6i plus endocrine therapy. The remaining 159 patients
were treated with endocrine therapy alone. Patients were excluded
if they had been treated with additional targeted therapies, such as
mammalian target of rapamycin (mTOR) or AKT inhibitors. Tumor
genomic data were converted to calls (0 = unaltered, 1 = altered) for
all gene mutation, CNA and CND features. Features used by NeST-VNN
that were not assessed in the clinical trial were represented as unaltered. We predicted patient response to CDK4/6 inhibition using the average
AUC over the five pretrained palbociclib models and then thresholded
this value as described in the main text (Fig. 2d). Patients whose status
label was ‘living’ at 120 months were censored. minw||Pkw −D||2
2 + α||w||2
2 where w is a vector of the coefficients of length N and α imposes an L2
penalty on coefficient complexity. Assembly ‘importance’ (Fig. 3 and
Extended Data Figs. 3 and 5) is the Spearman correlation (ρ) between Mk
and D. The mean correlation of the five NeST-VNN models was reported. A higher score indicates an assembly whose neuron values contrib-
uted more strongly to NeST-VNN predictions and can, therefore, be
considered important. To assess statistical significance, we gener-
ated a null distribution of assembly importance scores, as follows. We
randomly rearranged gene assembly memberships in the NeST-VNN
while preserving the assembly size and parent–child relationships. We trained 500 null models with these random rearrangements and
calculated assembly importance for each null. One-tailed t tests were
used to evaluate whether the assembly importance scores from the five
NeST-VNN models were greater than the assembly importance scores
from the nulls, with a Benjamini–Hochberg control for false discovery
rate (FDR; Fig. 3a). Finally, we defined ‘core assemblies’ as those with
an importance score of ≥0.5 and an FDR of ≤0.1, while excluding less
important redundant assemblies (Jaccard similarity > 0.5). To identify
specific genetic alterations in the NeST:85 assembly associated with
palbociclib resistance (Fig. 5c), we performed L1-norm regularized
logistic regression74,75. Translation to PDXs We analyzed a PDX dataset28, which contained 172 tumor samples
treated with a CDK4/6i (ribociclib) across five tumor types (breast
carcinoma, non-small cell lung carcinoma, cutaneous melanoma, colo-
rectal cancer and pancreatic ductal carcinoma). Treatment responses
had been measured by changes in the volume of the tumor xenograft
over time, with an accompanying determination of treatment time and
a classification according to the RECIST (Response Evaluation Criteria
in Solid Tumors) standard (including categories of progressive disease,
stable disease, partial response and complete response). PDX samples
had been genomically characterized, covering 660 of the 718 genes
in the NeST-VNN gene set. Similar to the procedure for cell lines and
patients, tumor genomic data were converted to calls (0 = unaltered,
1 = altered) for all gene mutation, CNA and CND features. Features used
by NeST-VNN that were not assessed in the PDX data were represented
as unaltered. We predicted the responses of PDX tumors to CDK4/6
inhibition as the average AUC over the five pretrained NeST-VNN mod-
els for palbociclib and then thresholded this score as described in the
main text (Fig. 2c). Model benchmarking 350 (similar to the number of genes characterized in the GENIE study),
the average performance is only slightly less than that obtained when
using all genes (ρ = 0.30 versus ρ = 0.33), with a more precipitous fall
in performance seen for 200 genes or fewer. A similar pattern was
observed when we compared the assembly importance scores with
their enrichments for gene KOs that modulate the response to palboci-
clib treatment (Extended Data Fig. 5b). Notably, we also found that the
precise panel of genes used by GENIE performs better than expected
compared to a random subsampling (Extended Data Fig. 5). For baseline benchmarking, we trained the RF67, ElasticNet68 and
black-box ANN69 (allotted the same number of neurons and layers
as the NeST-VNN model) models using the Python scikit-learn pack-
age70. For all models, including NeST-VNN, we used nested fivefold
cross-validation71, producing five models for each drug. For each fold
setting, we split 64% of cell lines as a training set, 16% as a validation set
(used for hyperparameter tuning) and 20% as a test set, ensuring that
cell line replicate measurements (for example, from different datasets)
were not split between the test and training sets. Hyperparameters were
optimized with Optuna72. NeST-VNN was implemented in PyTorch and
trained using five GPU (graphics processing unit) servers containing
four NVIDIA Tesla V100s, each with 5,120 CUDA (Compute Unified
Device Architecture) cores and 32-GB GDDR6 random access memory. All five NeST-VNN models were evaluated in downstream analyses. Article https://doi.org/10.1038/s43018-024-00740-1 Identifying important assemblies and genes (model
interpretation) To determine which assemblies were important for drug response
prediction in cell lines, PDX or clinical samples, we adopted a variation
of the ‘relative local improvement in predictive power’ method as previ-
ously reported13. Each assembly was modeled using linear regression,
with the aim of evaluating how well its NeST-VNN neuron values capture
the NeST-VNN overall drug response prediction. Each assembly k was
assigned a g × N matrix Pk, where g is the number of samples and N is
the number of neurons. Pk was then used in a linear ridge regression73
model to predict the NeST-VNN drug response D, creating models
M1, M2, …, Mk. The following function was minimized for each model: Translation to cancer patients Genetic alterations (mutations, CNAs, CNDs)
for the 15 assembly genes were used as regression features to predict
AUCs. AUC values in the top 30% were encoded as 1 to represent resist-
ance, whereas AUC values in the bottom 30% were encoded as 0 to
represent sensitivity. Nonzero coefficients from the fitted model were
recognized as important alterations governing drug response, with
the sign indicating whether the presence of alterations contributed
to resistance (plus) or sensitivity (minus). We used scikit-learn70 with
logistic regression settings of penalty = ‘l1’, C = 0.01 (default for other
parameters). Loss = MSE (Linear(Oroot), y) + α ∑
s≠root
MSE(Linear(Os), y) + β‖W‖ The parameter α was set to 0.3; β is a tuned hyperparameter. y represents the actual AUC.‘Linear’ denotes the linear function used
for transforming the vector Oi to a scalar. W denotes the weights of the
neural network. Weight optimization was performed using AdamW66. Nature Cancer | Volume 5 | July 2024 | 996–1009 1004 Article CRISPRa screen A custom panel of sgRNA expression plasmids targeting genes in the
NeST:85 assembly was obtained from Horizon Discovery (Fig. 5 and
Supplementary Table 4). Controls included an NTC sgRNA and an over-
expression (positive control) sgRNA targeting OCT4 (not a component
of NeST:85). dCas9–VPR stable A549 cells were plated in a complete
medium and transfected the next day with sgRNA plasmids for 24 h
using FuGENE HD (Promega). Cells were selected with puromycin
(0.44 µg ml−1) for 48 h and then lifted onto appropriate plates for fur-
ther experimentation, where they were permitted to recover for 72 h. RNA was collected using the TRIzol reagent (Invitrogen, 15596026)
and the RNeasy Mini kit (Qiagen, 74104). cDNA was synthesized using
the iScript cDNA kit (Bio-Rad, 1708891). qPCR was performed using
SYBR green, and cycle threshold (Ct) values were compared for genes
overexpressed by CRISPRa versus NTC samples (Extended Data Fig. 4c
and Supplementary Table 4). Dual CRISPR KO combinatorial screen The genome-wide chemogenetic data (above section) were comple-
mented by a de novo dual CRISPR screen performed in-house in MCF7
(HTB-22), MCF10A (CRL-10317) and MDAMB231 (CRM-HTB-26) cell lines
from American Type Culture Collection (ATCC) (Fig. 4a). Cells were
grown in DMEM with 10% FBS, screened for Mycoplasma contamina-
tion by PCR and verified by short tandem repeat (STR) testing (IDEXX
BioAnalytics). CRISPR–Cas9 nuclease was stably integrated by a lenti-
virus. LentiCas9-Blast (Addgene plasmid no. 52962) and lentiCRISPR
v2 (Addgene plasmid no. 52961) were gifts from F. Zhang79. Blasticidin
was used to select Cas9 stable integrants. Cas9 protein expression
was confirmed by capillary western blot analysis (Wes, ProteinSim-
ple). We constructed a library of double gRNA constructs targeting
druggable targets (such as CDK4 and CDK6), tumor suppressors and
oncogenes. Here, we analyzed a subset of data from individual genes
from core assemblies (sgRNA1) together with CDK4 or CDK6 (sgRNA2)
(Supplementary Table 3). The library was packaged into lentiviruses,
and cells were infected to achieve a multiplicity of infection of 0.3. Puromycin (2.5 mg ml−1) selection was started 2 days after transduction. Selection continued for 7 days, after which puromycin was removed
for the remainder of the screen. Cells were maintained in exponential
growth by isolating and removing a fraction of cells every 2–3 days. We
analyzed data from two time points at approximately 14 and 21 days. DNA was extracted from cells with a Blood and Cell Culture DNA Mini
kit (Qiagen). To assess the relative frequencies of gRNAs before and
after selection, we amplified gRNA sequences from genomic DNA by
PCR and prepared them for HiSeq 4000 sequencing (Illumina). Stand-
ard Illumina primers were used for library preparation, and 100-bp
paired-end reads were collected. Data quality was assessed with FastQC. The fitness effects of gene KOs at a time point were determined as the
fold enrichment of a construct compared to the relative abundance
of that construct in the plasmid library. Fitness measurements were
normalized to the median fitness for nontargeting guides. The mean
z score across two biological replicates, two time points and genes in
each assembly was then determined and plotted (Fig. 4f,g). Capillary western assays for RB status Transfected cells were treated with palbociclib for 24 h and then
trypsinized and washed in cold PBS; pellets were frozen at –80 °C. Protein was extracted in a hot 1× MES SDS running buffer (Invitrogen,
NP0002) for 10 min. Cooled samples were vortexed for 2 min with
glass beads (Sigma, G8772). cOmplete EDTA-free protease inhibitor
cocktail (Roche, 04693132001) and PhosSTOP (Roche, 4906845001)
were added to the cleared lysate. Protein was quantified using the
Pierce 660-nm protein assay reagent (Thermo Fisher Scientific, 22662). Protein analysis was performed on a capillary-based western blot
system (Wes, ProteinSimple, product no. 004-600) according to the
manufacturer’s instructions using the 12- to 230-kDa separation mod-
ule (ProteinSimple, SM-W001) and either the anti-rabbit detection
module (ProteinSimple, DM-001) or the anti-mouse detection module
(ProteinSimple, DM-002). Protein samples were diluted to 1 μg ml−1
in 0.1× sample buffer (ProteinSimple, 042-195) and then mixed with Article with a second-generation packaging plasmid (pCMV-dR8.2, Addgene
8455), vesicular stomatitis virus-G envelope-expressing plas-
mid (pMD2.G, Addgene 12259) and dCas9–VPR lentiviral plasmid
(hCMV-Blast-dCas9-VPR, Horizon Discovery) using Lipofectamine
3000 (Invitrogen, L3000015). Viral supernatant was collected and
cleared of cell debris by centrifugation and Steriflip column (Millipore,
SE1M003M00). Lentivirus was concentrated using Amicon Ultra-15
centrifugal filters (Millipore, Z706345). Viral titer was determined
through serial dilution. Subsequently, A549 cells (CCL-185, ATCC) were
grown in a virus-containing medium (DMEM/F12: 10% FBS, 100 IU ml−1
penicillin/streptomycin) with 8 µg ml−1 polybrene for 72 h, followed
by medium washout and selection with blasticidin (3.5 µg ml−1) for
6 days. After selection, cells were cultured with maintenance-dose
blasticidin (0.35 µg ml−1) every other passage. The identity of stable
dCas9 A549 cells was confirmed by STR testing (IDEXX BioAnalytics,
August 31, 2020). Assembly importances in the NeST-VNN versus RF models were mod-
erately but not completely correlated (ρ = 0.31; Extended Data Fig. 6a). Relevant to the differences, we found that the NeST-VNN importance
of an assembly was also moderately correlated with its enrichment
for gene KOs conferring palbociclib sensitivity or resistance (ρ = 0.33;
Extended Data Fig. 6b); in contrast, RF assembly importance showed
a correlation that was substantially weaker (ρ = 0.07; Extended Data
Fig. 6c). Thus, while RF models can achieve comparable predictive per-
formance by identifying individual gene mutations that are indicative
of drug response (Extended Data Fig. 2a,c), NeST-VNN demonstrates its
strength by integrating the effects of such mutations within predictive
cancer protein assemblies. Comparison of the interpretability of NeST-VNN and RF Comparison of the interpretability of NeST VNN and RF
We systematically evaluated the assembly importance scores provided
by NeST-VNN versus RFs67 using the genome-wide loss-of-function
screen for palbociclib treatment. To determine the assembly impor-
tance score for the RF models, we performed gene set enrichment
analysis (GSEA76, implemented using GSEApy77) on the gene list ranked
according to the gene-level feature importance scores derived from
the RF models. The absolute normalized enrichment scores generated
from GSEA were used as assembly importance scores for the RF models. Given the difference in the number of genes used for prediction in cell
lines (n = 718) versus GENIE analysis (n = 360) or PDX analysis (n = 660),
we systematically studied the dependence of model performance on
the number of genes for which genetic alteration data are provided. We computed the average predictive performance of the pretrained
NeST-VNN model when it is supplied with data for diminishing numbers
of genes (Extended Data Fig. 5a). We found that, at a gene set size of Nature Cancer | Volume 5 | July 2024 | 996–1009 1005 https://doi.org/10.1038/s43018-024-00740-1 Genome-wide CRISPR KO chemogenetic screen Core protein assemblies were validated using a genome-wide CRISPR–
Cas9 screen in MCF7 cells exposed to palbociclib treatment33 (Fig. 4a)
(Gene Expression Omnibus accession no. GSE192525). This screen had
been run previously using the GeCKO (genome-wide CRISPR KO) v2
library. Gene-level z scores (referred to as ‘normZ’) from that study were
used to indicate the effects of gene KO on cell fitness in the context of
CDK4/6 inhibition (Fig. 4b). As a reference, the cell fitnesses of gene KOs
(provided as Chronos scores78) in the MCF7 cell line in the absence of
CDK4/6i treatment (Fig. 4c) were obtained from the DepMap (Depend-
ency Map) project34 (https://depmap.org/portal/). EdU assays for S-phase entry Transfected cells were plated in collagen-coated glass-bottom 96-well
plates in a complete medium containing palbociclib (4 µM) for 24 h. Components of EdU Click-iT (Thermo Fisher Scientific, C10337)
were prepared as instructed. Cells were labeled for 4 h with 10 µM
EdU-labeling solution in the medium and then counterstained with
Hoechst dye (1:10,000) for 10 min. Cells were fixed in 3.75% formalde-
hyde for 10 min at room temperature and then washed, permeabilized
and stained according to the manufacturer’s instructions. Images
were collected using a Keyence microscope (BZ-X800) fitted with a 4×
objective and green fluorescent protein/fluorescein isothiocyanate
(Chroma, C209879) and DAPI (Chroma, C209877) filters. Images were
processed in bulk using scikit-image80. Cells were identified using Hoe-
chst counterstain and then assessed for EdU incorporation (Fig. 5e,f). Data availability The datasets used in this study are all publicly available. GDSC version 1:
https://www.cancerrxgene.org/downloads/bulk_download; GDSC
version 2: https://www.cancerrxgene.org/downloads/bulk_download;
CTRP version 1: https://portals.broadinstitute.org/ctrp.v1/; CTRP ver-
sion 2: https://portals.broadinstitute.org/ctrp.v2.1/; DepMap 22Q1:
https://doi.org/10.6084/m9.figshare.19139906.v1; PDX,: https://www. nature.com/articles/nm.3954; Project GENIE: https://genie.cbioportal. org/study/summary?id=brca_akt1_genie_2019; genome-wide CRISPR
KO chemogenetic screen: https://www.ncbi.nlm.nih.gov/geo/query/
acc.cgi?acc=GSE192525 (Gene Expression Omnibus accession no. GSE192525). The Cytoscape session containing the NeST-VNN hier-
archy and the pretrained models are available on GitHub. Cytoscape
session: https://github.com/idekerlab/nest_vnn/blob/main/misc/
NeST_VNN_Palbociclib.cys; pretrained models: https://github.com/
idekerlab/nest_vnn/tree/main/pretrained_models/palbociclib. Source
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cancer genomic profiling test based on massively parallel DNA
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for uncovering cancer-specific and drug-sensitive genes and
pathways. NAR Genom. Bioinform. 3, lqab097 (2021). https://doi.org/10.1038/s43018-024-00740-1 fluorescent master mix and heated at 95 °C for 5 min. Anti-phospho-RB
Ser807/811 (mouse monoclonal antibody, clone D20B12, 1:100, Cell
Signaling, 8516) or anti-actin (rabbit polyclonal antibody, 2 μM, Novus,
NB600-532) was used as the primary antibody, whereas a horseradish
peroxidase-conjugated anti-rabbit antibody (ProteinSimple, DM-001)
was used as a secondary antibody. Program settings were as follows:
separation at 375 V, 25 min; blocking reagent, 15 min; 20-s wash (for
runs with phospho-RB only); primary antibody blocking, 35 min; two
150-s washes; secondary antibody blocking, 35 min; 150-s wash; chemi-
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783–784 (2014). Author contributions S.P., E.S. and A.S. contributed equally to the paper. S.P., E.S., A.S. and
T.I. conceived the idea. S.P., E.S. and A.S. developed the methods. S.P., E.S., A.S., M.R.K. and T.I. interpreted the results. K.L., I.P. and C.F. conducted the CRISPRa experiment. S.F. and J.J.Y.L. conducted the
dual CRISPR KO combinatorial screen experiment. M.R.K., X.Z., R.B. and T.I. reviewed the experimental analyses. X.Z., B.A.P., K.T.Y. and T.I. reviewed the data analyses. S.P., E.S., A.S. and T.I. wrote the paper. All authors reviewed the paper. © The Author(s) 2024 Nature Cancer | Volume 5 | July 2024 | 996–1009 1009 https://doi.org/10.1038/s43018-024-00740-1 Article Extended Data Fig. 1 | NeST-VNN schematic. a, The first layer of NeST-VNN
incorporates gene-level features, including gene mutations, copy number
amplifications (CNA), and copy number deletions (CND). Subsequent assembly
layers aggregate gene-level features into assembly-level information, guided
by the hierarchical relationships defined by the NeST map. The output state of
each gene (g) and assembly (O) is represented by artificial neurons (one neuron
per gene, multiple neurons per assembly). b, Position of the assemblies detailed
in panel a within the greater NeST map. Each node indicates a protein assembly. An example path of information flow, from the neurons of CDK holoenzyme
complex to Cell cycle through to the model Root, is shown in red. each gene (g) and assembly (O) is represented by artificial neurons (one neuron
per gene, multiple neurons per assembly). b, Position of the assemblies detailed
in panel a within the greater NeST map. Each node indicates a protein assembly. An example path of information flow, from the neurons of CDK holoenzyme
complex to Cell cycle through to the model Root, is shown in red. Extended Data Fig. 1 | NeST-VNN schematic. a, The first layer of NeST-VNN
incorporates gene-level features, including gene mutations, copy number
amplifications (CNA), and copy number deletions (CND). Subsequent assembly
layers aggregate gene-level features into assembly-level information, guided
by the hierarchical relationships defined by the NeST map. The output state of Data Fig. 1 | NeST-VNN schematic. a, The first layer of NeST-VNN Nature Cancer Nature Cancer rticle
https://doi.org/10.1038/s43018-024-00740-1
xtended Data Fig. 2 | Supplemental model performance analysis. Dot plot of model performance for each of 51 drugs for NeST-VNN (red) versus
alternate models: ElasticNet (green), Random Forest (purple), and a
onventional Artificial Neural Network (ANN, blue). Palbociclib model
ghlighted in pink. b, Boxplot of performance for all drugs. Author contributions Box plots show the
th, 50th, and 75th percentiles of Pearson correlation. P-values reflect results of
one-tailed t-test assessing whether NeST-VNN outperforms baseline models. c,
ar chart of performance for palbociclib models. Error bars represent
95% confidence intervals with mean as the midpoint. P-values reflect results of one-
tailed t-test. d, ROC curves for predicting resistance (yellow) or sensitivity (blue)
to palbociclib. Marked points indicate cutoffs used to label samples as "resistant"
or "sensitive" at different stringencies. Values in parenthesis indicate the (x,y)
coordinate. e, Survival curves for CDK4/6i-treated patients stratified by somatic
mutations or copy number deletions in RB1. f, Survival curves for CDK4/6i-treated
patients stratified by somatic copy number amplifications in CCND1. Article https://doi.org/10.1038/s43018-024-00740-1 Extended Data Fig. 2 | Supplemental model performance analysis. 95% confidence intervals with mean as the midpoint. P-values reflect results of one-
tailed t-test. d, ROC curves for predicting resistance (yellow) or sensitivity (blue)
to palbociclib. Marked points indicate cutoffs used to label samples as "resistant"
or "sensitive" at different stringencies. Values in parenthesis indicate the (x,y)
coordinate. e, Survival curves for CDK4/6i-treated patients stratified by somatic
mutations or copy number deletions in RB1. f, Survival curves for CDK4/6i-treated
patients stratified by somatic copy number amplifications in CCND1. a, Dot plot of model performance for each of 51 drugs for NeST-VNN (red) versus
3 alternate models: ElasticNet (green), Random Forest (purple), and a
conventional Artificial Neural Network (ANN, blue). Palbociclib model
highlighted in pink. b, Boxplot of performance for all drugs. Box plots show the
25th, 50th, and 75th percentiles of Pearson correlation. P-values reflect results of
a one-tailed t-test assessing whether NeST-VNN outperforms baseline models. c,
Bar chart of performance for palbociclib models. Error bars represent Nature Cancer https://doi.org/10.1038/s43018-024-00740-1 Article Extended Data Fig. 3 | Supplemental model interpretation. a, NeST-VNN
interpretation of the Nutlin-3a response. Nodes indicate assemblies; node sizes
indicate assembly sizes in numbers of proteins; node colors indicate degrees of
importance for response prediction; squares inside the nodes indicate whether
the assembly contains TP53 and MDM2, drug targets of Nutlin-3a. b, Scatter plots
of gene importance based on copy number amplifications (CNA) in clinical vs. cell line contexts (x vs. y). c, same as panel b except the gene importance is based
on copy number deletions (CND). d, Alteration frequencies of genes within core
assemblies. Includes somatic mutations, CNA, and CND observed in the TCGA/
ICGC pan-cancer data. a Fig. Author contributions 3 | Supplemental model interpretation. a, NeST-VNN of gene importance based on copy number amplifications (CNA) in clinical vs. cell line contexts (x vs. y). c, same as panel b except the gene importance is based
on copy number deletions (CND). d, Alteration frequencies of genes within core
assemblies. Includes somatic mutations, CNA, and CND observed in the TCGA/
ICGC pan-cancer data. Extended Data Fig. 3 | Supplemental model interpretation. a, NeST-VNN
interpretation of the Nutlin-3a response. Nodes indicate assemblies; node sizes
indicate assembly sizes in numbers of proteins; node colors indicate degrees of
importance for response prediction; squares inside the nodes indicate whether
the assembly contains TP53 and MDM2, drug targets of Nutlin-3a. b, Scatter plots
of gene importance based on copy number amplifications (CNA) in clinical vs. cell line contexts (x vs. y). c, same as panel b except the gene importance is based
on copy number deletions (CND). d, Alteration frequencies of genes within core
assemblies. Includes somatic mutations, CNA, and CND observed in the TCGA/
ICGC pan-cancer data. Extended Data Fig. 3 | Supplemental model interpretation. a, NeST-VNN
interpretation of the Nutlin-3a response. Nodes indicate assemblies; node sizes
indicate assembly sizes in numbers of proteins; node colors indicate degrees of
importance for response prediction; squares inside the nodes indicate whether
the assembly contains TP53 and MDM2, drug targets of Nutlin-3a. b, Scatter plots Nature Cancer https://doi.org/10.1038/s43018-024-00740-1 Article Article
https://doi.org/10.1038/s43018-024-00740-1
Extended Data Fig. 4 | Supplemental analysis of Histone-mediated
transcription regulation (NeST:85). a, Alteration frequency of NeST:85 genes
across tumor types. Frequency (y-axis) and type (color) of genetic alterations in
NeST:85 genes KAT6A, MYC, RUNX1, TBL1XR1, and TERT, displayed across tumor
cohorts documented by the cBioPortal (x-axis). Downloaded from cbioportal.org
on 14 July 2023. b, Waterfall plot illustrating NeST:85 prediction (y-axis) in PDX
samples (x-axis, n = 41). The prediction was determined from the first principal
component (PC1) of the in-silico activity of the NeST:85 assembly, thresholded
(median ± stdev) to assign class labels (predicted sensitive/undefined/predicted
resistant). Bar color represents true response of a PDX sample on a CDK4/6
inhibitor (ribociclib) based on the RECIST categories (yellow, resistance (PD);
blue, sensitive (CR, PR, or SD)). c, Bar plot depicting fold increase in mRNA
expression level due to overexpression of specific gene targets relative to
non-targeting control (NTC). Bars indicate means of repeat experiments, with
technical replicate data points shown (n = 2). Author contributions d, Full capillary western blot image
of phospho-RB (pRB) level for nominal conditions (non-targeting control, NTC),
TBL1XR1 overexpression, or KAT6A overexpression. A representative image from
two independent experiments. Extended Data Fig. 4 | Supplemental analysis of Histone-mediated
transcription regulation (NeST:85). a, Alteration frequency of NeST:85 genes
across tumor types. Frequency (y-axis) and type (color) of genetic alterations in
NeST:85 genes KAT6A, MYC, RUNX1, TBL1XR1, and TERT, displayed across tumor
cohorts documented by the cBioPortal (x-axis). Downloaded from cbioportal.org
on 14 July 2023. b, Waterfall plot illustrating NeST:85 prediction (y-axis) in PDX
samples (x-axis, n = 41). The prediction was determined from the first principal
component (PC1) of the in-silico activity of the NeST:85 assembly, thresholded
(median ± stdev) to assign class labels (predicted sensitive/undefined/predicted resistant). Bar color represents true response of a PDX sample on a CDK4/6
inhibitor (ribociclib) based on the RECIST categories (yellow, resistance (PD);
blue, sensitive (CR, PR, or SD)). c, Bar plot depicting fold increase in mRNA
expression level due to overexpression of specific gene targets relative to
non-targeting control (NTC). Bars indicate means of repeat experiments, with
technical replicate data points shown (n = 2). d, Full capillary western blot image
of phospho-RB (pRB) level for nominal conditions (non-targeting control, NTC),
TBL1XR1 overexpression, or KAT6A overexpression. A representative image from
two independent experiments. Extended Data Fig. 4 | Supplemental analysis of Histone-mediated resistant). Bar color represents true response of a PDX sample on a CDK4/6
inhibitor (ribociclib) based on the RECIST categories (yellow, resistance (PD);
blue, sensitive (CR, PR, or SD)). c, Bar plot depicting fold increase in mRNA
expression level due to overexpression of specific gene targets relative to
non-targeting control (NTC). Bars indicate means of repeat experiments, with
technical replicate data points shown (n = 2). d, Full capillary western blot image
of phospho-RB (pRB) level for nominal conditions (non-targeting control, NTC),
TBL1XR1 overexpression, or KAT6A overexpression. A representative image from
two independent experiments. resistant). Bar color represents true response of a PDX sample on a CDK4/6
inhibitor (ribociclib) based on the RECIST categories (yellow, resistance (PD);
blue, sensitive (CR, PR, or SD)). c, Bar plot depicting fold increase in mRNA
expression level due to overexpression of specific gene targets relative to
non-targeting control (NTC). Bars indicate means of repeat experiments, with
technical replicate data points shown (n = 2). Author contributions d, Full capillary western blot image
of phospho-RB (pRB) level for nominal conditions (non-targeting control, NTC),
TBL1XR1 overexpression, or KAT6A overexpression. A representative image from
two independent experiments. g
pp
y
transcription regulation (NeST:85). a, Alteration frequency of NeST:85 genes
across tumor types. Frequency (y-axis) and type (color) of genetic alterations in
NeST:85 genes KAT6A, MYC, RUNX1, TBL1XR1, and TERT, displayed across tumor
cohorts documented by the cBioPortal (x-axis). Downloaded from cbioportal.org
on 14 July 2023. b, Waterfall plot illustrating NeST:85 prediction (y-axis) in PDX
samples (x-axis, n = 41). The prediction was determined from the first principal
component (PC1) of the in-silico activity of the NeST:85 assembly, thresholded
(median ± stdev) to assign class labels (predicted sensitive/undefined/predicted Nature Cancer Article https://doi.org/10.1038/s43018-024-00740-1 Article
https://doi.org/10.1038/s43018 024 00
xtended Data Fig. 5 | Supplemental analysis of NeST-VNN robustness. , Predictive performance of NeST-VNN according to the number of genes used
or prediction. Predictive performance is defined by the Pearson correlation
etween the predicted and actual drug responses. Each point represents the
verage predictive performance (y-axis) from 10 repeated experiments, with
ach experiment drawing a different random selection of genes of a given set
ize (x-axis). The error bar indicates the standard deviation of the predictive
erformance across these experiments. The orange point indicates the
predictive performance (Pearson ρ = 0.33) using the GENIE gene panel (n = 3
for prediction. b, Correlation (Pearson ρ) between the importance of protei
assemblies for model prediction and their enrichments for gene KOs that
modulate palbociclib response (y-axis) as a function of the number of genes
for prediction (x-axis). Each point represents the average Pearson correlatio
from 10 repeated experiments, with each experiment drawing a different
random selection of genes of a given set size. The error bar indicates the sta
deviation of the Pearson correlation across these experiments. Extended Data Fig. 5 | Supplemental analysis of NeST-VNN robustness. predictive performance (Pearson ρ = 0.33) using the GENIE gene panel (n = 360)
for prediction. b, Correlation (Pearson ρ) between the importance of protein
assemblies for model prediction and their enrichments for gene KOs that
modulate palbociclib response (y-axis) as a function of the number of genes used
for prediction (x-axis). Each point represents the average Pearson correlation
from 10 repeated experiments, with each experiment drawing a different
random selection of genes of a given set size. Reporting Summary Nature Portfolio wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency
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Our web collection on statistics for biologists contains articles on many of the points above. For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section. n/a Confirmed n/a Confirmed / n of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted
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Give P values as exact values whenever suitable. For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings Author contributions The error bar indicates the standard
deviation of the Pearson correlation across these experiments. a, Predictive performance of NeST-VNN according to the number of genes used
for prediction. Predictive performance is defined by the Pearson correlation
between the predicted and actual drug responses. Each point represents the
average predictive performance (y-axis) from 10 repeated experiments, with
each experiment drawing a different random selection of genes of a given set
size (x-axis). The error bar indicates the standard deviation of the predictive
performance across these experiments. The orange point indicates the Nature Cancer Nature Cancer Article https://doi.org/10.1038/s43018-024-00740-1 Extended Data Fig. 6 | Supplemental comparison of NeST-VNN versus
Random Forest models. a, Scatter plot of assembly importances from a Random
forest-GSEA approach (x-axis) versus a NeST-VNN approach (y-axis). b, Scatter
plot of assembly importance in the NeST-VNN model (y-axis) versus enrichment
of gene KOs modulating cell fitness under palbociclib treatment (x-axis). Each
dot represents an assembly (n = 130). Rho (ρ) indicates the Pearson correlation. c, Same as panel b except the y-axis indicating Random Forest-GSEA importance. Extended Data Fig. 6 | Supplemental comparison of NeST-VNN versus of gene KOs modulating cell fitness under palbociclib treatment (x-axis). Each
dot represents an assembly (n = 130). Rho (ρ) indicates the Pearson correlation. c, Same as panel b except the y-axis indicating Random Forest-GSEA importance. of gene KOs modulating cell fitness under palbociclib treatment (x-axis). Each
dot represents an assembly (n = 130). Rho (ρ) indicates the Pearson correlation. c, Same as panel b except the y-axis indicating Random Forest-GSEA importance. Random Forest models. a, Scatter plot of assembly importances from a Random
forest-GSEA approach (x-axis) versus a NeST-VNN approach (y-axis). b, Scatter
plot of assembly importance in the NeST-VNN model (y-axis) versus enrichment Nature Cancer Nature Cancer nature portfolio | reporting summary Software and code Policy information about availability of computer code
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https://openalex.org/W2039810463
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https://bmcecolevol.biomedcentral.com/counter/pdf/10.1186/1471-2148-14-150
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English
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An improved approximate-Bayesian model-choice method for estimating shared evolutionary history
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BMC evolutionary biology
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cc-by
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METHODOLOGY ARTICLE Open Access © 2014 Oaks; 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. Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 Open Access An improved approximate-Bayesian
model-choice method for estimating shared
evolutionary history Jamie R Oaks1,2 Abstract Background: To understand biological diversification, it is important to account for large-scale processes that affect
the evolutionary history of groups of co-distributed populations of organisms. Such events predict temporally
clustered divergences times, a pattern that can be estimated using genetic data from co-distributed species. I introduce a new approximate-Bayesian method for comparative phylogeographical model-choice that estimates the
temporal distribution of divergences across taxa from multi-locus DNA sequence data. The model is an extension of
that implemented in msBayes. Results: By reparameterizing the model, introducing more flexible priors on demographic and divergence-time
parameters, and implementing a non-parametric Dirichlet-process prior over divergence models, I improved the
robustness, accuracy, and power of the method for estimating shared evolutionary history across taxa. Conclusions: The results demonstrate the improved performance of the new method is due to (1) more appropriate
priors on divergence-time and demographic parameters that avoid prohibitively small marginal likelihoods for models
with more divergence events, and (2) the Dirichlet-process providing a flexible prior on divergence histories that does
not strongly disfavor models with intermediate numbers of divergence events. The new method yields more robust
estimates of posterior uncertainty, and thus greatly reduces the tendency to incorrectly estimate models of shared
evolutionary history with strong support. Keywords: Dirichlet-process prior, Approximate-Bayesian computation, Model choice, Phylogeography,
Biogeography Correspondence: joaks1@gmail.com
1Department of Ecology and Evolutionary Biology, University of Kansas, 1200
Sunnyside Avenue, Lawrence, Kansas 66045, USA
2Department of Biology, University of Washington, Box 351800, Seattle,
Washington 98195, USA Background
d
d The sample space of such a model-choice pro-
cedure would include all models ranging from a single
divergence-time parameter (i.e., simultaneous divergence
of all co-distributed taxa) to the fully generalized model in
which each taxon diverged at a unique time. The software package msBayes implements such
an approach in an approximate-Bayesian model-choice
framework [3,4]. The method models temporally clus-
tered divergences across taxa caused by a biogeographical
event (or a “divergence event”) as a single, instantaneous
occurrence. In other words, a divergence event causes
a set of taxa to share the same moment of divergence
along a continuous time scale (i.e., simultaneous diver-
gence). Given aligned sequence data for Y pairs of pop-
ulations, msBayes estimates the number of divergence
events shared among the pairs, the timing of the events,
and the assignment of pairs to the events, while integrat-
ing out uncertainty in demographic parameters and the
genealogical histories of the sequences. Thus, the method
samples over all possible divergence models of differing
dimensionality (i.e., all the possible partitions of Y pairs to
1, 2, . . . , Y divergence-time parameters), and, in so doing,
estimates the posterior probability of each model. Furthermore, msBayes uses a discrete uniform prior
over the number of divergence events 1, 2, . . . , Y. Because
there are many more possible assignments of population
pairs to intermediate numbers of divergence events, this
imposes a prior on divergence models that puts most
of the prior mass on models with either very few or
very many divergence-time parameters (see Figure five of
[7]; for brevity I will refer to this prior as “U-shaped”). Given that models with many divergence events can have
small marginal likelihoods due to the uniform priors
on divergence-time parameters, the U-shaped prior will
effectively create a strong prior preference for models with
very few divergence events. Recently, Oaks et al. [7,15] found via simulation that
msBayes will often strongly support models with a small
number of divergence events shared among taxa, even
when divergences were random over broad timescales. They suggested this behavior was due to the combination
of uniform priors on parameters causing small marginal
likelihoods of richer models and the U-shaped prior on
divergence models. Hickerson et al. [16] suggested the
problem was caused by sampling error, and proposed
as a solution an approximate-Bayesian model averaging
approach that samples over empirically informed uniform
priors. Background
d
d processes likely play a significant role in determining
diversification rates and patterns. At recent timescales,
temporal clusters of diversification caused by biogeo-
graphical events can leave a signature in the genetic
variation within and among the affected lineages. Thus,
methods for accurately estimating models of shared evo-
lutionary events across co-distributed taxa from genetic
data are important for better understanding how regional
and global biogeographical processes affect biodiversity. Understanding the processes that generate biodiversity
and regulate community assembly is a major goal of
evolutionary biology. Large-scale changes to the environ-
ment, including geological and climatic events, can affect
the evolutionary history of entire communities of co-
distributed species and their associated microbiota. For
example, by partitioning communities, such an event can
isolate groups of populations and cause a temporal clus-
ter of speciation events across co-distributed taxa. Given
the dynamic nature of our planet, such biogeographical This inference problem is challenging due to the
stochastic nature by which mutations occur in popula-
tions and how they are inherited over generations [1,2]. Thus, a method for estimating historical patterns of diver-
gences across taxa should explicitly model the stochastic
mutational and ancestral processes that generate and fil-
ter the genetic variation we observe in present-day genetic
data. An appealing approach would be a comparative, Correspondence: joaks1@gmail.com
1Department of Ecology and Evolutionary Biology, University of Kansas, 1200
Sunnyside Avenue, Lawrence, Kansas 66045, USA
2Department of Biology, University of Washington, Box 351800, Seattle,
Washington 98195, USA Page 2 of 23 Page 2 of 23 Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 fewer divergence events [7,15]. This is not a critique of
Bayesian model choice in general; comparing models by
their marginal likelihoods provides a “natural” penalty
for over-parameterization and can be a great strength
of the Bayesian approach. However, given the sensitiv-
ity of marginal likelihoods to the prior, care is needed
when selecting prior distributions [14]. Selecting distri-
butions that will often place high prior density in large
regions of parameter space with low likelihood can lead to
small marginal likelihoods of parameter-rich models even
if they are correct. Bayesian model-choice method for inferring the proba-
bility of competing divergence histories while integrating
over uncertainty in mutational and ancestral processes
via models of nucleotide substitution and lineage coa-
lescence. Background
d
d However, Oaks et al. [15] evaluated the approach
proposed by Hickerson et al. [16] using simulations and
found that it did not mitigate the method’s propensity
to incorrectly infer clustered divergences, and often pre-
ferred priors that excluded the true values of the model’s
parameters. Here, I describe a new approach that success-
fully mitigates spurious inference of co-divergence while
avoiding negative side effects of empirically informed uni-
form priors. y
msBayes has been used to address biogeographical
questions in a variety of empirical systems. Some exam-
ples include (1) whether the rise of the Isthmus of Panama
caused co-divergence among species of echinoids co-
distributed across the Pacific and Atlantic sides of the
isthmus [3], (2) if an historical seaway across the Baja
Peninsula caused co-divergence across species of squa-
mates and mammals co-distributed both north and south
of the putative seaway [5], (3) if species of gall-wasps
and their associated parasitoids share divergences across
putative glacial refugia [6], and (4) whether repeated frag-
mentation of the oceanic Islands of the Philippines during
Pleistocene sea-level fluctuations caused diversification of
vertebrate taxa distributed across the islands [7]. Such
applications of the method often result in strong posterior
support for co-divergence among all or subsets of the taxa
investigated (e.g., [3,5-12]). In this study, I introduce a new method, implemented
in the software dpp-msbayes, that extends the model
of msBayes. I use this method to test whether alterna-
tive parameterizations and priors improve the behavior
of the approximate-Bayesian model-choice approach to
estimating shared divergence events. The new approach
uses a Dirichlet-process prior (DPP) over all possible
models of divergence, and gamma and beta probability
distributions in place of uniform priors on many of the
model’s parameters. Using simulations, I show that the For priors on divergence-time and demographic param-
eters, msBayes uses continuous uniform probability dis-
tributions. This causes divergence models with more
divergence-time parameters to integrate over a much
greater parameter space with low likelihood yet high prior
density, which can result in small marginal likelihoods
relative to models with fewer divergence-time param-
eters [13,14]. Given that the marginal likelihood of a
model weighted by its prior is what determines its pos-
terior probability, this can cause support for models with Page 3 of 23 Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 new implementation has improved robustness, accuracy,
and power compared to the original model. The model In this section, I describe the model, which is a modifi-
cation of the model implemented in msBayes [4,7]. The
code implementing the new model is freely available in the
open-source software package dpp-msBayes (https://
github.com/joaks1/dpp-msbayes). To perform the anal-
yses
described
below,
I
used
the
freely
avaliable,
open-source software package PyMsBayes (https://
github.com/joaks1/PyMsBayes), which provides a multi-
processing interface to msBayes and dpp-msBayes. I
performed the work described below following the prin-
ciples of Open Notebook Science. Using version-control
software, I make progress in all aspects of the work freely
and publicly available in real-time at https://github.com/
joaks1/msbayes-experiments. All information necessary
to reproduce my results is provided there. I follow much
of the notation of Oaks et al. [7], but modify it to aid in the
description of the new model. A summary of my notation
can be found in Table 1. p(G, T, , υ, α|X, φ, ρ, ν)
= p(X|G, φ), p(G|T, , υ, ρ, ν)p(υ|α)p(α)p(T)p()
p(X|φ, ρ, ν)
, (2) where T = (T1, . . . , TY) is a vector of population diver-
gence times for each of the Y pairs of populations, =
(1, . . . , Y) is a vector of the demographic parameters
for each of the Y population pairs, υ = (υ1, . . . υK) is
a vector of locus-specific mutation-rate multipliers for
each of the K loci, α is the shape parameter of a gamma-
distributed prior on υ, and p(X|φ, ρ, ν), is the probability
of the data (or the marginal likelihood of the model) given
the fixed constants provided by the investigator. where T = (T1, . . . , TY) is a vector of population diver-
gence times for each of the Y pairs of populations, =
(1, . . . , Y) is a vector of the demographic parameters
for each of the Y population pairs, υ = (υ1, . . . υK) is
a vector of locus-specific mutation-rate multipliers for
each of the K loci, α is the shape parameter of a gamma-
distributed prior on υ, and p(X|φ, ρ, ν), is the probability
of the data (or the marginal likelihood of the model) given
the fixed constants provided by the investigator. Background
d
d The results
confirm that the improved performance of the new model
is due to a combination of (1) more flexible priors on
divergence-time and demographic parameters that avoid
placing high prior density in improbable regions of param-
eter space, and (2) a diffuse Dirichlet-process prior that
does not strongly disfavor divergence models with inter-
mediate numbers of divergence events. After reanalyzing
sequence data from 22 pairs of taxa from the Philippines
[7] under the new model, I find a large amount of pos-
terior uncertainty in the number of divergence events
shared among the taxa; a result in contrast with the orig-
inal msBayes model and congruent with intuition given
the richness of the model and the relatively small amount
of information in the data. vector of fixed constants ρ = (ρ1,1, . . . , ρY,kY) that scale
the population-size parameters for known differences in
ploidy among loci and/or differences in generation times
among population pairs. Lastly, the investigator must also
specify a vector of fixed constants ν = (ν1,1, . . . , νY,kY)
that scale the population-size parameters for known dif-
ferences in mutation rates among loci and/or among taxa. g
g
With X, φ, ρ, and ν in hand, the joint posterior distribu-
tion of the model is given by Bayes’ rule as p(G, T, , υ, α|X, φ, ρ, ν)
= p(X|G, T, , υ, α, φ, ρ, ν)p(G, T, , υ, α|φ, ρ, ν)
p(X|φ, ρ, ν)
(1) (1) which can be expanded using the chain rule of probability
into components that are assumed to be independent to
get The model To avoid calculating the likelihood terms of Equation 2,
I distill each sequence alignment X into a vector of insuf-
ficient summary statistics S, thus replacing the full dataset
X = (X1,1, . . . , XY,kY ) with vectors of summary statistics
for each alignment S∗=
S∗
1,1, . . . , S∗
Y,kY
Optionally, for
each population pair, the means of the summary statistics
can be calculated across the k loci, and the vector can be
further reduced to S∗=
S∗
1, . . . , S∗
Y
. With S∗in hand, we
can estimate the approximate joint posterior distribution I assume an investigator is interested in inferring the
distribution of divergence times among Y pairs of pop-
ulations. For each pair i, ni genome copies have been
sampled, with n1,i copies sampled from population 1, and
n2,i sampled from population 2. From these genomes, let
ki be the number of DNA sequence loci collected for pop-
ulation pair i, and K be the total number of unique loci
sampled across the Y pairs of populations. I use Xi, j to
represent the multiple sequence alignment of locus j for
population pair i. X = (X1,1, . . . , XY,kY ) is the full dataset,
i.e., a vector of sequence alignments for all pairs and loci. Let Gi, j represent the gene tree upon which Xi, j evolved
according to fixed HKY85 substitution model parame-
ters φi,j. The investigator must specify the parameters of
all φ = (φ1,1, . . . , φY,kY) substitution models by which
the alignments evolved along the G = (G1,1, . . . , GY,kY)
gene trees. Furthermore, the investigator must specify a (3) where Bϵ(S∗) is the multidimensional Euclidean space
around the vector of summary statistics, the radius of
which is the tolerance ϵ. The sources of approximation are
the insufficiency of the statistics and the ϵ being greater
than zero. I describe the full model in detail before delving Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 Page 4 of 23 Table 1 Summary of the notation used throughout this work; modified from Oaks et al. [7]
Symbol
Description
Y
Number of population pairs. ni
The number of genome copies sampled from population pair i, with n1,i sampled from population 1, and n2,i from populatio
ki
Number of loci sampled from population pair i. The model K
Total number of unique loci sampled. Xi, j
Sequence alignment of locus j sampled from population pair i. S∗
i, j
Population genetic summary statistics calculated from Xi, j. X
Vector containing the sequence alignments of each locus from each population pair:
X1,1, . . . , XY,kY
. S∗
Vector containing the summary statistics of each locus from each population pair:
S∗
1,1, . . . , S∗
Y,kY
. Bϵ(S∗)
Multi-dimensional Euclidean space around the observed summary statistics, S∗. ϵ
Radius of Bϵ(S∗), i.e., the tolerance of the ABC estimation. Gi, j
Gene tree of the sequences in Xi, j. G
Vector containing the gene trees of each locus from each population pair:
G1,1, . . . , GY,kY
. |τ|
Number of population divergence-time parameters shared among the Y population pairs. τ
Time of population divergence in 4NC generations. τ
Set of divergence-time parameters:
τ1, . . . , τ|τ|
. ti
The index of the divergence-time in τ to which population pair i is mapped. t
Vector of divergence-time indices: (t1, . . . , tY). Ti
Time of divergence in 4NC generations between the populations of pair i. T
Vector of divergence times for each of the population pairs: (T1, . . . , TY). Ti, j
Scaled time of divergence between the populations of pair i for locus j. T
Vector containing the scaled divergence times of each locus from each population pair: (T1,1, . . . , TY,kY ). θD1,i, θD2,i
Mutation-rate-scaled effective population size of the 1st and 2nd descendent population, respectively, of pair i. θA,i
Mutation-rate-scaled effective population size of the population ancestral to pair i. θD1, θD2
Vectors (θD1,1, . . . , θD1,Y) and (θD2,1, . . . , θD2,Y), respectively. θA
Vector containing the θA parameters for each population pair: (θA,1, . . . , θA,Y). υj
Mutation-rate multiplier of locus j. υ
Vector containing the locus-specific mutation-rate multipliers: (υ1, . . . , υK). α
The shape parameter of the gamma prior distribution on υ. ζD1,i, ζD2,i
θ-scaling parameters that determine the magnitude of the population bottleneck in the 1st and 2nd descendant population
respectively. The bottleneck in each descendant population begins immediately after divergence. ζD1, ζD2
Vectors (ζD1,1, . . . , ζD1,Y) and (ζD2,1, . . . , ζD2,Y), respectively. The model τB,i
Proportion of time between present and Ti when the bottleneck ends for the descendant populations of pair i. τB
Vector containing the τB parameters for each population pair: (τB,1, . . . , τB,Y). mi
Symmetric migration rate between the descendant populations of pair i. m
Vector containing the migration rates for each population pair: (mi, . . . , mY). ρi, j
θ-scaling constant provided by the investigator for locus j of pair i. This constant is required to scale θ for differences in ploid
or differences in generation times among taxa. νi, j
θ-scaling constant provided by the investigator for locus j of pair i. This constant is required to scale θ for differences in mut
among loci or among taxa. ρ
Vector of ploidy and/or generation-time scaling constants: (ρ1, 1, . . . , ρY,kY)
ν
Vector of mutation-rate scaling constants: (ν1, 1, . . . , νY,kY)
¯T
Mean of divergence times across the Y population pairs. s2
T
Variance of divergence times across the Y population pairs. DT
Dispersion index of divergence times across the Y population pairs
s2
T/¯T
. n
Number of samples from the joint prior. i
θ-scaling parameters that determine the magnitude of the population bottleneck in the 1st and 2nd descendant population of pair i,
respectively. The bottleneck in each descendant population begins immediately after divergence. Proportion of time between present and Ti when the bottleneck ends for the descendant populations of pair i. Vector containing the τB parameters for each population pair: (τB,1, . . . , τB,Y). Symmetric migration rate between the descendant populations of pair i. Vector containing the migration rates for each population pair: (mi, . . . , mY). θ-scaling constant provided by the investigator for locus j of pair i. This constant is required to scale θ for differences in ploidy among loci
or differences in generation times among taxa. θ scaling constant provided by the investigator for locus j of pair i This constant is required to scale θ for differences in mutation rates θ-scaling constant provided by the investigator for locus j of pair i. This constant is required to scale θ for differences in ploidy among loci
or differences in generation times among taxa. θ-scaling constant provided by the investigator for locus j of pair i. Likelihood and gene-tree prior terms of Equation 2 The likelihood and gene-tree prior terms of Equation 2
can be expanded out as a product over population pairs
and loci loci p(υ|α) =
K
j=1
p(υj|α),
(5) p(X|G, φ)p (G|T, , υ, ρ, ν)
=
Y
i=1
ki
j=1
p
Xi, j|Gi, j, φi, j
p
Gi, j|Ti, i, υj, ρi,j, νi,j
. (4) p(X|G, φ)p (G|T, , υ, ρ, ν) (5) =
Y
i=1
ki
j=1
p
Xi, j|Gi, j, φi, j
p
Gi, j|Ti, i, υj, ρi,j, νi,j
. where each υ is independently and identically distributed
(iid) as υ ∼Gamma(α, 1/α). If the recombination rate
r is allowed to be non-zero, the prior term p(r) would
be added to Equation 2, and the prior would be r ∼
Gamma(ar, br), where ar and br are specified by the inves-
tigator. (4) The first term, p
Xi, j|Gi, j, φi, j
, is the probability of the
sequence alignment of locus j for population pair i given
the gene tree and HKY85 [17] substitution model param-
eters [18, i.e., the “Felsenstein likelihood”]. The model
allows for an intra-locus recombination rate r, which,
for simplicity, is assumed to be zero in Equation 2. If
r is non-zero, this term requires an additional product
over the columns (sites) of each sequence alignment to
allow sites to have different genealogies. The second term,
p(Gi, j|Ti, i, υj, ρi, j, νi, j), is the probability of the gene tree
under a multi-population coalescent model (i.e., species
tree) where the ancestral population of pair i diverges
and gives rise to the two sampled descendant populations. Each contains the following demographic parameters:
The mutation-rate-scaled effective sizes (θ = 4Nμ) of
the ancestral, θA, and descendant populations, θD1 and
θD2; the proportion of the first, ζD1, and second pop-
ulation, ζD2, that persist during bottlenecks that begin
immediately after divergence in forward-time; the pro-
portion of time between present and divergence when
the bottlenecks end for both populations, τB; and the
symmetric migration rate between the descendant pop-
ulations, m. Likelihood and gene-tree prior terms of Equation 2 Thus, the probability of the ni −1 coales-
cence times (node heights) of gene tree Gi, j is given by a
multi-population Kingman-coalescent model [19] where
the ancestral population of size θA,iρi, jνi, jυj diverges
at time Ti into two descendant populations of con-
stant size θD1,iρi, jνi, jυjζD1,i and θD2,iρi, jνi, jυjζD2,i, which,
after time TiτB,i, grow exponentially to their present
size θD1,iρi, jνi, jυj and θD2,iρi, jνi, jυj, respectively. Following
divergence, the descendant populations of pair i exchange
migrants at a symmetric rate of mi. The prior term for the demographic parameters, p(),
expands out into its components and as a product over the
Y pairs of populations p() =
Y
i=1
p(θA,i)p(θD1,i)p(θD2,i)p(ζD1,i)p(ζD2,i)p(τB,i)p(mi). (6) (6) The
priors
for
the
demographic
parameters
are
θA ∼Gamma(aθA, bθA), θD1 ∼Gamma(aθD, bθD), θD2 ∼
Gamma(aθD, bθD), ζD1 ∼Beta(aζD, bζD), ζD2 ∼Beta(aζD,
bζD), τB ∼U(0, 1), and m ∼Gamma(am, bm), where
the hyper-parameters of each prior distribution can be
specified by the investigator. By default, θA, θD1, and θD2
share the same prior (i.e., aθA = aθD and bθA = bθD),
but a separate gamma-distributed prior can be assigned
to θA. Also, the ζD1, ζD2, and m parameters are optional
(i.e., the investigator can assume that there has been no
migration between populations of each pair and/or the
population size of each descendant population has been
constant through time). Table 1 Summary of the notation used throughout this work; modified from Oaks et al. [7] (Continued) Table 1 Summary of the notation used throughout this work; modified from Oaks et al. [7] (Continued) Table 1 Summary of the notation used throughout this work; modified from Oaks et al. [7] (Continued)
Vector of parameter values drawn from the joint prior. S
Vector containing the summary statistics calculated from data simulated under parameter values drawn from the prior ( ). Random sample of 1, . . . , n drawn form the prior. S
Summary statistic vectors S1, . . . , Sn for each 1, . . . , n drawn from the prior. Vector of parameter values drawn from the joint prior. S
Vector containing the summary statistics calculated from data simulated under parameter values drawn from the prior ( ). Random sample of 1, . . . , n drawn form the prior. S
Summary statistic vectors S
S for each
drawn from the prior ector containing the summary statistics calculated from data simulated under parameter values drawn from the prior ( ). Random sample of 1, . . . , n drawn form the prior. into the numerical method of estimating the approximate
model. Additional prior terms of Equation 2 The term p(α) is the prior density function for the shape
parameter of the gamma-distributed prior on rate hetero-
geneity among loci. This prior is α ∼U(1, 20). The prior
probability of the vector of locus-specific mutation-rate
multipliers given α then expands out as a product over the
loci The model This constant is required to scale θ for differences in mu
among loci or among taxa. constant provided by the investigator for locus j of pair i. This constant is required to scale θ for differences in mutation rates
ci or among taxa. Vector of ploidy and/or generation-time scaling constants: (ρ1, 1, . . . , ρY,kY) Dispersion index of divergence times across the Y population pairs
s2
T/¯T
. n Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 Page 5 of 23 Priors on divergence models The prior term for the vector of divergence times for each
of the Y pairs of populations, T, can be expanded as p(T) = p(t)p(τ|t),
(7) (7) p(T) = p(t)p(τ|t), where τ is an ordered set of divergence-time parameters
{τ1, . . . , τ|τ|} whose length |τ| can range from 1 to Y, and
t is a vector of indices (t1, . . . , tY), where ti ∈{1, . . . , |τ|}. Oaks BMC Evolutionary Biology 2014, 14:150
Page 6 of 23
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http://www.biomedcentral.com/1471-2148/14/150 Page 6 of 23 mutations, they must be scaled by the realized population
size for locus j of population-pair i These indices map each of the Y pairs of populations to
a divergence-time parameter in τ. Thus, T is the result of
applying the mapping function Ti, j = Ti ×
θC
¯θD,iρi,j
,
(12) (12) f (τ, t, i) = τti
(8) f (τ, t, i) = τti
(8)
to each population pair i, such that T=(T1 =f (τ, t, 1), . . . ,
TY = f (τ, t, Y)). (8) f (τ, t, i) = τti to each population pair i, such that T=(T1 =f (τ, t, 1), . . . ,
TY = f (τ, t, Y)). where ¯θD,i is the mean of θD1 and θD2 for pair i. This gives
us the vector of scaled divergence times T = (T1,1, . . . ,
TY,kY). Biologically speaking, τ contains the times of divergence
events, the length of which |τ| is the number of diver-
gence events shared across the Y pairs of populations. For
example, if τ contains a single divergence-time parame-
ter τ1, all Y pairs of populations are constrained to diverge
at this time (i.e., t would contain the index 1 repeated
Y times, and T would contain the value τ1 repeated Y
times), whereas if it contains Y divergence-time parame-
ters, the model is fully generalized to allow all of the pairs
to diverge at unique times. As for the prior term p(t), the total sample space of t
is all the possible partitionings of the Y pairs of popu-
lations into 1 to Y divergence-time classes, where each
partitioning consists of non-overlapping and non-empty
subsets whose union is the Y pairs. Priors on divergence models Hereinafter, I refer to
these partitionings as “ordered” divergence models or par-
titions. The total number of possible partitions is a sum of
the Stirling numbers of the second kind over all possible
numbers of categories |τ| Unlike the model implemented in msBayes, here I
place priors on t and τ, rather than |τ| and τ. As a result,
t determines the number of divergence-time parameters
(|τ|) in the model. Below, I first describe the prior used
for τ and the timescale it imposes on the model before
discussing the priors implemented for t. BY =
Y
|τ|=1
⎡
⎣1
|τ|! |τ|−1
j=0
(−1)j
|τ|
j
|τ| −j
Y
⎤
⎦,
(13) (13) which is the Bell number [20]. The original msBayes
model samples over the unordered realizations of t, such
that the sample space is reduced to all the possible inte-
ger partitions of Y [4,7,21-23] (Additional file 1: Table S1). I denote the set of all possible integer partitions of the
Y pairs of populations as a(Y) and the length of that set
as |a(Y)|, and I hereinafter refer to these integer parti-
tions as “unordered” divergence models or partitions. The
advantages, disadvantages, and justification of ignoring
the order of t is discussed in detail below. Each τ within τ is iid as τ ∼Gamma(aτ, bτ), where
aτ and bτ are specified by the investigator. Thus, given
the number of unique divergence-time classes in t, this
determines the probability of prior term p(τ|t). The diver-
gence times are in coalescent units relative to the size of
a constant reference population, which I denote θC, that
is equal to the expectation of the prior on the size of the
descendant populations I implement two prior probability distributions over the
space of all possible divergence models (t). The first simply
gives all possible unordered partitions of Y elements equal
probability θC = E(θD),
(9) (9) θC = E(θD), Given the size of the descendant populations are iid as
θD1 ∼Gamma(aθD, bθD) and θD2 ∼Gamma(aθD, bθD),
this becomes Given the size of the descendant populations are iid as
θD1 ∼Gamma(aθD, bθD) and θD2 ∼Gamma(aθD, bθD),
this becomes p(t) =
1
|a(Y)|,
(14) (14) (10) θC = aθDbθD. (10) θC = aθDbθD. i.e., a discrete uniform prior over all the integer partitions
of Y (unordered divergence models). I denote this prior as
t ∼DU{a(Y)}. The priors on nuisance parameters I have replaced the use of continuous uniform dis-
tributions for priors on many of the model’s param-
eters (τ, θA, θD1, θD2, ζD1, ζD2, r, m) with more flexible
parametric distributions from the exponential family. I introduce gamma-distributed priors for rate parame-
ters that have a sample space of all positive real num-
bers (τ, θA, θD1, θD2, r, m), and beta-distributed priors for
parameters that are proportions bounded by zero and one
(ζD1 and ζD2). These priors provide an investigator with
much greater flexibility in expressing prior uncertainty
regarding the parameters of the model. p(ti = tj) =
1
1 + χ ,
(15) (15) and also the prior probability of the number of divergence-
time parameters p (|τ| | χ, Y) =
c(Y, |τ|)χ|τ|
Y
i=1 (χ + i −1)
,
(16) (16) In addition, I have modified the prior on the sizes of the
descendant populations of each pair. As described by Oaks
et al. [7], msBayes uses the joint prior where c(·, ·) are the unsigned Stirling numbers of the first
kind. Equations 15 and 16 show that smaller values of
χ will favor fewer divergence-time parameters, and thus
more clustered models of divergence, whereas larger val-
ues will favor more divergence-time parameters, and thus
less clustered models of divergence. θD1, θD2 ∼Beta(1, 1) × 2 × U(aθ, bθD),
(17) (17) such that the user-specified uniform prior on descendant
population size is a prior on the mean size of the two
descendant populations of each pair. Under my model, the
sizes of the descendant populations of each pair are iid
as θD1 ∼Gamma(aθD, bθD) and θD2 ∼Gamma(aθD, bθD). This relaxes the assumption that the sizes of the two
descendant populations are interdependent and nega-
tively correlated. Flexibility in parameterizing the model In the new implementation, I provide the ability to control
the richness of the model. For the θ parameters, by default,
the model is fully generalized to allow each population
pair to have three parameters: θA, θD1, and θD2. Further-
more, if an investigator prefers to reduce the number
of parameters, any model of θ parameters nested within
this general model can also be specified, including the
most restricted model where the ancestral and descendant
populations of each pair share a single θ parameter. I also provide the option of eliminating the parame-
ters associated with the post-divergence bottlenecks in
the descendant populations of each pair (τB, ζD1, and
ζD2), which constrains the descendant populations to be of
constant size from present back to the divergence event. Also, rather than eliminate the bottleneck parameters, I
allow ζD1 and ζD2 to be constrained to be equal, which
removes one free parameter from the model for each of
the population pairs. Priors on divergence models More specifically, the τ parameters are in units of θC/μ
generations, which I denote as 4NC generations. Thus,
each τ within τ is proportional to time and can be con-
verted to the number of generations of the reference
population, which I denote τGC, by assuming a mutation
rate and multiplying by the effective size of the reference
population The second prior is based on the Dirichlet process,
which is a stochastic process that groups random vari-
ables into an unknown number of discrete parameter
classes [24,25]. The Dirichlet process has been used as
a non-parametric Bayesian approach to many inference
problems in evolutionary biology [26-31]. Here, I use
the Dirichlet process to place a prior over all possible
ordered partitions of Y population pairs into divergence-
time parameter classes (i.e., “divergence events”). As dis-
cussed above, the time of each divergence-time parameter
is drawn from the base distribution τ ∼Gamma(aτ, bτ). The partitioning of the population pairs to divergence-
time classes is controlled by the concentration parameter τGC = τ × θC
μ = τ × aθDbθD
μ
. (11) (11) Thus, for each of the divergence times in τ to be on the
same scale, the relative mutation rates among the pairs of
populations are assumed to be known and fixed according
to the user-provided values in ν. As described by Oaks et al. [7], to get the divergence
times in units proportional to the expected number of Page 7 of 23 Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 χ, which determines how clustered the process will be. I
take a hierarchical approach and use a prior probability
distribution (i.e., hyperprior) for χ [32]. More specifically,
I use a gamma-distributed prior χ ∼Gamma(aχ, bχ),
where aχ and bχ are specified by the investigator. I use
t ∼DP(χ) to denote this Dirichlet-process prior. pairs of populations). That is, the identity of each popu-
lation pair, and all the information associated with it, is
discarded. In my implementation, inference can be done
on either unordered or ordered models of divergence. This
is discussed in more detail in the description of the ABC
implementation below. This provides a great deal of flexibility for specifying the
prior uncertainty regarding divergence models. The con-
centration parameter χ determines the prior probability
that any two pairs of populations i and j will be assigned
to the same divergence-time parameter The prior on divergence models One of the key differences between this model and that of
msBayes [4] is the prior distribution on divergence mod-
els. As discussed in Oaks et al. [7], in msBayes the prior
used for t is a combination of a discrete uniform prior
over the possible number of divergence events |τ| from
1 to Y with a multinomial distribution on the number of
times each index of τ appears in t, with the constraint
that all τ parameters are represented at least once (see
Equation two of [7]). I denote this prior used by msBayes
as t ∼DU{1, . . . , Y}. Oaks et al. [7] discuss how placing a
uniform prior over the number of divergence parameters
(denoted |τ| here, and as in [4]) imposes an “U-shaped”
prior over divergence models (t; see Figure five(B) of [7]). To avoid this, I place priors directly on the sample space
of divergence models, thus eliminating the parameter
from the model. I introduce two priors on divergence
models: (1) a prior that is uniform over all unordered
divergence models, and (2) a Dirichlet-process prior on all
ordered divergence models. The latter provides an inves-
tigator with a great deal of flexibility in expressing their
prior beliefs about models of divergence. Time scale As described above, divergence times are in units of θC/μ
generations, where θC is the expectation of the prior on
descendant-population size. As described by Oaks et al. [7], in msBayes, θC is half of the upper limit of the con-
tinuous uniform prior on the mean of the descendant
population sizes. This is only equal to the expectation of
the prior if the lower limit of the prior is zero. Estimating ordered divergence models As mentioned above, msBayes samples over unordered
divergence models (i.e., unordered partitions of the Y Overall, my implementation allows an investigator to
specify a model that has as many as seven parameters per Oaks BMC Evolutionary Biology 2014, 14:150
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http://www.biomedcentral.com/1471-2148/14/150 divergence-time parameters is no longer a parameter in
the new implementation. population pair (θA, θD1, θD2, τB, ζD1, ζD2, and m) or as few
as one parameter per pair (θ), in addition to the ni −1
coalescence-time parameters (i.e., the node heights of the
gene tree). Obtaining an approximate posterior from the prior samples
I use a rejection algorithm to retain an approximate poste-
rior sample of from the prior sample
= ( 1, . . . , n). First, the observed summary statistics S∗, and the sum-
mary statistics of the prior samples S = (S1, . . . , Sn), are
standardized using the means and standard deviations of
the statistics from the prior sample (i.e., the prior mean
is subtracted from each statistic, and the difference is
divided by the prior standard deviation). After all statis-
tics are standardized, the Euclidean distance between S∗
and each S within S is calculated. The samples that fall
within a range of tolerance ϵ around S∗are retained. The
range of tolerance is determined by specifying the desired
number of posterior samples to be retained. Post-hoc
adjustment of the posterior sample can also be performed
with a number of regression techniques [37-39]. For anal-
yses below, I use the general linear model (GLM) regres-
sion adjustment [39] as implemented in ABCtoolbox
v1.1 [40], which Oaks et al. [7] showed performs very
similarly to weighted local-linear regression and multi-
nomial logistic regression adjustments [37] for msBayes
posteriors. ABC estimation of the posterior of the model
Sampling from the prior To estimate the approximate posterior of Equation 3, I use
an ABC rejection algorithm. The first step of this algo-
rithm entails collecting a random sample of parameter
values from the joint prior and their associated sum-
mary statistics. Each sample is generated by (1) drawing
values of all the model’s parameters, which I denote ,
from their respective prior distributions; (2) simulating
gene trees G = (G1,1, . . . , GY,kY ) for each locus of each
population pair by drawing coalescent times from a multi-
population Kingman-coalescent model given the demo-
graphic parameters; (3) simulating sequence alignments
X = (X1,1, . . . , XY,kY ) along the gene trees under the
HKY85 substitution parameters φ = (φ1,1, . . . , φY,kY ) that
have the same number of sequences and sequence lengths
as the observed dataset; and (4) calculating population
genetic summary statistics S = (S1,1, . . . , SY,kY ) from the
simulated sequence alignments. Optionally, an additional
step can be performed to reduce the summary statistics
to the means across loci for each population pair to get
S = (S1, . . . , SY). Either way, S contains the same sum-
mary statistics as those estimated from the observed data
S∗. After repeating this procedure n times, we have a ran-
dom sample of parameter vectors
= ( 1, . . . , n) from
the model prior and their associated vectors of summary
statistics S = (S1, . . . , Sn). Assessing model-choice behavior and robustness Following the simulation-based approach of Oaks et al. [7], I characterize the behavior of several models under
the ideal conditions where the data are generated from
parameters drawn from the same prior distributions used
for analysis (i.e., the prior is correct). I selected the follow-
ing four model priors for these analyses (Table 2). 1. The MmsBayes model represents the original
msBayes implementation with the U-shaped prior
on unordered divergence models and uniform priors
on divergence-time and demographic parameters;
t ∼DU{1, . . . , Y}, τ ∼U(0, 10), θA ∼U(0, 0.05),
and ¯θD ∼U(0, 0.05). 2. The MUshaped model with the U-shaped prior of
msBayes on unordered divergence models, but with
exponential priors on divergence-time and
demographic parameters; t ∼DU{1, . . . , Y},
τ ∼Exp(mean = 2.887), θA ∼Exp(mean = 0.025),
θD1 ∼Exp(mean = 0.025), and θD2 ∼Exp(mean =
0.025). 2. The MUshaped model with the U-shaped prior of
msBayes on unordered divergence models, but with
exponential priors on divergence-time and
demographic parameters; t ∼DU{1, . . . , Y}, g
p
p
τ ∼Exp(mean = 2.887), θA ∼Exp(mean = 0.025),
θD1 ∼Exp(mean = 0.025), and θD2 ∼Exp(mean =
0.025). The magnitude of the affect of this violation of
exchangeability is not known. Huang et al. [4] demon-
strated that the reordering of the summary statistic vec-
tors can greatly increase the method’s tendency to infer
a single divergence event. By definition, if the summary
statistic vectors were exchangeable, the reordering would
not change the likelihood or posterior (barring sampling
error). Thus, the results of Huang et al. [4] suggest the
reordering of the statistics is potentially introducing size-
able error to the analysis. 3. The MUniform model with a uniform prior over
unordered divergence models and exponential priors
on divergence-time and demographic parameters;
t ∼DU{a(Y)}, τ ∼Exp(mean = 2.887), θA ∼
Exp(mean = 0.025), θD1 ∼Exp(mean = 0.025), and
θD2 ∼Exp(mean = 0.025). 4. The MDPP model with a Dirichlet-process prior on
ordered divergence models and exponential priors on
divergence-time and demographic parameters; 4. The MDPP model with a Dirichlet-process prior on
ordered divergence models and exponential priors on
divergence-time and demographic parameters; Table 2 The models evaluated in the simulation-based analyses
Priors
Model
t
τ
θ
MmsBayes
t ∼DU{1, . . . , Y}
τ ∼U(0, 10 [25 MGA] )
θA ∼U(0, 0.05)
¯θD ∼U(0, 0.05)
MUshaped
t ∼DU{1, . . . Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 Page 9 of 23 (2) length of alignments, (3) numbers of loci (k), (4)
HKY85 model parameters (φ), (5) mutation-rate multipli-
ers (ν), and (6) ploidy multipliers (ρ). (2) length of alignments, (3) numbers of loci (k), (4)
HKY85 model parameters (φ), (5) mutation-rate multipli-
ers (ν), and (6) ploidy multipliers (ρ). For comparability with msBayes, I maintain the option
for re-ordering taxon-specific summary statistics by πb. However, by default, the order is preserved, and ordered
divergence models are estimated. In all of the simulation-
based analyses described below, the summary statistic
vectors are exchangeable, because the simulated datasets
have the same (1) numbers of sequences, (2) length of
sequences, (3) numbers of loci, (4) HKY85 model parame-
ters, (5) mutation-rate multipliers, and (6) ploidy multipliers. p
y
p
In the original descriptions of the msBayes method
[3,4], this re-ordering is justified by the fact that the
expected value of πb is unrelated to sample size n1 and
n2 and thus exchangeable among pairs. This is incorrect
for two reasons. First, the entire vector of summary statis-
tics S for each pair of populations is re-ordered across
pairs, which implies that the justification for re-ordering
πb applies to all the statistics within each S. However,
the expectations for statistics that estimate gross diversity
(e.g., π and θW) are not independent of sample size for
structured populations (e.g., the divergent pairs of popu-
lations modeled by msBayes), and other statistics are not
independent of sample size in general (e.g., SD(π −θW)). Second, and more importantly, having the same expecta-
tion does not ensure random variables are exchangeable. Rather, for variables to be exchangeable their marginal
distributions must be the same (i.e., they must be iden-
tically distributed). None of the summary statistics used
by msBayes, including πb, have this property when there
is any variation among taxa or loci in the (1) numbers
of sequences (n1 and n2), (2) length of alignments, (3)
numbers of loci (k), (4) HKY85 model parameters (φ), (5)
mutation-rate multipliers (ν), or (6) ploidy multipliers (ρ). Whenever such variation is present (i.e., nearly all empir-
ical applications), the taxon-specific summary statistics S
are not exchangeable, and the reshuffling of the summary
statistic vectors is not mathematically valid. Assessing model-choice behavior and robustness , Y}
τ ∼Exp(mean = 2.887[7.22MGA] )
θA ∼θD1 ∼θD2 ∼Exp(mean = 0.025)
MUniform
t ∼DU{a(Y)}
τ ∼Exp(mean = 2.887[7.22MGA] )
θA ∼θD1 ∼θD2 ∼Exp(mean = 0.025)
MDPP
t ∼DP(χ ∼Gamma(·, ·))
τ ∼Exp(mean = 2.887[7.22MGA] )
θA ∼θD1 ∼θD2 ∼Exp(mean = 0.025)
For the MDPP model, the prior on the concentration parameter, χ ∼Gamma(·, ·), was set to Gamma(2,2) for the validation analyses and Gamma(1.5,18.1) for the
power analyses. The distributions of divergence times are given in units of 4NC generations followed in brackets by units of millions of generations ago (MGA), with
the former converted to the latter assuming a per-site rate of 1 × 10−8 mutations per generation. For model MmsBayes, the priors for theta parameters are
θA ∼U(0, 0.05) and θD1, θD2 ∼Beta(1, 1) × 2 × U(0, 0.05). The later is summarized as ¯θD ∼U(0, 0.05). For the MDPP and MUniform, and MUshaped models, θA, θD1, and θD2
are independently and exponentially distributed with a mean of 0.025. Table 2 The models evaluated in the simulation-based analyses Table 2 The models evaluated in the simulation-based analyses For the MDPP model, the prior on the concentration parameter, χ ∼Gamma(·, ·), was set to Gamma(2,2) for the validation analyses and Gamma(1.5,18.1) for the
power analyses. The distributions of divergence times are given in units of 4NC generations followed in brackets by units of millions of generations ago (MGA), with
the former converted to the latter assuming a per-site rate of 1 × 10−8 mutations per generation. For model MmsBayes, the priors for theta parameters are
θA ∼U(0, 0.05) and θD1, θD2 ∼Beta(1, 1) × 2 × U(0, 0.05). The later is summarized as ¯θD ∼U(0, 0.05). For the MDPP and MUniform, and MUshaped models, θA, θD1, and θD2
are independently and exponentially distributed with a mean of 0.025. For the MDPP model, the prior on the concentration parameter, χ ∼Gamma(·, ·), was set to Gamma(2,2) for the validation analyses and Gamma(1.5,18.1) for the
power analyses. The distributions of divergence times are given in units of 4NC generations followed in brackets by units of millions of generations ago (MGA), with
the former converted to the latter assuming a per-site rate of 1 × 10−8 mutations per generation. For model MmsBayes, the priors for theta parameters are
θA ∼U(0, 0.05) and θD1, θD2 ∼Beta(1, 1) × 2 × U(0, 0.05). The later is summarized as ¯θD ∼U(0, 0.05). Ordering of taxon-specific summary statistics As alluded to in the model description, msBayes does not
maintain the order of the taxon-specific summary statis-
tics S within each S. Rather, the summary statistics are
re-ordered by descending values of average pairwise dif-
ferences between the descendant populations (πb) [4,41]. This has the advantage of reducing the sample space of
possible divergence models t, but there are at least two
disadvantages. First, additional information in the data is
lost. By discarding the identity of the Y pairs of popula-
tions, all pair-specific information about the amount of
data (e.g., the number of gene copies collected from each
of the populations [n1 and n2], the number of loci, and
the length of the loci), and the taxon- and locus-specific
parameters (φ, ν, ρ, and υ) is lost. Second, the results are
more difficult to interpret, because divergence models and
parameter estimates cannot be directly associated to the
taxa under study. For all of the analyses below, I use four summary statis-
tics for each pair of populations: π [33], θW [34], πnet [35],
and SD(π −θW) [36]. Furthermore, in addition to model
parameters, each sample also contains four statistics
that summarize T: the mean ( ¯T), variance
s2
T
, dispersion
index (DT = s2
T/ ¯T), and the number of divergence time
parameters (|τ|). Previously, these have been denoted as
E(τ)Var(τ), , and , respectively [3,4,7]. I use ¯T and s2
T
in place E(τ) and Var(τ) to make clear that these values do
not represent the prior or posterior expectation/variance
of divergence times. I use DT in place of to clarify that
this is a statistic rather than a parameter of the model. Lastly, I use |τ| in place of , because the number of The re-ordering of the summary statistic vectors also
has an important implication for the ABC algorithm. When calculating the Euclidean distance between the
observed data and each simulated dataset, the summary
statistics being compared often represent sequence align-
ments of different taxon pairs and/or loci. More specifi-
cally, the summary statistics calculated from the observed
sequence alignments are being compared to summary
statistics calculated from datasets simulated with poten-
tially different (1) numbers of sequences (n1 and n2), Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 Assessing power
l
d h Assessing power
I evaluated the power of the same four models (Table 2)
to detect random variation in divergence times using
methods similar to Oaks et al. [7]. For all power simu-
lations, I used a data structure identical to that of the
empirical dataset of Philippine vertebrates analyzed by
Oaks et al. [7], which consists of 22 pairs of populations. Due to the larger number of pairs, I used a different hyper-
prior on the concentration parameter for the MDPP model;
I used a prior of t ∼DP(χ ∼Gamma(1.5, 18.1)) over
divergence models for the model MDPP. All other aspects
of the four models in Table 2 were identical to those used
in the validation analyses described above. For each of the
four models, I generated 2 × 106 samples from the prior. Next, I simulated datasets from three series of models in
which the divergence times of the 22 pairs were random
(i.e., no clustering; |τ| = 22). The models comprising each
series differ in the variance of the distribution from which
the divergence times are randomly drawn. When the vari-
ance of random divergence times is small, all of the models
in Table 2 are expected to struggle to detect this varia-
tion and will often incorrectly estimate highly clustered
models of divergence (i.e., few divergence events). The
goal is to assess how much temporal variation in random
divergence times is necessary before the behavior of the
models of Table 2 begins to improve. This will determine I evaluated the power of the same four models (Table 2)
to detect random variation in divergence times using
methods similar to Oaks et al. [7]. For all power simu-
lations, I used a data structure identical to that of the
empirical dataset of Philippine vertebrates analyzed by
Oaks et al. [7], which consists of 22 pairs of populations. Due to the larger number of pairs, I used a different hyper-
prior on the concentration parameter for the MDPP model;
I used a prior of t ∼DP(χ ∼Gamma(1.5, 18.1)) over
divergence models for the model MDPP. All other aspects
of the four models in Table 2 were identical to those used
in the validation analyses described above. For each of the
four models, I generated 2 × 106 samples from the prior. Assessing model-choice behavior and robustness For the MDPP and MUniform, and MUshaped models, θA, θD1, and θD2
are independently and exponentially distributed with a mean of 0.025. Page 10 of 23 Page 10 of 23 Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 t∼DP(χ ∼Gamma(2, 2)), τ ∼Exp(mean = 2.887),
θA ∼Exp(mean = 0.025), θD1 ∼Exp(mean = 0.025),
and θD2 ∼Exp(mean = 0.025). given the data, then the one-divergence model should be
the true model in approximately 900 of the 1000 repli-
cates. Any trend away from the identity line indicates the
method is biased in the sense that it is not accurately esti-
mating the probability that the one-divergence model is
the correct model. I selected the exponential prior on divergence time used
in models MDPP, MUniform, and MUshaped to have the same
variance as the uniform prior in model MmsBayes. I selected
the exponential prior on population size used in mod-
els MDPP, MUniform, and MUniform to have the same mean
as the uniform prior in model MmsBayes, so that all four
models have the same θC and thus the same units of
time. All of the models were the same in other respects,
with three free θ parameters for each population pair, two
uniformly distributed (beta(1, 1)) ζD parameters per pair,
no migration, no recombination, and re-sorting of taxon-
specific summary statistics by πb (i.e., sampling unordered
divergence models). For all simulations, I used a data
structure of eight population pairs, with a single 1000
base-pair locus sampled from 10 individuals from each
population. I constructed these plots using two criteria for the
one-divergence model: (1) the number of divergence-time
parameters (|τ| = 1) and (2) the dispersion index of diver-
gence times (DT
< 0.01). For the latter, DT
< 0.01
has been commonly used as an arbitrary criterion for
a single “simultaneous” divergence event (e.g., [3,5,6]). I
focused on the one-divergence model to assess model-
choice behavior, because it is often of biogeographic inter-
est and is easily comparable among the three different
priors used on divergence models. In addition to the four models above, I also assessed
the behavior of a model that samples over ordered diver-
gence models (i.e., the order of the taxon-specific sum-
mary statistic vectors were maintained for the observed
and simulated datasets); all other settings were identical
to the MDPP model. I denote this model as M◦
DPP. Assessing model-choice behavior and robustness I sim-
ulated 1 × 106 prior samples and 50,000 datasets, and
analyzed them as above. I was not able to analyze the
simulated datasets of the other models under the ordered
model, because the identity of the population pairs is not
contained in the simulations of the other models. For each of the four models, I simulated 1 × 106 sam-
ples from the prior and 50,000 datasets, also drawn from
the prior. I then analyzed each of the simulated datasets,
retaining a posterior of 1000 samples from the respective
prior. A GLM-regression adjusted posterior was also esti-
mated from each of the posterior samples [39]. To assess
the robustness of each of the four models, I also ana-
lyzed the datasets simulated under the other three models. Overall, for each model, I produced 200,000 posterior
estimates, 50,000 from the datasets simulated under that
model, and 150,000 from the datasets simulated under the
other three models. An empirical application I also assessed the behavior of the newly implemented
models when applied to the empirical dataset of Oaks
et al. [7], which is comprised of sequence data from 22
pairs of taxa from the Philippine Islands ([43]; Dryad DOI:
10.5061/dryad.5s07m). I analyzed these data under five
different models, which are detailed in Table 4. All of these
models except one (Msimple
DPP ) have six free demographic
parameters per pair of taxa (θA, θD1, θD2, τB, ζD1, and ζD2),
in addition to the ni −1 coalescent times. Three of
these models use a Dirichlet-process prior on divergence
models: MDPP, Minform
DPP , and Msimple
DPP . The MDPP model rep-
resents the priors that Oaks et al. [7] would have selected
to reflect their prior uncertainty about the parameters
of the model if provided the more flexible distributions
that are now implemented. To assess prior sensitivity, 2. The MUniform models are identically distributed as
MUniform and MDPP except the 22 divergence times
are randomly drawn from the same series of uniform
priors as above. 3. The MExp models are also identically distributed
as MUniform and MDPP except the 22 divergence
times are randomly drawn from a series of of
exponential distributions: Exp(mean = 0.058),
Exp(mean = 0.115), Exp(mean = 0.173),
Exp(mean = 0.231), Exp(mean = 0.289), and
Exp(mean = 0.577). These exponential distributions
have the same variance as their uniform counterparts
in the first two series of models. Assessing power
l
d h I also estimated
a GLM-regression adjusted posterior from each of the
posterior samples [39]. For each of the six models in each of the three series
of models, I simulated 1000 datasets (18,000 datasets
in total). I then analyzed each simulated dataset under
all four prior models (Table 2), producing 72,000 poste-
rior estimates, each with 1000 samples. I also estimated
a GLM-regression adjusted posterior from each of the
posterior samples [39]. Specifically, I simulated datasets from the following
three series of six models (Table 3). 1. The MmsBayes models are identically distributed as
MmsBayes except the divergence times for each of the
22 pairs of populations are randomly drawn from a
series of uniform distributions, U(0, τmax), where
τmax was set to: 0.2, 0.4, 0.6, 0.8, 1.0, and 2.0, in 4NC
generations. The distributions of divergence times are given in units of 4NC generations followed in brackets by units of millions of generations ago (MGA), with the former
converted to the latter assuming a per-site rate of 1 × 10−8 mutations per generation. For all of the MmsBayes models, the priors for theta parameters are
θA ∼U(0, 0.05) and θD1, θD2 ∼Beta(1, 1) × 2 × U(0, 0.05. The later is summarized as ¯θD ∼U(0, 0.05). For the MUniform and MExp models, θA, θD1, and θD2 are
independently and exponentially distributed with a mean of 0.025. An empirical application Table 3 The models used to simulate pseudo-replicate datasets for assessing the power of the models in Table 2
Priors
Model series
t
τ
θ
MmsBayes
|τ| = 22
τ ∼U(0, 0.2 [0.5 MGA])
θA ∼U(0, 0.05)
¯θD ∼U(0, 0.05)
|τ| = 22
τ ∼U(0, 0.4 [1.0 MGA])
θA ∼U(0, 0.05)
¯θD ∼U(0, 0.05)
|τ| = 22
τ ∼U(0, 0.6 [1.5 MGA])
θA ∼U(0, 0.05)
¯θD ∼U(0, 0.05)
|τ| = 22
τ ∼U(0, 0.8 [2.0 MGA])
θA ∼U(0, 0.05)
¯θD ∼U(0, 0.05)
|τ| = 22
τ ∼U(0, 1.0 [2.5 MGA])
θA ∼U(0, 0.05)
¯θD ∼U(0, 0.05)
|τ| = 22
τ ∼U(0, 2.0 [5.0 MGA])
θA ∼U(0, 0.05)
¯θD ∼U(0, 0.05)
MUniform
|τ| = 22
τ ∼U(0, 0.2 [0.5 MGA])
θA ∼θD1 ∼θD2 ∼Exp(mean = 0.025)
|τ| = 22
τ ∼U(0, 0.4 [1.0 MGA])
θA ∼θD1 ∼θD2 ∼Exp(mean = 0.025)
|τ| = 22
τ ∼U(0, 0.6 [1.5 MGA])
θA ∼θD1 ∼θD2 ∼Exp(mean = 0.025)
|τ| = 22
τ ∼U(0, 0.8 [2.0 MGA])
θA ∼θD1 ∼θD2 ∼Exp(mean = 0.025)
|τ| = 22
τ ∼U(0, 1.0 [2.5 MGA])
θA ∼θD1 ∼θD2 ∼Exp(mean = 0.025)
|τ| = 22
τ ∼U(0, 2.0 [5.0 MGA])
θA ∼θD1 ∼θD2 ∼Exp(mean = 0.025)
MExp
|τ| = 22
τ ∼Exp(mean = 0.058 [0.14 MGA])
θA ∼θD1 ∼θD2 ∼Exp(mean = 0.025)
|τ| = 22
τ ∼Exp(mean = 0.115 [0.29 MGA])
θA ∼θD1 ∼θD2 ∼Exp(mean = 0.025)
|τ| = 22
τ ∼Exp(mean = 0.173 [0.43 MGA])
θA ∼θD1 ∼θD2 ∼Exp(mean = 0.025)
|τ| = 22
τ ∼Exp(mean = 0.231 [0.58 MGA])
θA ∼θD1 ∼θD2 ∼Exp(mean = 0.025)
|τ| = 22
τ ∼Exp(mean = 0.289 [0.72 MGA])
θA ∼θD1 ∼θD2 ∼Exp(mean = 0.025)
|τ| = 22
τ ∼Exp(mean = 0.577 [1.44 MGA])
θA ∼θD1 ∼θD2 ∼Exp(mean = 0.025)
The distributions of divergence times are given in units of 4NC generations followed in brackets by units of millions of generations ago (MGA), with the former
converted to the latter assuming a per-site rate of 1 × 10−8 mutations per generation. For all of the MmsBayes models, the priors for theta parameters are
θA ∼U(0, 0.05) and θD1, θD2 ∼Beta(1, 1) × 2 × U(0, 0.05. The later is summarized as ¯θD ∼U(0, 0.05). Assessing power
l
d h For each set of 50,000 simulated datasets, I used the
posterior estimates to assess the model-choice behavior
of each model. I did this by assigning the 50,000 esti-
mates of the posterior probability of one-divergence event
to 20 bins of width 0.05, and plotted the estimated pos-
terior probability of each bin against the proportion of
replicates in that bin with a true value consistent with one
divergence event [7,42]. Ideally, the estimated posterior
probability of the one-divergence model should estimate
the probability that the one-divergence model is correct. For large numbers of simulation replicates, the proportion
of the replicates in each bin for which the one-divergence
model is true will approximate the probability that the
one-divergence model is the correct model. Thus, if the
method has the desirable behavior such that the esti-
mated posterior probability of the one-divergence model
is an unbiased estimate of the probability that the one-
divergence model is correct, the points should fall near the
identity line. For example, let us say the method estimates
a posterior probability of 0.90 for 1000 datasets simu-
lated from the prior. If the method is accurately estimating
the probability that the one-divergence model is correct Next, I simulated datasets from three series of models in
which the divergence times of the 22 pairs were random
(i.e., no clustering; |τ| = 22). The models comprising each
series differ in the variance of the distribution from which
the divergence times are randomly drawn. When the vari-
ance of random divergence times is small, all of the models
in Table 2 are expected to struggle to detect this varia-
tion and will often incorrectly estimate highly clustered
models of divergence (i.e., few divergence events). The
goal is to assess how much temporal variation in random
divergence times is necessary before the behavior of the
models of Table 2 begins to improve. This will determine Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 Page 11 of 23 the timescales over which the models can reliably detect
random variation in divergence times and avoid spurious
inference of clustered divergence models. For each of the six models in each of the three series
of models, I simulated 1000 datasets (18,000 datasets
in total). I then analyzed each simulated dataset under
all four prior models (Table 2), producing 72,000 poste-
rior estimates, each with 1000 samples. g
p
p
g
MmsBayes
,
p
p
D1, θD2 ∼Beta(1, 1) × 2 × U(0, 0.05. The later is summarized as ¯θD ∼U(0, 0.05). For the MUniform and MExp models, θA, θD1, and θD2 are
xponentially distributed with a mean of 0.025. gence times are given in units of 4NC generations followed in brackets by units of millions of generations ago (MGA), with the former
suming a per-site rate of 1 × 10−8 mutations per generation. For all of the MmsBayes models, the priors for theta parameters are An empirical application For the MUniform and MExp models, θA, θD1, and θD2 are Table 3 The models used to simulate pseudo-replicate datasets for assessing the power of the m dels used to simulate pseudo-replicate datasets for assessing the power of the models in Table 2 The distributions of divergence times are given in units of 4NC generations followed in brackets by units of millions of generations ago (MGA), with the former
converted to the latter assuming a per-site rate of 1 × 10−8 mutations per generation. For all of the MmsBayes models, the priors for theta parameters are
θA ∼U(0, 0.05) and θD1, θD2 ∼Beta(1, 1) × 2 × U(0, 0.05. The later is summarized as ¯θD ∼U(0, 0.05). For the MUniform and MExp models, θA, θD1, and θD2 are
independently and exponentially distributed with a mean of 0.025. Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 Page 12 of 23 Table 4 The models used to analyze the data from the 22 pairs of taxa from the Philippines (M), and a subset of nine of
those pairs from the Islands of Negros and Panay (M)
Model
Priors
MmsBayes
t ∼DU{1, . . . , Y}
τ ∼U(0, 34.64 [17.3 MGA])
θA ∼U(0, 0.01)
θD1, θD2 ∼Beta(1, 1) × 2 × U(0, 0.01)
ζD1 ∼U(0, 1)
ζD2 ∼U(0, 1)
MUniform
t ∼DU{a(Y)}
τ ∼Exp(mean = 10 [5 MGA])
θA ∼Exp(mean = 0.005)
θD1 ∼Exp(mean = 0.005)
θD2 ∼Exp(mean = 0.005)
ζD1 ∼Beta(5, 1)
ζD2 ∼Beta(5, 1)
MDPP
t ∼DP(χ ∼Gamma(1.5, 18.1))
τ ∼Exp(mean = 10 [5 MGA] )
θA ∼Exp(mean = 0.005)
θD1 ∼Exp(mean = 0.005)
θD2 ∼Exp(mean = 0.005)
ζD1 ∼Beta(5, 1)
ζD2 ∼Beta(5, 1)
Minform
DPP
t ∼DP(χ ∼Gamma(1.5, 18.1))
τ ∼Exp(mean = 6 [3 MGA])
θA ∼Exp(mean = 0.005)
θD1 ∼Exp(mean = 0.005)
θD2 ∼Exp(mean = 0.005)
ζD1 ∼Beta(5, 1)
ζD2 ∼Beta(5, 1)
Msimple
DPP
t ∼DP(χ ∼Gamma(1.5, 18.1))
τ ∼Exp(mean = 10 [5 MGA])
θA = θD1 = θD2 ∼Exp(mean = 0.005)
ζD1 = ζD2 = 1.0
MDPP
t ∼DP(χ ∼Gamma(1.5, 5.0))
τ ∼Exp(mean = 10 [5 MGA])
θA ∼Exp(mean = 0.005)
θD1 = θD2 ∼Exp(mean = 0.005)
ζD1 = ζD2 = 1.0
In addition to the n −1 coalescent times, the Msimple
DPP
has only a single θ parameter for each taxon pair. The remaining M models have three θ, two ζ D, and one τ B
parameter. An empirical application The distributions of divergence times are given in units of 4NC generations followed in brackets by units of millions of generations ago (MGA), with the
former converted to the latter assuming a per-site rate of 1 × 10−8 mutations per generation. The MDPP model (and its M◦
DPP counterpart that samples over ordered
divergence models) has only two θ parameters (the descendant populations of each pair share the same θ parameter, and there are no bottleneck parameters). Table 4 The models used to analyze the data from the 22 pairs of taxa from the Philippines (M), and a subset of nine of
those pairs from the Islands of Negros and Panay (M) In addition to the n −1 coalescent times, the Msimple
DPP
has only a single θ parameter for each taxon pair. The remaining M models have three θ, two ζ D, and one τ B
parameter. The distributions of divergence times are given in units of 4NC generations followed in brackets by units of millions of generations ago (MGA), with the
former converted to the latter assuming a per-site rate of 1 × 10−8 mutations per generation. The MDPP model (and its M◦
DPP counterpart that samples over ordered
divergence models) has only two θ parameters (the descendant populations of each pair share the same θ parameter, and there are no bottleneck parameters). In addition to the n −1 coalescent times, the Msimple
DPP
has only a single θ parameter for each taxon pair. The remaining M models have three θ, two ζ D, and one τ B
parameter. The distributions of divergence times are given in units of 4NC generations followed in brackets by units of millions of generations ago (MGA), with the
former converted to the latter assuming a per-site rate of 1 × 10−8 mutations per generation. The MDPP model (and its M◦
DPP counterpart that samples over ordered
divergence models) has only two θ parameters (the descendant populations of each pair share the same θ parameter, and there are no bottleneck parameters). 5 × 107 samples from the prior and retained an approxi-
mate posterior of 10,000 samples. the Minform
DPP
model uses a more informative exponentially
distributed prior on divergence times, but otherwise is
identical to MDPP. An empirical application To assess sensitivity to parameteriza-
tion, I also applied the simplest possible model under
the new implementation
Msimple
DPP
with only a single
demographic parameter (θ) per taxon pair, in addition
to the ni −1 coalescent times. I also applied the orig-
inal msBayes model (MmsBayes) with priors selected to
make the results directly comparable to those of the MDPP
model; the uniform prior on divergence times was selected
to have the same variance as the exponential prior of
the MDPP model, and the prior on population size was
selected to have the same mean so that the models are
on the same timescale. I also applied a model with a
uniform distribution over divergence models (MUniform). For each of these models, I simulated 2 × 107 samples
from the prior, and retained an approximate posterior of
the 10,000 samples with the smallest Euclidean distance
from the summary statistics calculated from the empirical
sequence alignments. Validation analyses: Estimation accuracy The
MmsBayes model performs slightly better when applied to Page 13 of 23 Page 13 of 23 Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 taxa. Thus, it is critical to assess the method’s ability to
accurately estimate the posterior probabilities of diver-
gence models. Consistent with the findings of Oaks
et al. [7], my results demonstrate that the unadjusted
estimates of divergence-model posterior probabilities are
generally more accurate than regression-adjusted esti-
mates (compare the plots along the upper-left to lower-
right diagonal for Figure 1 versus Additional file 1: Figure
S7 and Figure 2 versus Additional file 1: Figure S8). Regression adjustment results in biased estimates of the
posterior probability of the one-divergence model when
all model assumptions are satisfied, which is well illus-
trated in Additional file 1: Figure S8. As a result, I will
focus my discussion of the results on the unadjusted
estimates. its own data, but is the worst performer when applied
to data generated under other models (Additional file 1:
Figure S5). There is a trend of MDPP > MUniform >
MUshaped > MmsBayes in terms of estimation accuracy as
measured by RMSE when the models are applied to data
generated under most of the models (Additional file 1:
Figure S5). Unlike for the continuous statistics, regression
adjustment of this discrete statistic tends to increase esti-
mation bias; all of the models tend to underestimate |τ|
after the GLM-adjustment (Additional file 1: Figure S6). Validation analyses: Estimation accuracy y
y
In terms of estimating the variance of divergence times
(DT), the models with exponentially distributed priors
(MUshaped, MUniform, and MDPP) perform similarly when
applied to datasets generated under all four of the mod-
els in Table 2 (Additional file 1: Figure S1). The MmsBayes
model performs similarly to these models when applied to
its own datasets, but is sensitive to model violations and
is more biased when applied to data generated under the
other three models (Additional file 1: Figure S1). Results
are similar for the GLM-adjusted estimates of DT, albeit
the regression adjustment tends to improve estimates of
this continuous statistic for all the models (Additional
file 1: Figure S2). The same general pattern is seen for estimates of ¯T,
with (1) all four models performing similarly when applied
to the data generated under the MmsBayes model, (2) the
models with exponentially distributed priors perform-
ing similarly when applied to data generated under the
other three models, and (3) the MmsBayes model is sen-
sitive to model violations and is more biased whenever
applied to data generated under other models (Additional
file 1: Figure S3). Also, the regression adjustment tends to
slightly improve estimates of this continuous statistic for
all of the models (Additional file 1: Figure S4). To compare models that sample over ordered versus
unordered models of divergence, I also analyzed the data
from the subset of nine-taxon pairs that are sampled
from the Islands of Negros and Panay in the Philippines. The model I used for these analyses had a Dirichlet-
process prior over divergence models and two demo-
graphic parameters (θA and (θD) for each pair of taxa, in
addition to the ni −1 coalescent times (see Table 4 for
details). One of the models, which I denote M◦
DPP, main-
tained the identity of the taxon pairs and sampled over
ordered models of divergence, while the other (MDPP) re-
sorted the summary statistics of the pairs by πb, losing
the identity of the taxa and thus sampled over unordered
models of divergence. For both analyses, I simulated In terms of estimating the number of divergence events
(|τ|), the MDPP model has the lowest root mean square
error (RMSE) when applied to data generated under most
of the models of Table 2 (Additional file 1: Figure S5). Validation analyses: Model-choice accuracy The msBayes model, and my modification of it, is a
model-choice method with the primary purpose of esti-
mating the probabilities of models of divergence across Figure 1 Comparison of model-choice accuracy. Model-choice accuracy for models (A–D) MmsBayes, (E–H) MUshaped, (I–L) MUniform, and (M–P)
MDPP when analyzing data generated under models (A, E, I, and M) MmsBayes, (B, F, J, and N) MUshaped, (C, G, K, and O) MUniform, and (D, H, L, and
P) MDPP. The unadjusted posterior probability of a single divergence event, based on |τ| = 1, from 50,000 posterior estimates are assigned to bins
of width 0.05 and plotted against the proportion of replicates in each bin where the truth is |τ| = 1. Figure 1 Comparison of model-choice accuracy. Model-choice accuracy for models (A–D) MmsBayes, (E–H) MUshaped, (I–L) MUniform, and (M–P)
MDPP when analyzing data generated under models (A, E, I, and M) MmsBayes, (B, F, J, and N) MUshaped, (C, G, K, and O) MUniform, and (D, H, L, and
P) MDPP. The unadjusted posterior probability of a single divergence event, based on |τ| = 1, from 50,000 posterior estimates are assigned to bins
of width 0.05 and plotted against the proportion of replicates in each bin where the truth is |τ| = 1. Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 Page 14 of 23 Figure 2 Comparison of model-choice accuracy using DT threshold. Model-choice accuracy for models (A–D) MmsBayes, (E–H) MUshaped, (I–L)
MUniform, and (M–P) MDPP when analyzing data generated under models (A, E, I, and M) MmsBayes, (B, F, J, and N) MUshaped, (C, G, K, and O)
MUniform, and (D, H, L, and P) MDPP. The unadjusted posterior probability of a single divergence event, based on DT < 0.01, from 50,000 posterior
estimates are assigned to bins of width 0.05 and plotted against the proportion of replicates in each bin where the truth is DT < 0.01. Figure 2 Comparison of model-choice accuracy using DT threshold. Model-choice accuracy for models (A–D) MmsBayes, (E–H) MUshaped, (I–L)
MUniform, and (M–P) MDPP when analyzing data generated under models (A, E, I, and M) MmsBayes, (B, F, J, and N) MUshaped, (C, G, K, and O)
MUniform, and (D, H, L, and P) MDPP. Validation analyses: Model-choice accuracy The unadjusted posterior probability of a single divergence event, based on DT < 0.01, from 50,000 posterior
estimates are assigned to bins of width 0.05 and plotted against the proportion of replicates in each bin where the truth is DT < 0.01. Figure 2 Comparison of model-choice accuracy using DT threshold. Model-choice accuracy for models (A–D) MmsBayes, (E–H) MUshaped, (I–L)
MUniform, and (M–P) MDPP when analyzing data generated under models (A, E, I, and M) MmsBayes, (B, F, J, and N) MUshaped, (C, G, K, and O)
MUniform, and (D, H, L, and P) MDPP. The unadjusted posterior probability of a single divergence event, based on DT < 0.01, from 50,000 posterior
estimates are assigned to bins of width 0.05 and plotted against the proportion of replicates in each bin where the truth is DT < 0.01. I find that all four models accurately estimate the poste-
rior probability of the one-divergence model when applied
to their own datasets (i.e., when the prior is correct; see
diagonal of Figures 1 and 2). The MUniform and MDPP
models show robustness to prior violations and perform
well when applied to data generated under other models
(Figures 1 and 2). However, both are less accurate and tend
to underestimate the probability of the one-divergence
model when applied to the data generated under MUshaped
(Figures 1 and 2). In contrast, the MmsBayes model is
biased toward overestimating the posterior probability of
the one-divergence model when applied to data gener-
ated under the other three models (Figures 1 and 2). This
bias is particularly strong whenever divergence models are
not distributed under its U-shaped prior (Figure 1C–D). The other model with the U-shaped prior on divergence
models, but exponential priors on parameters (MUshaped),
performs similarly to the MmsBayes model in that it per-
forms well when applied to its own data, but overestimates
the probability of the one-divergence model when applied
to data generated by the other models (Figures 1 and 2). However, the bias is stronger in the MmsBayes model than
MUshaped. The other model with the U-shaped prior on divergence
models, but exponential priors on parameters (MUshaped),
performs similarly to the MmsBayes model in that it per-
forms well when applied to its own data, but overestimates
the probability of the one-divergence model when applied
to data generated by the other models (Figures 1 and 2). Validation analyses: Ordered divergence models The results show that the method performs similarly when
sampling over ordered models of divergence (Additional
file 1: Figures S9 and S10). This suggests that the method
is not adversely affected by the increase in the number
of possible discrete models (from 22 unordered to 4140
ordered models) when there are eight pairs of populations. This is encouraging, because, as discussed above, esti-
mating unordered models of divergence by shuffling the
summary statistic vectors calculated from the sequence
alignments is not valid for most empirical datasets. Given these results, estimation of unordered divergence
models should be avoided for empirical applications of the
method. Power analyses: Model choice The modifications of the msBayes model decrease
the method’s bias toward clustered divergences when
applied to data generated under random divergence times
(Figure 3 and Additional file 1: Figures S14–16). The
MmsBayes model performs the worst of the four models
across all three series of data-generating models, infer-
ring a single divergence event across most of the 18,000
simulations (Figure 3A–D and plots A–F of Additional
file 1: Figures S14–16). Importantly, the MmsBayes model
tends to strongly support these estimates of one diver-
gence across most of the simulations (Figure 4A–D and
plots A–F of Additional file 1: Figures S17–19). The MDPP
model also prefers the one-divergence model when diver-
gences are random over narrow windows of time, but
performs much better when divergences are random over
a timescale of 1–2 coalescent units (Figure 3M–P and
plots S–X of Additional file 1: Figures S14–16). How-
ever, even when MDPP infers the one-divergence model
over narrow timescales, the posterior probability support
is always low (Figure 4M–P and plots S–X of Additional
file 1: Figures S17–19). The MUniform model never infers
the one-divergence model in any of the simulation repli-
cates but still tends to infer relatively few (4–6) divergence
events when divergences are random over longer periods
(Figure 3I–L and plots M–R of Additional file 1: Figures
S14–16). Using exponential priors on divergence-time and
demographic parameters does increase the power of the
MUshaped model compared to MmsBayes across all three
series of data models, but the U-shaped prior still pre-
vents the model from performing as well as the MDPP and
MUniform models (Figure 3 and Additional file 1: Figures
S14–16). Validation analyses: Model-choice accuracy However, the bias is stronger in the MmsBayes model than
MUshaped. Overall, the results suggest that the MDPP and MUniform
models are relatively robust in terms of model-choice
accuracy, and when model violations do cause them to
be biased, they tend to under-estimate the probability of
the model with a single, shared divergence event. In con-
trast, the MmsBayes model is very sensitive to model vio-
lations, and strongly over-estimates the probability of the Page 15 of 23 Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 and MDPP/MUniform models (Additional file 1: Figure S12). The results are very similar when the four models are
applied to the data simulated under the MmsBayes series of
models (Additional file 1: Figure S13). one-divergence model whenever the model is misspec-
ified. Furthermore, the results suggest that using expo-
nentially distributed priors on nuisance parameters rather
than uniform priors helps the MUshaped model perform
better than MmsBayes, but it is still hindered by the
U-shaped prior on divergence models and tends to over-
estimate the probability of the one-divergence model
whenever there are violations of the model. Power analyses: Estimation accuracy The 22 divergence
times were randomly drawn as indicated above each column of plots, where time is respresented as millions of generations ago (MGA) according to
a per-site rate of 1 × 10−8 mutations per generation. Four of the six data-generating models of the MmsBayes series are shown; please see
Additional file 1: Figure S14 for all results. Figure 3 Power to avoid spurious estimation of clustered divergences when divergence times are random. The power of models (A–D)
MmsBayes, (E–H) MUshaped, (I–L) MUniform, and (M–P) MDPP to detect random variation in divergence times as simulated under the MmsBayes series of
models. The plots illustrate the estimated number of divergence events (|ˆτ|) from analyses of 1000 datasets simulated under each of the MmsBayes
models, with the the estimated probability of the model inferring one divergence event, p(|ˆτ| = 1), given for each combination. The 22 divergence
times were randomly drawn as indicated above each column of plots, where time is respresented as millions of generations ago (MGA) according to
a per-site rate of 1 × 10−8 mutations per generation. Four of the six data-generating models of the MmsBayes series are shown; please see
Additional file 1: Figure S14 for all results. the simulations. This is in contrast to the MmsBayes
model, which infers high posterior probabilities of a
single divergence for most replicates across all simula-
tions (Figure 4 and Additional file 1: Figures S17–19). The
exponential priors on divergence-time and demographic
parameters (model MUshaped) result in lower estimates
of the probability of one divergence when compared
to MmsBayes, but higher estimates when compared to
MUniform and MDPP (Figure 4 and Additional file 1: Figures
S17–19). The MDPP and MUniform models do frequently
support the one-divergence model according to a Bayes
factor criterion of greater than 10, but still less frequently
than the MmsBayes model. This result is not surprising power than the models with Dirichlet-process or uniform
priors across divergence models (Figure 5 and Additional
file 1: Figure S20–22). Although the DT threshold of 0.01
is arbitrary, Oaks et al. [7] did show via simulation that
the true value of DT will almost always be greater than
0.01 when divergences are random over periods of 0.1
coalescent units or more (see Figure Sfour of [7]). Power analyses: Estimation accuracy All of the models I evaluated (Table 2) struggle to esti-
mate the variance of divergence times DT regardless of
which of the three series of models (Table 3) the data were
generated under (Additional file 1: Figures S11–13). The
models with the U-shaped prior on divergence models
(MmsBayes and MUshaped) tend to underestimate the vari-
ance in divergence times (Plots A–L of Additional file 1:
Figures S11–13). whereas the models with Uniform or
Dirichlet-process priors over divergence models tend to
overestimate variance in divergence times (Plots M–X of
Additional file 1: Figures S11–13). When the divergence times of the 22 population pairs
are randomly drawn from a series of exponential priors
(MExp), the MDPP model is the best estimator of DT, fol-
lowed by MUniform (Additional file 1: Figure S11). The
MmsBayes model is strongly biased toward underestimat-
ing DT, estimating values of zero for most of the replicates
across all the data models of MExp (Additional file 1:
Figure S11). The results of the MUshaped model are inter-
mediate between those of MmsBayes and the new models
MDPP and MUniform (Additional file 1: Figure S11). The improved power of the new models is even more
pronounced when looking at estimates of the variance of
divergence times (DT) across the simulations (Figure 5
and Additional file 1: Figures S20–22). The performance
among the models is so different, that the histograms of
DT estimates cannot be plotted along a shared x-axis. The
MDPP and MUniform models perform similarly across all
three series of data models, inferring values of DT con-
sistent with one divergence event (DT < 0.01) in almost
none of the replicates across all the simulations. In con-
trast, the MmsBayes model infers values consistent with a
single divergence event in most of the replicates across all
the simulations. Using exponential priors on divergence-
time and demographic parameters greatly increases the
power of the MUshaped model to detect variation in
divergence times relative to MmsBayes, but it still has less f
Similarly, when the true divergence times are ran-
domly drawn from a series of uniform priors (MUniform),
the MDPP and MUniform models tend to over-estimate
the variance in divergence times, whereas the MmsBayes
model underestimates DT, estimating values of zero for
most replicates across all the data models of MUniform
(Additional file 1: Figure S12). Power analyses: Estimation accuracy Again, the performance of
the MUshaped model is intermediate between the MmsBayes Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 Page 16 of 23 Figure 3 Power to avoid spurious estimation of clustered divergences when divergence times are random. The power of models (A–D)
MmsBayes, (E–H) MUshaped, (I–L) MUniform, and (M–P) MDPP to detect random variation in divergence times as simulated under the MmsBayes series of
models. The plots illustrate the estimated number of divergence events (|ˆτ|) from analyses of 1000 datasets simulated under each of the MmsBayes
models, with the the estimated probability of the model inferring one divergence event, p(|ˆτ| = 1), given for each combination. The 22 divergence
times were randomly drawn as indicated above each column of plots, where time is respresented as millions of generations ago (MGA) according to
a per-site rate of 1 × 10−8 mutations per generation. Four of the six data-generating models of the MmsBayes series are shown; please see
Additional file 1: Figure S14 for all results. Figure 3 Power to avoid spurious estimation of clustered divergences when divergence times are random. The power of models (A–D)
MmsBayes, (E–H) MUshaped, (I–L) MUniform, and (M–P) MDPP to detect random variation in divergence times as simulated under the MmsBayes series of
models. The plots illustrate the estimated number of divergence events (|ˆτ|) from analyses of 1000 datasets simulated under each of the MmsBayes
models, with the the estimated probability of the model inferring one divergence event, p(|ˆτ| = 1), given for each combination. The 22 divergence
times were randomly drawn as indicated above each column of plots, where time is respresented as millions of generations ago (MGA) according to
a per-site rate of 1 × 10−8 mutations per generation. Four of the six data-generating models of the MmsBayes series are shown; please see
Additional file 1: Figure S14 for all results. Figure 3 Power to avoid spurious estimation of clustered divergences when divergence times are random. The power of models (A–D)
MmsBayes, (E–H) MUshaped, (I–L) MUniform, and (M–P) MDPP to detect random variation in divergence times as simulated under the MmsBayes series of
models. The plots illustrate the estimated number of divergence events (|ˆτ|) from analyses of 1000 datasets simulated under each of the MmsBayes
models, with the the estimated probability of the model inferring one divergence event, p(|ˆτ| = 1), given for each combination. Power analyses: Estimation accuracy The 22 divergence times were randomly drawn as
indicated above each column of plots, where time is respresented as millions of generations ago (MGA) according to a per-site rate of 1 × 10−8
mutations per generation. Four of the six data-generating models of the MmsBayes series are shown; please see Additional file 1: Figure S17 for all
results. upport for one divergence event when divergence times are random. The tendency of models (A–D)
d (M P) M
d
h
h
d
d Figure 4 Power to avoid spurious support for one divergence event when divergence times are random. The tendency of models (A–D)
MmsBayes, (E–H) MUshaped, (I–L) MUniform, and (M–P) MDPP to support one divergence event when there is random variation in divergence times as
simulated under the MmsBayes series of models. The plots illustrate histograms of the estimated posterior probability of the one divergence model,
p(|τ| = 1|Bϵ(S∗)), from analyses of 1000 datasets simulated under each of the MmsBayes models. The 22 divergence times were randomly drawn as
indicated above each column of plots, where time is respresented as millions of generations ago (MGA) according to a per-site rate of 1 × 10−8
mutations per generation. Four of the six data-generating models of the MmsBayes series are shown; please see Additional file 1: Figure S17 for all
results high probabilities, and MUshaped inferring intermediate
values (Figure 6 and Additional file 1: Figures S23–25). given the extremely small prior probability of the one-
divergence model under the MDPP and MUniform models
(i.e., very few posterior samples of the one-divergence
model will result in a large Bayes factor under these mod-
els). However, the small posterior probability of the one-
divergence model estimated under MDPP and MUniform
should prevent an investigator from overinterpreting the
Bayes factor as strong support for clustered divergences. Power analyses: Estimation accuracy As mentioned above, the increased power of the new
models is also evident when looking at the estimated
posterior probability of the one-divergence model across
the power analyses (Figure 4 and Additional file 1:
Figures S17–19). The MDPP and MUniform models estimate
low posterior probability of |τ|
=
1 across all of Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 Page 17 of 23 Figure 4 Power to avoid spurious support for one divergence event when divergence times are random. The tendency of models (A–D)
MmsBayes, (E–H) MUshaped, (I–L) MUniform, and (M–P) MDPP to support one divergence event when there is random variation in divergence times as
simulated under the MmsBayes series of models. The plots illustrate histograms of the estimated posterior probability of the one divergence model,
p(|τ| = 1|Bϵ(S∗)), from analyses of 1000 datasets simulated under each of the MmsBayes models. The 22 divergence times were randomly drawn as
d
d b
h
l
f l
h
d
ll
f
( G )
d
f
8 Figure 4 Power to avoid spurious support for one divergence event when divergence times are random. The tendency of models (A–D)
MmsBayes, (E–H) MUshaped, (I–L) MUniform, and (M–P) MDPP to support one divergence event when there is random variation in divergence times as
simulated under the MmsBayes series of models. The plots illustrate histograms of the estimated posterior probability of the one divergence model,
p(|τ| = 1|Bϵ(S∗)), from analyses of 1000 datasets simulated under each of the MmsBayes models. The 22 divergence times were randomly drawn as
indicated above each column of plots, where time is respresented as millions of generations ago (MGA) according to a per-site rate of 1 × 10−8
mutations per generation. Four of the six data-generating models of the MmsBayes series are shown; please see Additional file 1: Figure S17 for all
results. Figure 4 Power to avoid spurious support for one divergence event when divergence times are random. The tendency of models (A–D)
MmsBayes, (E–H) MUshaped, (I–L) MUniform, and (M–P) MDPP to support one divergence event when there is random variation in divergence times as
simulated under the MmsBayes series of models. The plots illustrate histograms of the estimated posterior probability of the one divergence model,
p(|τ| = 1|Bϵ(S∗)), from analyses of 1000 datasets simulated under each of the MmsBayes models. Empirical results The 22 divergence times were randomly drawn as indicated above each column of plots, where time is respresented as millions of
generations ago (MGA) according to a per-site rate of 1 × 10−8 mutations per generation. Four of the six data-generating models of the MmsBayes
series are shown; please see Additional file 1: Figure S20 for all results. Figure 5 Power to avoid spurious estimation of small temporal variance in divergences when divergence times are random. The power of
models (A–D) MmsBayes, (E–H) MUshaped, (I–L) MUniform, and (M–P) MDPP to detect random variation in divergence times as simulated under the
MmsBayes series of models. The plots illustrate the estimated dispersion index of divergence times (|ˆτ|) from analyses of 1000 datasets simulated
under each of the MmsBayes models, with the the estimated probability of the model inferring one divergence event, p(|ˆτ| < 0.01), given for each
combination. The 22 divergence times were randomly drawn as indicated above each column of plots, where time is respresented as millions of
generations ago (MGA) according to a per-site rate of 1 × 10−8 mutations per generation. Four of the six data-generating models of the MmsBayes
series are shown; please see Additional file 1: Figure S20 for all results. shared among the taxa, especially under the DPP models
(Figure 7B–E). Figure 7 also shows the prior distribution
across the number of divergence events (|τ|) for each
model, as well as the average prior probability of an
unordered and ordered model of divergence (t) across |τ|. Estimates under the new models tend to be similar to
the prior, which is expected under such a parameter-rich
model when there is limited information from the data
(four summary statistics from a single locus for each pair
of taxa). have the highest probability under each of the models
(Additional file 1: Figures S26–30). Again, the new models
estimate more divergences, a large amount of poste-
rior uncertainty, and an order of magnitude smaller
probability for their respective MAP-divergence model
when compared to the MmsBayes model (Additional file 1:
Figures S26–30). Figure 8 shows the estimated posterior probability dis-
tribution over the number of divergence events when the
data from the nine-taxon pairs from the Islands of Negros
and Panay are analyzed under DPP models that sample
over unordered (MDPP) and ordered (M◦
DPP) models of
divergence. Empirical results As expected based on the results of Oaks et al. [7], when
the Philippines data are analyzed under the MmsBayes
model, there is strong support for very few divergence
events shared among all 22 pairs of taxa, with a maximum
a posteriori (MAP) estimate of one-shared divergence
(Figure 7A). When these data are analyzed using mod-
els allowed by the new implementation, there is much
less support for highly clustered models and much greater
uncertainty regarding the number of divergence events Lastly, when looking at the estimated posterior prob-
ability of DT being consistent with one shared diver-
gence (p(DT < 0.01|Bϵ(S∗))), I find the same pattern
of model behavior, with MDPP and MUniform inferring
low probabilities across all simulations, MmsBayes inferring Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 Page 18 of 23 Figure 5 Power to avoid spurious estimation of small temporal variance in divergences when divergence times are random. The power of
models (A–D) MmsBayes, (E–H) MUshaped, (I–L) MUniform, and (M–P) MDPP to detect random variation in divergence times as simulated under the
MmsBayes series of models. The plots illustrate the estimated dispersion index of divergence times (|ˆτ|) from analyses of 1000 datasets simulated
under each of the MmsBayes models, with the the estimated probability of the model inferring one divergence event, p(|ˆτ| < 0.01), given for each
combination. The 22 divergence times were randomly drawn as indicated above each column of plots, where time is respresented as millions of
generations ago (MGA) according to a per-site rate of 1 × 10−8 mutations per generation. Four of the six data-generating models of the MmsBayes
series are shown; please see Additional file 1: Figure S20 for all results. Figure 5 Power to avoid spurious estimation of small temporal variance in divergences when divergence times are random. The power of
models (A–D) MmsBayes, (E–H) MUshaped, (I–L) MUniform, and (M–P) MDPP to detect random variation in divergence times as simulated under the
MmsBayes series of models. The plots illustrate the estimated dispersion index of divergence times (|ˆτ|) from analyses of 1000 datasets simulated
under each of the MmsBayes models, with the the estimated probability of the model inferring one divergence event, p(|ˆτ| < 0.01), given for each
combination. Empirical results The results are similar under both models The disparity between the results of the MmsBayes model
and the new models is even more pronounced when
looking at the 10 divergence models (t) estimated to Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 Page 19 of 23 Figure 6 Power to avoid spurious support for no temporal variance in divergences (i.e., DT < 0.01) when divergence times are random. The tendency of models (A–D) MmsBayes, (E–H) MUshaped, (I–L) MUniform, and (M–P) MDPP to support one divergence event when there is random
variation in divergence times as simulated under the MmsBayes series of models. The plots illustrate histograms of the estimated posterior
probability of the one divergence model, p(DT < 0.01|Bϵ(S∗)), from analyses of 1000 datasets simulated under each of the MmsBayes models. The
22 divergence times were randomly drawn as indicated above each column of plots, where time is respresented as millions of generations ago
(MGA) according to a per-site rate of 1 × 10−8 mutations per generation. Four of the six data-generating models of the MmsBayes series are shown;
please see Additional file 1: Figure S23 for all results. Figure 6 Power to avoid spurious support for no temporal variance in divergences (i.e., DT < 0.01) when divergence times are random. The tendency of models (A–D) MmsBayes, (E–H) MUshaped, (I–L) MUniform, and (M–P) MDPP to support one divergence event when there is random
variation in divergence times as simulated under the MmsBayes series of models. The plots illustrate histograms of the estimated posterior
probability of the one divergence model, p(DT < 0.01|Bϵ(S∗)), from analyses of 1000 datasets simulated under each of the MmsBayes models. The
22 divergence times were randomly drawn as indicated above each column of plots, where time is respresented as millions of generations ago
(MGA) according to a per-site rate of 1 × 10−8 mutations per generation. Four of the six data-generating models of the MmsBayes series are shown;
please see Additional file 1: Figure S23 for all results. Figure 6 Power to avoid spurious support for no temporal variance in divergences (i.e., DT < 0.01) when divergence times are random. The tendency of models (A–D) MmsBayes, (E–H) MUshaped, (I–L) MUniform, and (M–P) MDPP to support one divergence event when there is random
variation in divergence times as simulated under the MmsBayes series of models. Empirical results The plots illustrate histograms of the estimated posterior
probability of the one divergence model, p(DT < 0.01|Bϵ(S∗)), from analyses of 1000 datasets simulated under each of the MmsBayes models. The
22 divergence times were randomly drawn as indicated above each column of plots, where time is respresented as millions of generations ago
(MGA) according to a per-site rate of 1 × 10−8 mutations per generation. Four of the six data-generating models of the MmsBayes series are shown;
please see Additional file 1: Figure S23 for all results. Figure 6 Power to avoid spurious support for no temporal variance in divergences (i.e., DT < 0.01) when divergence times are random. The tendency of models (A–D) MmsBayes, (E–H) MUshaped, (I–L) MUniform, and (M–P) MDPP to support one divergence event when there is random
variation in divergence times as simulated under the MmsBayes series of models. The plots illustrate histograms of the estimated posterior
probability of the one divergence model, p(DT < 0.01|Bϵ(S∗)), from analyses of 1000 datasets simulated under each of the MmsBayes models. The
22 divergence times were randomly drawn as indicated above each column of plots, where time is respresented as millions of generations ago
(MGA) according to a per-site rate of 1 × 10−8 mutations per generation. Four of the six data-generating models of the MmsBayes series are shown;
please see Additional file 1: Figure S23 for all results. and, again, yield a large amount of uncertainty about the
number of divergence events that is similar to the prior
uncertainty. taxa is maintained, and thus a fully marginalized esti-
mate of divergence times across the taxa can be obtained
(Additional file 1: Figure S33), and (3) the probability of
co-divergence among any set of taxa can be estimated
from the posterior sample. The small difference between the results of the MDPP
and M◦
DPP models is consistent across multiple analy-
ses, and thus could be due to error introduced to the
MDPP model by the invalid shuffling of the summary
statistic vectors. Both models estimate a similar set of 10
unordered divergence models with the highest posterior
probability (Additional file 1: Figures S31 and S32). Discussion My results demonstrate that using alternative priors on
parameters and divergence models improved the behav-
ior of the msBayes model. In the new implementation,
model-choice estimation is more accurate and shows
greater robustness to model violations (Figures 1 and 2). The main advantages of the M◦
DPP model over the
MDPP are that (1) the incorrect shuffling of the sum-
mary statistic vectors is avoided, (2) the identity of the Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 Page 20 of 23 Figure 7 Estimated number of divergence events for 22 taxa from the Philippines. The (A–E) posterior and (F–J) prior probabilities of the
number of divergence events (|τ|) when the data of the 22 pairs of taxa from the Philippines are analyzed under the five models indicated at the
top of each column of plots (Table 4). The average prior probability of an (K–O) unordered and (P–T) ordered model of divergence (t) with |τ|
divergence-time parameters is also shown. The posterior median of the dispersion index of divergence times (DT) is also given for each model,
followed by the 95% highest posterior density interval in parentheses. Figure 7 Estimated number of divergence events for 22 taxa from the Philippines. The (A–E) posterior and (F–J) prior probabilities of the
number of divergence events (|τ|) when the data of the 22 pairs of taxa from the Philippines are analyzed under the five models indicated at the
top of each column of plots (Table 4). The average prior probability of an (K–O) unordered and (P–T) ordered model of divergence (t) with |τ|
divergence-time parameters is also shown. The posterior median of the dispersion index of divergence times (DT) is also given for each model,
followed by the 95% highest posterior density interval in parentheses. across a range of conditions underlying the data. The new
method, dpp-msBayes, has greater power to detect ran-
dom temporal variation in divergences, is less prone to
spurious inference of clustered divergence models, and
much less likely to incorrectly infer such models with
strong support (Figures 3, 4, 5 and 6). The original model is very sensitive to violations and,
when present, strongly over-estimates the probability of
one-divergence event shared across all taxa (Figures 1
and 2). Discussion The posterior median of the dispersion
index of divergence times (DT) is also given for each model, followed
by the 95% highest posterior density interval in parentheses. divergence models. This suggests, as predicted by Oaks
et al. [7,15], that the tendency of msBayes to erroneously
support models of temporally clustered divergences is
caused by a combination of (1) small marginal likelihoods
of models with more τ parameters due to uniform pri-
ors on divergence-time and demographic parameters and
(2) the U-shaped prior on divergence models giving low
prior density to models with intermediate numbers of
divergence parameters. The former essentially rules out
models with many τ parameters, which causes the latter
to act like an "L-shaped" prior with a spike of prior den-
sity on the one-divergence model. Given the parameter
richness of the model and the relatively small amount of
information in the summary statistics, it is not surpris-
ing that the combination of these two factors can create a
strong tendency to infer clustered models of divergence. While the modifications improve the behavior of the
model, I urge caution when using the method and inter-
preting its results. The method attempts to approximate
the posterior of a very parameter-rich model using rel-
atively little information from the data. For example,
when applied to the dataset of 22 taxon pairs from the
Philippines [7], the model has as many as 604–625 free
parameters (depending on |τ|), and samples over 1002
unordered divergence models. Even under the simplest
possible model allowed under the new implementation,
the model still has 471–492 free parameters. Furthermore,
the stochastic coalescent and mutational processes being
modeled predict a large amount of variation in possi-
ble datasets even when the parameter values are known. The richness and stochastic nature of the model makes
for a difficult inference problem, especially when using a
small number of summary statistics calculated from the
sequence alignments of each taxon pair. The population-
genetic summary statistics used by the method contain
little information about many of the free parameters in
the model. Thus, I expect the improved method will still
be sensitive to priors, and the power, while improved,
may still be low. While there is much less prior sensitiv-
ity under the new model compared to those observed by
Oaks et al. Discussion When more appropriate priors are used for
divergence-time and demographic parameters, and either
a Dirichlet-process or uniform prior applied across diver-
gence models, the model is less sensitive to violations,
and, when violations do cause bias, the method tends
to underestimate the probability of models with tempo-
rally clustered divergences (Figures 1 and 2). Given that
clustered models are often of particular interest to bio-
geographers, this behavior of the new method can be
considered conservative. By evaluating a model intermediate between the old
and new implementation (MUshaped), I was able to deter-
mine the relative affects of my modifications to the model. Across all of the analyses, the results show that using bet-
ter priors on divergence-time and demographic parame-
ters alone does improve the performance of the method. The magnitude of the bias toward inferring support for
the one-divergence model when there are model viola-
tions is reduced when the exponential priors are used in
place of the uniform priors (Figures 1 and 2). Furthermore,
using exponential priors improves the method’s power
to detect temporally random divergences (Figures 3, 4, 5
and 6). Throughout the analyses, the intermediate model
(MUshaped) performs better than the msBayes model,
but not as well as the models with alternative priors on The modifications also improve the method’s power to
detect random variation in divergence times, reducing the
tendency to estimate clustered divergences (Figures 3, 4, 5
and 6). My results are similar to those of Oaks et al. [7]
in that I find msBayes will often infer strong support for
clustered divergences when divergences are random over
quite broad timescales (Figures 3, 4, 5 and 6). My results
expand on this by showing that this behavior is consistent Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 Page 21 of 23 Page 21 of 23 Figure 8 Estimated number of divergence events for 9 taxa from
the Philippines. The posterior probabilities of the number of
divergence events, |τ|, when the data of the 9 pairs of taxa from
Negros and Panay Islands are analyzed under the DPP model that
samples over (A) unordered and (B) ordered models of divergence
(Table 4). Both models share the same (C) prior probability of the
number of divergence events, and the average prior probability of an
(D) unordered and (E) ordered model of divergence (t) with |τ|
divergence-time parameters. Discussion [7], there is still an effect when comparing
the results of the empirical data analyzed under a dif-
fuse (MDPP) and informative
Minform
DPP
divergence-time
prior (Figure 7C versus D). The fact that the posterior
shifts toward the prior under the informative prior sug-
gests that the shift away from the prior toward fewer
divergence events under the diffuse prior might still be
caused by small marginal likelihoods of models with more
divergence-time parameters (Figure 7). Figure 8 Estimated number of divergence events for 9 taxa from
the Philippines. The posterior probabilities of the number of
divergence events, |τ|, when the data of the 9 pairs of taxa from
Negros and Panay Islands are analyzed under the DPP model that
samples over (A) unordered and (B) ordered models of divergence
(Table 4). Both models share the same (C) prior probability of the
number of divergence events, and the average prior probability of an
(D) unordered and (E) ordered model of divergence (t) with |τ|
divergence-time parameters. The posterior median of the dispersion
index of divergence times (DT) is also given for each model, followed
by the 95% highest posterior density interval in parentheses. Figure 8 Estimated number of divergence events for 9 taxa from Figure 8 Estimated number of divergence events for 9 taxa from
the Philippines. The posterior probabilities of the number of
divergence events, |τ|, when the data of the 9 pairs of taxa from
Negros and Panay Islands are analyzed under the DPP model that
samples over (A) unordered and (B) ordered models of divergence
(Table 4). Both models share the same (C) prior probability of the
number of divergence events, and the average prior probability of an
(D) unordered and (E) ordered model of divergence (t) with |τ|
divergence-time parameters. The posterior median of the dispersion
index of divergence times (DT) is also given for each model, followed
by the 95% highest posterior density interval in parentheses. Nonetheless, it is reassuring to see a large amount of
posterior uncertainty when the new implementation is
applied to the empirical datasets (Figures 7 and 8). Appli-
cations of the msBayes model often result in strong pos-
terior support for estimated scenarios (e.g., [3,5-12]), as I Page 22 of 23 Oaks BMC Evolutionary Biology 2014, 14:150
http://www.biomedcentral.com/1471-2148/14/150 found here (Figure 7). Authors’ contributions All aspects of this work were done by JRO. Discussion Given the richness of the model, the
variance of the processes being modeled, and the relatively
small amount of information in the summary statistics
calculated from the sequence data, finding strong pos-
terior support for any scenario is unexpected. Based on
results of the empirical and power analyses (Figures 4, 6, 7
and 8), the new implementation more accurately reflects
posterior uncertainty and avoids spurious support for
biogeographical scenarios. the new approach better estimates posterior uncertainty,
which greatly reduces the chances of incorrectly esti-
mating biogeographical scenarios of shared divergence
events. This is important, because models of shared diver-
gence events are often ofparticular interest to researchers
who employ these methods. This new tool will allow
evolutionary biologists to better leverage comparative
genetic data to assess the affects of regional and global
biogeographical processes on biodiversity. I also urge caution when using dpp-msBayes due
to the lack of theoretical validation of Bayesian model
choice when the full data are replaced by summary statis-
tics that are insufficient for discriminating across models
under comparison [44], which is certainly the case here. Robert et al. [44] demonstrated that ABC estimates of
model posterior probabilities can be inaccurate when such
across-model insufficient statistics are used. Acknowledgements I am greatful to Melissa Callahan, Mark Holder, Emily McTavish, Daniel Money,
Jordan Koch, Adam Leaché, Peter Foster, two anonymous reviewers, and
Christian Robert and his blog (http://xianblog.wordpress.com/) for insightful
comments that greatly improved this work. I thank the National Science
Foundation for supporting this work (DEB 1011423 and DBI 1308885). This
work was also supported by the University of Kansas (KU) Office of Graduate
Studies, Society of Systematic Biologists, Sigma Xi Scientific Research Society,
KU Department of Ecology and Evolutionary Biology, and the KU Biodiversity
Institute. I also thank Mark Holder, the KU Information and Telecommunication
Technology Center, KU Computing Center, and the iPlant Collaborative for the
computational support necessary to conduct the analyses presented herein. Given the difficulty of this estimation problem, I antic-
ipate that full-likelihood methods that can leverage all of
the information present in the sequence data will become
increasingly important for robustly estimating shared
evolutionary history across taxa [45]. With improving
numerical methods for sampling over models of differing
dimensionality [46,47], advances in Monte Carlo tech-
niques [48], and increasing efficiency of likelihood calcu-
lations [49], analyzing rich comparative phylogeograpical
models in a full-likelihood Bayesian framework is becom-
ing computationally practical, especially when consider-
ing that simulating millions of random datasets from the
prior under the simple ABC rejection approach is ineffi-
cient and computationally nontrivial. Received: 7 March 2014 Accepted: 10 June 2014
Published: 3 July 2014 1.
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Table S1 and Figures S1-S33. As referenced in the main text. Competing interests
The author declare that he has no competing interests. Competing interests
The author declare that he has no competing interests. Competing interests Given all of these caveats, I encourage investigators
to view this method as a means of exploring their data
for general temporal patterns of divergences across taxa,
rather than a rigorous means of evaluating hypothe-
ses. As recommended by Oaks et al. [7], any results
from the method should be accompanied by (1) analy-
ses under a variety of priors to assess the assumptions
underlying model inference and the prior sensitivity of the
results, and (2) simulation-based power analyses to pro-
vide insight into the temporal resolution of the method. Both approaches are important to help guide the interpre-
tation of results. Authors’ contributions
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introduced
a
new
model
for
estimating
shared
divergence histories across taxa from DNA sequence
data
within
an
approximate-Bayesian
model-choice
framework. The new method, dpp-msBayes, takes
a non-parametric approach to the problem by using
a Dirichlet-process prior on the temporal distribution
of divergences across taxa. The new method shows
improved robustness, accuracy, and power compared to
the existing method, msBayes. Compared to msBayes, ,
( )
j
8. Barber BR, Klicka J: Two pulses of diversification across the Isthmus of
Tehuantepec in a montane Mexican bird fauna. Proc R Soc B-Biol Sci
1694, 277:2675–2681. doi:10.1098/rspb.2010.0343. Oaks BMC Evolutionary Biology 2014, 14:150
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Stability predicts genetic diversity in the Brazilian Atlantic forest
Hotspot. Science 2009, 323(5915):785–789. doi:10.1126/science.1166955. 10. Chan LM, Brown JL, Yoder AD: Integrating statistical genetic and
geospatial methods brings new power to phylogeography. Mol
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via sequential Monte Carlo. Syst Biol 2012, 61(4):579–593. doi:10.1093/sysbio/syr131. 21. Sloan NJA: The on-line encyclopedia of integer sequences, sequence
A000041. http://oeis.org/A000041. 22. Sloan NJA: The on-line encyclopedia of integer sequences, Sequence
A008284. http://oeis.org/A008284. 49. Ayres DL, Darling A, Zwickl DJ, Beerli P, Holder MT, Lewis PO, Huelsenbeck
JP, Ronquist F, Swofford DL, Cummings MP, Rambaut A, Suchard MA:
BEAGLE: an application programming interface and
high-performance computing library for statistical phylogenetics. Syst Biol 2012, 61(1):170–173. 49. Ayres DL, Darling A, Zwickl DJ, Beerli P, Holder MT, Lewis PO, Huelsenbeck
JP, Ronquist F, Swofford DL, Cummings MP, Rambaut A, Suchard MA: 23. Malenfant J: Finite, closed-form expressions for the partition
function and for Euler, Bernoulli, and Stirling numbers. 2011. arXiv:1103.1585v6 [math.NT] http://arxiv.org/abs/1103.1585. Conclusions BEAGLE: an application programming interface and
high-performance computing library for statistical phylogenetics. Syst Biol 2012, 61(1):170–173. high performance computing library for statistical phylogenetics. Syst Biol 2012, 61(1):170–173. 24. Ferguson TS: A Bayesian analysis of some nonparametric problems. Ann Stat 1973, 1(2):209–230. 25. Antoniak CE: Mixtures of Dirichlet processes with applications to
Bayesian nonparametric problems. Ann Stat 1974, 2(6):1152–1174. doi:10.1186/1471-2148-14-150
Cite this article as: Oaks: An improved approximate-Bayesian
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concordance among gene trees. Mol Biol Evol 2007, 24(2):412–426. doi:10.1093/molbev/msl170. 29. Ané C, Larget B, Baum DA, Smith SD, Rokas A: Bayesian estimation of
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29(3):939–955. doi:10.1093/molbev/msr255. Conclusions Submit your next manuscript to BioMed Central
and take full advantage of:
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and take full advantage of:
• Convenient online submission
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• Immediate publication on acceptance
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and take full advantage of: 36. Tajima F: Statistical method for testing the neutral mutation
hypothesis by DNA polymorphism. Genetics 1989, 123(3):585–595. 36. Tajima F: Statistical method for testing the neutral mutation
hypothesis by DNA polymorphism. Genetics 1989, 123(3):585–595.
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The prevalence of SCT in China, its comorbidity with ADHD and its association with life events and parental-rearing behaviors
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Scientific reports
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cc-by
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The prevalence of SCT in China,
its comorbidity with ADHD and its
association with life events
and parental‑rearing behaviors
Fenghua Li 1,3, Jie Luo 2,3, Yanjie Qi 2, Huanhuan Huang 2, Yuanzhen Wu 2, Gaoyang Xu 2,
Zhengkui Liu 1, Fan He 2* & Yi Zheng 2*
OPEN Although sluggish cognitive tempo (SCT) symptoms are often observed in children with attention
deficit hyperactivity disorder (ADHD), an increasing number of studies have highlighted its
uniqueness. Nevertheless, no national survey on SCT among children and adolescents has been
conducted in China. Hence, this research aims to study SCT in China and to evaluate the differences
between SCT and ADHD symptoms by comparing their risk factors in terms of life events (LE) and
parental rearing behaviors (PRB). This cross-sectional study used data from a survey on 71,929
children and adolescents in 5 province-level regions in China to study the incidence and demographic
information of SCT in the Chinese population. Subsequently, the study investigated the comorbidity
of ADHD and SCT, and conducted three logistic regressions on the LE and PRB scores to predict
whether participants develop symptoms of ADHD or SCT, or neither symptom. 6658 participants
were allocated into the SCT group, and the weighted point prevalence of SCT was 9.78%. 36.34% of
participants with ADHD (n = 676) were found to demonstrate SCT symptoms, whereas no statistically
significant difference was observed in its comorbidity to the three ADHD subtypes (χ2 = 1.668, p > 0.05,
Δ = 2). The regression results on the presence or absence of ADHD revealed paternal excessive-
interference and rejection, and maternal favoring were associated with ADHD diagnosis, whereas
paternal punishment and favoring and maternal emotional warmth was related to the absence of
ADHD symptoms. Academic stress and maternal excessive-interference were associated with SCT
symptoms, and maternal emotional warmth associated with SCT absence. Concerning the presence
of ADHD-only or SCT-only symptoms, LE adaptation was found to relate to SCT-only symptoms,
while PRB paternal rejection and maternal favoring were associated with ADHD-only symptoms. While evidencing the high prevalence of SCT in China, our findings supported that although ADHD
and SCT were highly comorbid, they may be considered two independent disorders with different risk
factors. Specifically, participants with SCT symptoms are more vulnerable to stress from LE and tend
to face more maladjustment than ADHD and normally-developing participants, and maternal rearing
behaviours are the key factors to SCT symptoms. SCT brings global challenges in its diagnosis and
treatment, and the challenge is more severe in a mentally stressful environment. Therefore, stress
management and SCT etiology studies are recommended. Sluggish cognitive tempo (SCT) was recognized as a sub-phenotype of attention deficit hyperactivity disorder
(ADHD) inattentive subtype. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ Indeed, symptoms of SCT were found poorly correlated with symptoms of hyperactive-impulsive type ADHD4. A study demonstrated once symptoms of hyperactive-impulsive type ADHD were removed, no statistically
significant correlation could be observed between ADHD and SCT5.fi gi
Meanwhile, existing studies have further highlighted that exclusive symptoms of SCT as lethargy, difficulty
in initiating and sustaining effort, physical underactivity, excessive daydreaming, poor memory retrieval, and
easy confusion or mental fogginess6,7, and none of them are key symptoms of ADHD. Moreover, the symptom
differences between SCT and ADHD were observed via multiple measurements, such as ratings of parents and
teachers, school-based behavior monitoring, clinical behavior monitoring, etc8. Although no study has investi-
gated the extent to which SCT can be regarded as an independent disorder in China, studies in both Western and
Eastern cultures have been establishing the cross-cultural validity of distinctive SCT symptoms9–14. Particularly,
SCT has been studied not only in cultures rooted in individualism, but also in cultures that are similar to China in
terms of the roots in Confucianism and collectivism (e.g., South Korea10). Notably, the distinctive characteristics
of SCT were found based on both children and the self-reported assessment of senior groups, such as college stu-
dents and adults, further indicating that SCT is a stable independent disorder to a greater extent15. Results from a
recent meta-analysis demonstrated strong evidence for both internal and external validity of SCT by employing
demographic information, internalization/externalization problems assessment results, cognitive dysfunctions
and connections between cognition and neuropsychological functions16. More importantly, many studies have
proffered the biological differences between SCT and ADHD. For instance, research has provided evidence that
SCT symptoms are relevant to abnormal activities in the networks on orienting and attention shifting, whereas
ADHD was found to be associated with abnormalities in the network on executive functions17. In terms of brain
structures, SCT symptoms were relevant to altered frontal lobe anatomy in terms of the increased volume in
terms of the anomalously large cortical regions, which indicates immaturities in functional connectivity18,19. This
contrasted significantly with the brain anatomy and functional abnormalities of individuals developing ADHD,
highlighting the distinctions between the two disorders. Taken together, it may be considered that the uniqueness
of SCT has been widely evidenced by neuroimaging and heart rate variability studies20,21. y
y
g
g
y
Beyond neurocognitive and biological explanations for SCT and ADHD, nurture factors to the development
of their symptoms have received increasing attention. www.nature.com/scientificreports/ Particularly, although ADHD is largely considered a neuro-
cognitive condition, factors of school and family environment have been evidenced to play significant roles (i.e.,
either directly or indirectly through gene-environment interactions) in whether the symptoms develop to be more
impairing. Based on existing findings, life events (LE) and parental rearing behaviors (PRB) have been underlined
as two environmental risk factors for ADHD22–25. To explain, LE refers to events related to threatening stressors or
the adjustments for restoring normal life. Prior studies showed children with ADHD experienced more conflicts
in LE than normally developing children26. As a result, the elevated level of stress imposed by LE, such as suffering
from a serious illness or transferring to another school, would have a great impact on the development of children
with ADHD. In PRB, earlier studies reported positive and gentle PRB correlated with improved psychological
resilience children and reduced occurrence of mental disorders, including ADHD27. While negative and careless
PRB were also found to be positively associated with increased occurrence of ADHD28. p
y
Regarding SCT, twin studies have evidenced that SCT could be explained by environmental factors more
prominently than neurocognitive factors, highlighting its partially distinct etiological patterns to ADHD29. Therefore, it is reasonable to hypothesize that LE and PRB would affect patients with ADHD and SCT differently. However, no literature to our knowledge has studied specific environmental risk factors such as LE or PRB for
SCT. Based on the generally high relevance of PRB and LE to mental disorder symptoms27, PRB and LE may be
appropriate perspectives to explore SCT. In addition, as stated above, despite the increasing cross-cultural valid-
ity and internal reliability of SCT established from samples in countries with various cultural backgrounds9–14,
limited research has studied whether SCT can be considered independent within Chinese cultural backgrounds.i p
g
Overall, although SCT has not yet been defined as an independent disorder, existing evidence has suggested
its potential independence. However, limited research has studied SCT in China. This suggests that the prevalence
of SCT and the demographic information of the Chinese population developing this disorder remains largely
unexplored, leaving difficulties in evaluating the importance of SCT research in China. Therefore, the first aim
of the current study was (1) to investigate the prevalence and the demographic characteristics of SCT in Chinese
children and adolescents by age, gender and comorbidity to the three ADHD subtypes. www.nature.com/scientificreports/ In addition, considering
limited research has investigated the distinctions between SCT and ADHD by the environmental risk factors,
we also aimed (2) to evaluate if SCT can be considered as an independent disorder in China by assessing the
associations between the presence or absence of ADHD or SCT and the two primary independent variables in
terms of LE and PRB. The prevalence of SCT in China,
its comorbidity with ADHD and its
association with life events
and parental‑rearing behaviors
Fenghua Li 1,3, Jie Luo 2,3, Yanjie Qi 2, Huanhuan Huang 2, Yuanzhen Wu 2, Gaoyang Xu 2,
Zhengkui Liu 1, Fan He 2* & Yi Zheng 2*
OPEN However, although SCT has not yet been defined an independent disorder1, evi-
dence from factor analyses of prior studies has revealed significant differences between symptoms of ADHD
and SCT2. Specifically, a mutual variance of 25–36% was observed in ADHD-Inattentive and SCT, which implies
the symptom clusters are related but not collinearly correlated3. Barkley reported that 59% of children with SCT
were diagnosed to develop ADHD symptoms, while 39% of children with ADHD scored high in SCT symptoms2. 1Key Lab of Mental Health, Institute of Psychology, Chinese Academy of Sciences, Beijing, China. 2National Clinical
Research Center for Mental Disorders, Beijing Key Laboratory of Mental Disorders, Beijing Anding Hospital, Beijing
Institute for Brain Disorders Capital Medical University, De Sheng Men Wai An Kang Hu Tong 5 Hao, Xi Cheng Qu,
Beijing 100088, China. 3These authors contributed equally: Fenghua Li and Jie Luo. *email: hf981207@163.com;
yizheng@ccmu.edu.cn | https://doi.org/10.1038/s41598-023-43225-4 Scientific Reports | (2023) 13:16946 www.nature.com/scientificreports/ Materials and methods
Study designh This study used data from a large-scale mental disorder survey for children and adolescents in China. There
were two phases in this study (Fig. 1). In Phase I, researchers collected data by using the Sluggish Cognitive
Tempo-Children Behavior Checklist (SCT-CBCL) from 71,929 participants aged 6–16 in 5 province-level regions
(Beijing, Liaoning, Jiangsu, Hunan, Sichuan). We obtained the incidence and distribution of age and gender of
SCT. In Phase II, the diagnosis of ADHD was made following the DSM-IV by two psychiatrists with at least the
title of deputy chief physician. Based on the diagnoses, we investigated the comorbidity of ADHD and SCT. LE
and PRB data were also collected from Hunan and Sichuan. After excluding participants developing comorbid
ADHD and SCT symptoms, we allocated participants into either the ADHD-only group (i.e., participants with
only ADHD symptoms), the SCT-only group (i.e., participants with only SCT symptoms) or the control group
(i.e., participants who had not been diagnosed to develop any physical or psychological disorder). According to https://doi.org/10.1038/s41598-023-43225-4 Scientific Reports | (2023) 13:16946 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ Beijing
10215
Liaoning
11757
Jiangsu
13082
Hunan
16712
Sichuan
20341
Total 71,929 completed SCT-CBCL
3618 diagnosed ADHD/positive for SCT-
CBCLͧ1860 ADHD diagonsed by DSM-Ⅳ,
2434 SCT classified by SCT-CBCL)
119
SCT-only
1184
ADHD-only
676
ADHD+SCT
Excluded 6892 with no
diagnosis of ADHD, or
SCT-CBCL was negative
According to DSM-Ⅳ
interview excluded co-
morbidities
ADHD 676,
Anxiety disorders 864,
Depressive disorders 541,
Other disorders 234. A total of 61,419 people did
not continue to complete the
ASLEC and EMBU scales,
including a total of 35,054
people in Beijing, Liaoning
and Jiangsu provinces,
15,805 people in Sichuan
and 10,748 people in Hunan. Total 10,510 completed SCT-CBCL, ASLEC,
EMBU
Phase I
Phase II
Sichuan 4536, Hunan 5974 completed
ASLEC, EMBU
The prevalence of SCT
symptoms was calculated
using the top 5% of the
SCT-CBCL raw score as
the cut-off score. logistic regressions for
ADHD-only vs. Healthy,
SCT-only vs. Healthy
ADHD-only vs. SCT-only
were performed with LE
and PRB factors
Figure 1. Study profile. logistic regressions for
ADHD-only vs. Healthy,
SCT-only vs. Healthy
ADHD-only vs. SCT-only
were performed with LE
and PRB factors logistic regressions for
ADHD-only vs. Healthy,
SCT-only vs. Healthy
ADHD-only vs. SCT-only
were performed with LE
and PRB factors Figure 1. Study profile. the second research aim, the dependent variable was the three groups, and the independent variables were the
dimension scores of LE and PRB assessments. Materials and methods
Study designh To keep the homogeneity and the 1:1 sample size, participants were matched by province, age and gender
in Phase II investigations on SCT and ADHD presence or absence. In matching every pair of participants, we
focused on the group with a relatively small size, and all participants in the group with a relatively large size were
considered as candidates to be matched. Specifically, we matched one individual from the larger group to the one
participant selected from the smaller group. The selection followed a condition priority order of province, age and
gender. Once a participant was selected, they would be removed from the candidate list to ensure no participant
would be selected repeatedly. This process continued until we found the final pair of participants with all the
conditions matched. If the matching conditions of province or gender cannot be fulfilled, the condition would
be ignored, and the next condition selection would be performed in the one of the prior levels to the current
subset. If the age condition is not fulfilled, individuals with the closest age would be selected for the next condi-
tion matching. All participants and their parents signed consent forms before taking part in the study. This study
was approved by the Ethics Committee of Beijing Anding Hospital, Capital Medical University (2012BAI01B02). SCT‑CBCL SCT-CBCL (Sluggish Cognitive Tempo-Children Behavior Checklist) has been widely used in prior SCT studies. It has 4 items selected from CBCL30, including: 13 (confused/seems in a fog), 17 (daydreams), 80 (stares blankly)
and 102 (underactive). The items were presented on a 3-point scale of 0 (not true), 1 (somewhat or sometimes
true) and 2 (very true or often true). The total score ranged from 0 to 8, and larger total scores indicate more
severe SCT symptoms. In our study, the Cronbach’s Alpha of this scale was 0.7. EMBU
( EMBU (Egna Minnen Betraffande Uppfostran) is an assessment tool for PRB. The Chinese version of EMBU is a
4-point Likert scale with 66 items31. 58 items on 6 factors reflected the rearing behaviors of the father, including
Emotional Warmth (i.e., acceptance and recognition, frequent praise, unconditional love, support and affection
for the child), Severe Punishment (i.e., harsh disciplinary behavior, often through verbal or physical violence, to
restrain the child’s behavior), Excessive interference (i.e., controlling behavior, excessive demands on the child),
Favoring (i.e., more special care for the child, usually with more parental attention and indulgence than other
siblings), Rejecting (i.e., hostility, punishment, derogation and blame towards the child) and Overly-protecting
(i.e., fears and anxieties, feelings of guilt and intrusions about the child’s safety and health). There were also 57
items on 5 factors related to maternal rearing behaviours. The 5 factors are warm and affectionate, excessively
interfering, rejecting, punishing and severe, and favoring. Participants rated 1 for “never”, 2 for “yes but seldom”,
3 for “yes, often” and 4 for “yes, always” on each item, and the score on each factor was calculated by the sum
of all relevant items under it. The Cronbach’s α was between 0.717 and 0.893, and the test–retest reliability was
between 0.725 and 0.871. https://doi.org/10.1038/s41598-023-43225-4 Scientific Reports | (2023) 13:16946 | www.nature.com/scientificreports/ Data analytic plan y
p
In Phase I, we used the SCT-CBCL score at the top 5 percentile as the cutoff score18. Participants who scored
greater than the cutoff score were considered to develop SCT symptoms. The point-weighted prevalence of SCT
was also acquired. The overall sampling weights for participants in Phase I were the products of the sampling
weights of each participants’ provincial region, prefectural division, county/district, school, and class. Individuals
who withdrew or whose primary caregivers failed to finished the rating scale were treated as nonrespondents,
and relevant adjustment was included in the poststratification process. The reciprocal of response rate in the
corresponding demographic sub-group of each participant were used as their nonresponse weights33. SCT preva-
lence distributions in age spans and genders were obtained, and a chi-square test was carried out to find if the
prevalence was different in males and females. In Phase II, the age difference between ADHD and SCT groups
was inspected with a t-test. Comorbidity information of ADHD and SCT was acquired, and the SCT comorbid-
ity rates in subtypes of ADHD were compared by using chi-square tests. Finally, three logistic regressions were
conducted for LE and PRB scores on the presence or absence of ADHD and/or SCT symptoms. Specifically,
the associations between the two primary independent variables and the presence or absence of ADHD-only
symptoms, SCT-only symptoms and the presence of ADHD-only or SCT-only symptoms were assessed respec-
tively. Dimension scores on ALSEC (punishment, loss, interpersonal relationship, academic stress, adaptation)
and factor scores on EMBU (emotional warmth, severe punishment, excessive interference, favoring, rejecting
and overly-protecting of father and mother) were used as independent variables, and the dependent variables
were the groups. The backward step-wise method was applied in the fittings of the regression models to find the
variable combinations for the best-fit models. Analyses of variance were also carried out for the regressions. All
the statistics were done with the R language, version 3.5.1. ASLEC ASLEC (Adolescents Self-Rating Life Events Checklist) was adapted from the psychological and physiological
characteristics of Chinese youth in 1987 by Liu et al.32. There were 27 items in total, and all items were designed
to measure the impact of common stress sources referring to participants’ experiences during the last 12 months. Five factors were assessed by this 5-point Likert scale, including Adaptation (change in habits, discomfort with
the process of leaving or rebuilding intimate relationships), Loss (loss of finances; death of a friend or relative),
Punishment (punishment and criticism at school or home), Interpersonal Relationship (being ostracized in
interpersonal relationships, encountering disputes, and having unfavorable interactions with people), Academic
Stress (study pressure, exam failure, strict study requirements from parents). High scores on the scales indicate
more intense stress. ASLEC has been widely used in LE impact assessment in China. The Cronbach α of ASLEC
is 0.91, and the comparative fit index is 0.9. Ethics approval and consent to participateh pp
p
p
The project was approved by the Ethics Committee of Beijing Anding Hospital (201743FS-2) and performed in
accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. All subjects and their parents signed an informed consent form before joining the trial. Results A total of 71,929 children and adolescents aged 6–16 were recruited (MAge = 11.48 ± 2.84), and the sample con-
sisted of 36,430 male and 35,499 female participants. 6658 participants (MAge = 12.68 ± 2.56) were allocated into
the SCT group. The mean score on SCT-CBCL was found to be 0.736 (SD = 1.19), and the weighted point
prevalence of SCT was 9.78%. Among participants in the SCT group, 3442 were male (MAge = 12.56 ± 2.64) and
3216 were female (MAge = 12.81 ± 2.48). There were significantly more male participants with SCT compared to
female participants, χ2 = 4.047, p = 0.044, Δ = 2. The distributions of males and females stratified by age spans are
presented in Fig. 2. g
In Sichuan and Hunan provinces, 1860 participants (MAge = 10.08 ± 2.81) were diagnosed to have ADHD
symptoms. Among them, 551, 930 and 379 participants were diagnosed for ADHD-combined type, ADHD-
Inattentive type ADHD-hyperactive-impulsive respectively. Among participants with ADHD, 676 were found
to develop SCT symptoms, contributing to 36.34% of all ADHD diagnoses. In terms of the comorbidity, SCT
symptoms were found in 202 participants with ADHD-combined, 339 with ADHD-inattentive and 135 with 45
95
116
159
175
206
369
488
658
545
350
49
152
154
184
244
250
372
525
587
514
411
700
500
300
100
100
300
500
700
6
8
10
12
14
16
Age
female
male
Figure 2. Distributions of male and female stratified by age spans. Figure 2. Distributions of male and female stratified by age spans. Scientific Reports | (2023) 13:16946 | https://doi.org/10.1038/s41598-023-43225-4 www.nature.com/scientificreports/ Variables
Estimate
SE
z
p
LE
Punishment
− 0.055
0.031
− 1.779
0.075
Academic stress
0.119
0.052
2.296
0.022*
PRB
F-emotional warmth
0.030
0.020
1.489
0.137
M-emotional warmth
− 0.044
0.021
− 2.050
0.040*
M-excessive interference
0.064
0.023
2.804
0.005**
M-rejection
− 0.041
0.026
− 1.586
0.113 Table 1. Demographic information and comparisons for the participants. Significance code: *p < 0.05,
**p < 0.01, ***p < 0.001. ADHD-CON
SCT-CON
SCT-ADHD
ADHD
CON
t/χ2
p
SCT
CON
t/χ2
p
SCT
ADHD
t/χ2
p
Age (M ± SD)
10.51 ± 2.75
10.50 ± 2.31
0.086
0.931
12.83 ± 2.66
12.75 ± 2.66
0.301
0.763
12.83 ± 2.66
10.51 ± 2.75
11.26
< 0.001***
Gender (male/female)
1071/426
1077/420
0.059
0.808
104/95
103/96
0.01
0.92
104/95
1071/426
30.69
< 0.001*** Table 1. Demographic information and comparisons for the participants. Significance code: *p < 0.05,
**p < 0.01, ***p < 0.001. Table 2. Regression results on ADHD presence or absence LE life events, PRB parental rearing behaviors, F
father, M mother, SE standard error. Significance code: *p < 0.05, **p < 0.01, ***p < 0.001. Variables
Estimate
SE
z
p
LE
Interpersonal relationship
0.061
0.012
4.841
< 0.001***
PRB
F-emotional warmth
0.015
0.008
1.848
0.065
F-severe punishment
− 0.021
0.009
− 2.389
0.017*
F-excessive-interference
0.061
0.015
4.054
< 0.001***
F-favoring
− 0.123
0.041
− 2.986
0.003**
F-rejection
0.039
0.019
2.017
0.044*
M-emotional warmth
− 0.028
0.008
− 3.443
< 0.001***
M-favoring
0.100
0.034
2.984
0.003** Table 2. Regression results on ADHD presence or absence LE life events, PRB parental rearing behaviors, F
father, M mother, SE standard error. Significance code: *p < 0.05, **p < 0.01, ***p < 0.001. Variables
Estimate
SE
z
p
LE
Interpersonal relationship
0.061
0.012
4.841
< 0.001***
PRB
F-emotional warmth
0.015
0.008
1.848
0.065
F-severe punishment
− 0.021
0.009
− 2.389
0.017*
F-excessive-interference
0.061
0.015
4.054
< 0.001***
F-favoring
− 0.123
0.041
− 2.986
0.003**
F-rejection
0.039
0.019
2.017
0.044*
M-emotional warmth
− 0.028
0.008
− 3.443
< 0.001***
M-favoring
0.100
0.034
2.984
0.003** Table 2. Regression results on ADHD presence or absence LE life events, PRB parental rearing behaviors, F
father, M mother, SE standard error. Significance code: *p < 0.05, **p < 0.01, ***p < 0.001. Table 2. Regression results on ADHD presence or absence LE life events, PRB parental rearing behaviors, F
father, M mother, SE standard error. www.nature.com/scientificreports/ www.nature.com/scientificreports/ ADHD-hyperactive-impulsive symptoms. No statistically significant difference was observed in the comorbidity
of SCT and the three ADHD subtypes (χ2 = 1.668, p > 0.05, Δ = 2). Based on the SCT-CBCL scale, we identified
2434 patients with SCT and the co-morbidity rate of SCT to ADHD was found to be 27.77%. Participants with
SCT symptoms (MAge = 12.83 ± 2.66) were found to be significantly older than those who developed ADHD
symptoms (MAge = 10.51 ± 2.75), p < 0.001. ADHD-hyperactive-impulsive symptoms. No statistically significant difference was observed in the comorbidity
of SCT and the three ADHD subtypes (χ2 = 1.668, p > 0.05, Δ = 2). Based on the SCT-CBCL scale, we identified
2434 patients with SCT and the co-morbidity rate of SCT to ADHD was found to be 27.77%. Participants with
SCT symptoms (MAge = 12.83 ± 2.66) were found to be significantly older than those who developed ADHD
symptoms (MAge = 10.51 ± 2.75), p < 0.001. y
g
p
Among participants from the two provinces, 1184 were allocated to the ADHD-only group and 119 were
allocated to the SCT-only group. Table 1 shows the demographic information and comparisons for the partici-
pants in these groups and their matched control groups. Subsequently, Table 2 shows the regression results of
ADHD-only and control groups. It reveals high scores on PRB factors of excessive interference and rejection
from the father and favoring from the mother were associated with ADHD diagnosis; whereas low scores on
punishment and favoring from the father, emotional warmth from the mother, and LE factors of interpersonal
relationship were associated with ADHD diagnosis. Table 3 shows the regression results of SCT-only and control
groups. It indicates high scores on the LE factor of academic stress and PRB factor of excessive-interference from
the mother were associated with SCT symptoms, and low scores on the PRB factor of emotional warmth from
the mother were related to SCT symptom. Table 4 depicts the regression results of ADHD-only and SCT-only. High scores on the LE factor of adaptation were observed to be associated with SCT-only symptoms, while high
scores on PRB factors of rejection from the father and warmth and affection from the mother were associated
with ADHD-only diagnosis. Table 1. Demographic information and comparisons for the participants. Significance code: *p < 0.05,
**p < 0.01, ***p < 0.001. www.nature.com/scientificreports/ CON
SCT-CON
SCT-ADHD
CON
t/χ2
p
SCT
CON
t/χ2
p
SCT
ADHD
t/χ2
p
75
10.50 ± 2.31
0.086
0.931
12.83 ± 2.66
12.75 ± 2.66
0.301
0.763
12.83 ± 2.66
10.51 ± 2.75
11.26
< 0.001***
1077/420
0.059
0.808
104/95
103/96
0.01
0.92
104/95
1071/426
30.69
< 0.001***
Table 2. Regression results on ADHD presence or absence LE life events, PRB parental rearing behaviors, F
father, M mother, SE standard error. Significance code: *p < 0.05, **p < 0.01, ***p < 0.001. Variables
Estimate
SE
z
p
LE
Interpersonal relationship
0.061
0.012
4.841
< 0.001***
PRB
F-emotional warmth
0.015
0.008
1.848
0.065
F-severe punishment
− 0.021
0.009
− 2.389
0.017*
F-excessive-interference
0.061
0.015
4.054
< 0.001***
F-favoring
− 0.123
0.041
− 2.986
0.003**
F-rejection
0.039
0.019
2.017
0.044*
M-emotional warmth
− 0.028
0.008
− 3.443
< 0.001***
M-favoring
0.100
0.034
2.984
0.003** 5
Scientific Reports |
(2023) 13:16946 |
https://doi.org/10.1038/s41598-023-43225-4
Table 1. Demographic information and comparisons for the participants. Significance code: *p < 0.05,
**p < 0.01, ***p < 0.001. ADHD-CON
SCT-CON
SCT-ADHD
ADHD
CON
t/χ2
p
SCT
CON
t/χ2
p
SCT
ADHD
t/χ2
p
Age (M ± SD)
10.51 ± 2.75
10.50 ± 2.31
0.086
0.931
12.83 ± 2.66
12.75 ± 2.66
0.301
0.763
12.83 ± 2.66
10.51 ± 2.75
11.26
< 0.001***
Gender (male/female)
1071/426
1077/420
0.059
0.808
104/95
103/96
0.01
0.92
104/95
1071/426
30.69
< 0.001***
Table 2. Regression results on ADHD presence or absence LE life events, PRB parental rearing behaviors, F
father, M mother, SE standard error. Significance code: *p < 0.05, **p < 0.01, ***p < 0.001. Variables
Estimate
SE
z
p
LE
Interpersonal relationship
0.061
0.012
4.841
< 0.001***
PRB
F-emotional warmth
0.015
0.008
1.848
0.065
F-severe punishment
− 0.021
0.009
− 2.389
0.017*
F-excessive-interference
0.061
0.015
4.054
< 0.001***
F-favoring
− 0.123
0.041
− 2.986
0.003**
F-rejection
0.039
0.019
2.017
0.044*
M-emotional warmth
− 0.028
0.008
− 3.443
< 0.001***
M-favoring
0.100
0.034
2.984
0.003**
Table 3. Regression results on SCT presence or absence. LE life events, PRB parental rearing behaviors, F
father, M mother, SE standard error. Significance code: *p < 0.05, **p < 0.01, ***p < 0.001. Discussion
h
d In this study, we employed a nation-wide mental health survey dataset and investigated the prevalence and age
distribution of SCT, as well as its comorbidity with ADHD symptoms. We also compared the differences between
SCT and ADHD in terms of LE and PRB measured by ASLEC and EMBU with data from two provinces. The
result showed that, with the score at the top 5 percentile as the cutoff score, the point prevalence of SCT was
9.78% in China. In addition, our results were in line with prior findings that although ADHD and SCT had a
great overlap in participants, there are significant differences between ADHD and SCT particularly in terms of
LE and PRB.h The high point prevalence of 9.78% underlined SCT as one of the most prevalent mental disorders in Chinese
children and adolescents33. Also, 9.78% can be regarded as one of the highest point prevalence data among all
surveys on SCT worldwide. Meanwhile, we found no statistically significant difference between the comorbid-
ity rates of SCT and the three ADHD subtypes, corresponding to the result of an SCT survey conducted in the
United States7. In addition, in line with prior studies16, the average age of participants with ADHD was found
to be younger than that of participants with SCT in this study. Hence, our findings support that although SCT
is closely related to ADHD, it demonstrates uniqueness to a greater extent. In China, SCT has not been well
recognized as a distinct disorder. Instead, it has been treated as ADHD in most cases. However, no evidence has
suggested that the first-line medication treatment for ADHD, such as methylphenidate, was also effective for
SCT. Notably, this indicates serious problems of misdiagnosis and mistreatment under the condition of high
SCT prevalence are to be solved34,35. Although this study was conducted in China, based on the cross-cultural
validity illustrated in our introduction, we also highlight the distinction of SCT from ADHD as a problem to be
raised for global mental health.i g
A new finding from the regression results for LE on SCT absence or presence was that greater academic stress
was associated with SCT symptoms. Learning ability decrease has been proposed as inevitable for individuals
with SCT36, and it was within our expectations to find participants with SCT being sensitive to academic stress. Discussion
h
d While Becker et al.37 reported lower study ability in college students was associated with SCT, one study on cog-
nitive ability also reported that lower performance in mathematics and writing were correlated to symptoms of
SCT except for daydreaming38 Moreover, another study conducted with college students found that SCT may be
relevant to distinctive self-regulated learning strategies39. Taken together, our research confirms academic stress
as a risk factor for the presence of SCT symptoms in Chinese children and adolescents, extending the external
validity of previous findings. More importantly, the findings of the current study highlight that adaptation
scores demonstrated a positive relationship to SCT exclusively for ADHD symptoms. A potential explanation
for this may be that SCT is dominated by internalization rather than externalization symptoms. As previously
known, maladjustment is strongly correlated with anxiety, which is often manifested as worries and fears of
external uncontrollable factors. Existing studies have also revealed that SCT was more associated with anxiety
and depression symptoms occurrences than ADHD40. Corresponding to Sevincok et al. who proposed that while
the externalization symptoms were related to ADHD41, SCT was more associated with internalization symptoms
more prominently, findings in our research provide further empirical support on the distinctions between ADHD
and SCT. Results from the regression analysis on LE and ADHD presence or absence also highlighted interper-
sonal relationship as another risk factor for ADHD diagnosis. This corresponds to results from existing studies
evidencing that children and adolescents with ADHD have a greater chance of encountering problems in social
communication. Specifically, children with ADHD are more vulnerable to attacks from peers and have poorer-
quality friendship42. On one hand, this could be related to the symptoms of ADHD, that inhibition deficiency may
lead to impulsivity and overacting to unpleasant stimulations, bringing conflicts and worsening interpersonal
relationships43. On the other hand, impairments in peer relationships also in turn lead to increases in inattention
and hyperactivity or impulsivity in children with ADHD irrespective of age and gender44. Taken together, our
findings provided further support for the vicious cycle of ADHD symptoms and poor interpersonal relationship. Regarding PRB, both fathers’ and mothers’ parenting styles were associated with ADHD diagnosis, while
only maternal parenting factors (i.e., emotional warmth and excessive interference) were associated with SCT
symptoms. Furthermore, no PRB factor was observed to predict SCT diagnosis based on the logistic regression
results to the SCT-only and ADHD-only groups. www.nature.com/scientificreports/ Significance code: *p < 0.05, **p < 0.01, ***p < 0.001. Table 3. Regression results on SCT presence or absence. LE life events, PRB parental rearing behaviors, F
father, M mother, SE standard error. Significance code: *p < 0.05, **p < 0.01, ***p < 0.001. Variables
Estimate
SE
z
p
LE
Punishment
− 0.055
0.031
− 1.779
0.075
Academic stress
0.119
0.052
2.296
0.022*
PRB
F-emotional warmth
0.030
0.020
1.489
0.137
M-emotional warmth
− 0.044
0.021
− 2.050
0.040*
M-excessive interference
0.064
0.023
2.804
0.005**
M-rejection
− 0.041
0.026
− 1.586
0.113 Table 3. Regression results on SCT presence or absence. LE life events, PRB parental rearing behaviors, F
father, M mother, SE standard error. Significance code: *p < 0.05, **p < 0.01, ***p < 0.001. Variables
Estimate
SE
z
p
LE
Punishment
− 0.055
0.031
− 1.779
0.075
Academic stress
0.119
0.052
2.296
0.022*
PRB
F-emotional warmth
0.030
0.020
1.489
0.137
M-emotional warmth
− 0.044
0.021
− 2.050
0.040*
M-excessive interference
0.064
0.023
2.804
0.005**
M-rejection
− 0.041
0.026
− 1.586
0.113 Table 3. Regression results on SCT presence or absence. LE life events, PRB parental rearing behaviors, F
father, M mother, SE standard error. Significance code: *p < 0.05, **p < 0.01, ***p < 0.001. Table 3. Regression results on SCT presence or absence. LE life events, PRB parental rearing behaviors, F
father, M mother, SE standard error. Significance code: *p < 0.05, **p < 0.01, ***p < 0.001. https://doi.org/10.1038/s41598-023-43225-4 Scientific Reports | (2023) 13:16946 | www.nature.com/scientificreports/ Table 4. Regression results on SCT-only presence or ADHD-only presence. LE life events, PRB parental
rearing behaviors, F father, M mother, SE standard error. Significance code: *p < 0.05, **p < 0.01, ***p < 0.001. Variables
Estimate
SE
z
p
LE
Punishment
− 0.051
0.029
− 1.772
0.076
Interpersonal relationship
− 0.068
0.042
− 1.620
0.105
Adaptation
0.091
0.037
2.444
0.015*
PRB
F-favoring
0.152
0.078
1.953
0.051
F-rejection
− 0.061
0.028
− 2.178
0.029*
M-favoring
− 0.139
0.061
− 2.247
0.025* Table 4. Regression results on SCT-only presence or ADHD-only presence. LE life events, PRB parental
rearing behaviors, F father, M mother, SE standard error. Significance code: *p < 0.05, **p < 0.01, ***p < 0.001. Table 4. Regression results on SCT-only presence or ADHD-only presence. LE life events, PRB parental
rearing behaviors, F father, M mother, SE standard error. Significance code: *p < 0.05, **p < 0.01, ***p < 0.001 www.nature.com/scientificreports/ important than that of fathers to SCT children and adolescents. Also, the results of our study highlight the lack
of emotional warmth and excessive interference from mothers as critical risk factors for SCT symptoms.f y
p
Similar to many other countries and regions, father and mother play different roles in Chinese families. Specifically, previous studies have summarized the family roles in the traditional Chinese culture as “severe
father and kind mother” (SFKM)45. This could be a possible explanation for the strong impact of fathers’ PRB on
ADHD as well as its missing impact on SCT symptoms. To explain, children and adolescents with ADHD usually
exhibit more externalization symptoms such as impulsivity and disobedience to the punishment and disciplines
of “severe fathers”, whereas internalization symptoms demonstrated by individuals with SCT may result in fewer
conflicts with parents, and so appear more relevant to the cares and interference received from “kind mothers”. The SFKM positioning may also explain why the PRB factors of favoring from father and mother were oppositely
associated with ADHD diagnosis. Specifically, the differences in terms of parental role allocations may gener-
ate an observation baseline bias that favoring from the father is much rarer than that from the mother. That is,
more favoring from the mother may also indicate spoiling and indulging impulsivity in the child46. By contrast,
the association between high levels of paternal favoring and ADHD diagnosis may be explained by the usual
lack of special care from fathers in Chinese culture. Another unexpected result from the logistic regression is
that both favoring and punishment from the father were relevant to the absence of ADHD diagnosis. However,
this association may be explained by the positivity of stringent paternal regulations in improving children’s
academic performance. To elaborate, as one of the countries being categorized into the middle and low-end of
the international division of labor47, jobs in China require more skill and knowledge to be fulfilled, whereas the
payment levels remain relatively low. Indeed, this phenomenon has been further intensified by the huge size of
the Chinese population. www.nature.com/scientificreports/ Hence, it may be implied from our results that children and adolescents in China are
expected to follow strict discipline driven by punishment and rewards set by their parents, which allows them
to achieve better academic outcomes in schools and colleges to secure their future.i g
One clinical implication based on our findings and knowledge from prior studies is that SCT patients are
vulnerable to stress and tend to demonstrate maladjustment. Unlike ADHD, SCT symptoms are mostly internal-
izing, and this suggests that the feelings of individuals with SCT may be more easily overlooked48. Hence, our
findings call for attention to the emotional feelings and adjustment states of children and adolescents developing
SCT symptoms, particularly when they are under stressful conditions. Regarding directions for future research,
the results of this study demonstrated the importance of stress as a risk factor for the developing symptoms of
ADHD and SCT in multiple ways. From the perspective of etiology, long-term exposure to stress could overly
activate the hypothalamic–pituitary–adrenal (HPA) axis49. HPA axis is a system of humoral regulation and stress
response. It makes adjustments to deal with external threatening conditions by increasing adrenaline levels. This
stress response helps promote adjustment level for external conditions of individuals, but consistent activation
of the HPA axis is harmful to the development of nerve cells and accelerate the apoptosis process of nerve cells50,
leading to more impairing symptoms of mental disorders51. As the etiological mechanisms of the different types
of stress events remain unclear52, future studies are needed to answer this important question. In addition, a prior
study reported the impact of LE on ADHD-related DNA methylation and participate in ADHD occurrence via
epigenetic mechanism53. Concerning the commonality of ADHD and SCT, whether this process explains SCT
symptoms is another important question to be studied. Several limitations of this study should be acknowledged. Firstly, all SCT diagnoses in the current study were
conducted based on the cutoff line on SCT-CBCL, whereas the SCT-CBCL was finished by families without
clinician diagnosis. This may indicate a rating bias that could affect the study’s validity. However, professional
diagnosis can only be available after SCT is well-recognized by the mental health community. Secondly, without
prior specification of SCT, there is a possible increased type I error in statistics results. Nevertheless, findings
from Phase II of our research may provide more evidence for the distinction of SCT as an independent disorder. www.nature.com/scientificreports/ With the current large sample size and good culture representativeness of the study, we proffer based on the
findings of our study that identifying the uniqueness of SCT may not only contribute to the understanding of
SCT and ADHD, but also inspire the development of their treatments. Lastly, it should be noted that Phase II of
our research included participants from two rather than all five provinces being studied in Phase I to ensure a
standardized procedure in LE and PRB data collection. Particularly, excluding data from the other three provinces
reduced the number of participants in Phase II, and this may affect the population validity to an extent. However,
the number of remaining participants was still at a satisfactory level. Discussion
h
d This indicates that mothers’ parenting behaviors are more i
Regarding PRB, both fathers’ and mothers’ parenting styles were associated with ADHD diagnosis, while
only maternal parenting factors (i.e., emotional warmth and excessive interference) were associated with SCT
symptoms. Furthermore, no PRB factor was observed to predict SCT diagnosis based on the logistic regression
results to the SCT-only and ADHD-only groups. This indicates that mothers’ parenting behaviors are more https://doi.org/10.1038/s41598-023-43225-4 Scientific Reports | (2023) 13:16946 | www.nature.com/scientificreports/ Conclusionh This study used a nation-wide mental health survey dataset to investigate the point prevalence and the demo-
graphic characteristics of individuals developing SCT symptoms in China. Specifically, we found the point
prevalence of SCT in China was 9.78%. Moreover, we employed SCT-CBCL, LE and PRB survey results from
two provinces, which share the participants with the national survey, to investigate the comorbidity of ADHD
and SCT, as well as the associations between LE, PRB, ADHD and SCT symptoms. The findings of our study
suggested that although ADHD and SCT were highly comorbid, they may be considered as two independent
disorders. Particularly, our results indicated SCT patients are vulnerable to stress from LE and tend to demon-
strate maladjustments. It is underlined that their emotional states need to be taken good care of, and maternal
rearing behavior is the key factor. Overall, SCT brings global challenges in diagnosis and treatment, and the
challenge is more severe in tight cultures such as China. Hence, we recommend further empirical research on
stress management and SCT etiology. https://doi.org/10.1038/s41598-023-43225-4 Scientific Reports | (2023) 13:16946 | www.nature.com/scientificreports/ Data availability
Th d
h y
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. Received: 13 May 2023; Accepted: 21 September 2023 Received: 13 May 2023; Accepted: 21 September 2023 References e e e ces
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The authors declare no competing interests. Author contributions All authors were involved in data collection. J.L. performed data analysis and wrote the final manuscript. F.H.L. made important revisions to the first draft of the manuscript. Y.J.Q., H.H.H., Y.Z.W., and G.Y.X. were involved
throughout the data collection process. Z.K.L. and F.H. made practical contributions to the conduct and advance-
ment of the trial. Y.Z. was the leader of the entire team and directed and supervised the entire trial. The authors
have declared that they have no competing or potential conflicts of interest. Participants gave consent for data
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9. Shelton, C. R., Addison, W. E. & Hartung, C. M. ADHD and SCT symptomatology in relation to college students’ use of self-
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10.1080/08039488.2019.1697746 (2020). 42. Rokeach, A. & Wiener, J. Friendship quality in adolescents with ADHD. J. Atten. Disord. 24(8), 1156–1168 (2020). 43. Lee, Y., Mikami, A. Y. & Owens, J. S. Children’s ADHD symptoms and friendship patterns across a school year. Res. C
Psyhopathol. 49, 643–656 (2021).i 42. Rokeach, A. & Wiener, J. Friendship quality in adolescents with ADHD. J. Atten. Disord. 24(8), 1156–1168 (2020). Fundingh g
This study was funded by Research on prevention and control of major chronic non-communicable diseases in
the Ministry of Science and Technology (No: 2016YFC1306100), Beijing Hospitals Authority Clinical Medicine
Development of Special Funding Support, code: ZYLX202128. Acknowledgementsh g
The authors are grateful to all participants for their support. 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
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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/. Additional information Correspondence and requests for materials should be addressed to F.H. or Y.Z. Correspondence and requests for materials should be addressed to F.H. or Y.Z. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Scientific Reports | (2023) 13:16946 | https://doi.org/10.1038/s41598-023-43225-4 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International
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https://www.gvaa.com.br/revista/index.php/RVADS/article/download/5892/6538
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Resistência mecânica à penetração em sistemas de manejo do solo
|
Revista Verde de Agroecologia e Desenvolvimento Sustentável
| 2,019
|
cc-by
| 5,206
|
Recebido: 06/08/2018
Aprovado: 29/11/2018 Os sistemas de manejo do solo podem resultar em alterações físicas no solo como a
formação de camadas compactadas. Um dos atributos físicos mais adotados como indicativo
da compactação do solo têm sido a resistência mecânica do solo à penetração, por
apresentar relações diretas com o crescimento das plantas e por ser mais eficiente na
identificação de estados de compactação do solo. Nesse sentido, objetivou-se avaliar a
resistência mecânica do solo à penetração em dois sistemas de manejo do solo, por meio de
utilização da técnica de penetrometria. O experimento foi realizado em área da
Universidade Federal Rural da Amazônia, Campus de Parauapebas. O delineamento
experimental utilizado foi inteiramente casualizado com dois tipos de manejo: sistema de
plantio direto (SPD) e sistema de plantio convencional (SPC), com 40 repetições para cada
tratamento. A resistência do solo à penetração (RP) foi mensurada utilizando-se o Medidor
Eletrônico de compactação do solo por pressão, modelo PenetroLOG – PLG 1020 da marca
Falker, na profundidade de 0,00 a 0,40m a qual foi subdividida em oito camadas de 0,05 m
para avaliação. A adoção dos sistemas de plantio direto e plantio convencional ocasionaram
oscilação na resistência mecânica do solo à penetração ao longo das camadas do solo. No
SPC foi observada menor RP quando comparado ao outro tratamento, na profundidade 0,00
- 0,10 m. Diferente disso, no SPD as subcamadas de 0,15 - 0,25 m apresentaram menores
valores de RP e maior umidade do solo. A partir da profundidade de 0,20 m ambos os
sistemas apresentaram indicativo de camadas compactadas do solo. Recebido: 06/08/2018
Aprovado: 29/11/2018 Palavras-chave:
Compactação
Máquina agrícola
Penetrômetro Mechanical strength to penetration in different management systems Mechanical strength to penetration in different management systems
Gislayne Farias Valente1; Vicente Filho Alves Silva2; José Nilton da Silva3; Daiane Rodrigues da Silva
Pinto4; Jessivaldo Rodrigues Galvão5
1Mestranda em Agronomia, Universidade Federal Rural da Amazônia, Belém, Pará, gisllaynnefv@hotmail.com; 2Doutor em Agronomia, Professor Adjunto II,
Universidade Federal Rural da Amazônia, Parauapebas, Pará, vicente.silva@ufra.edu.br; 3Doutor em Agronomia, Professor Adjunto I, Universidade Federal Mechanical strength to penetration in different management systems
Gislayne Farias Valente1; Vicente Filho Alves Silva2; José Nilton da Silva3; Daiane Rodrigues da Silva
Pinto4; Jessivaldo Rodrigues Galvão5 1Mestranda em Agronomia, Universidade Federal Rural da Amazônia, Belém, Pará, gisllaynnefv@hotmail.com; 2Doutor em Agronomia, Professor Adjunto II,
Universidade Federal Rural da Amazônia, Parauapebas, Pará, vicente.silva@ufra.edu.br; 3Doutor em Agronomia, Professor Adjunto I, Universidade Federal
Rural da Amazônia, Parauapebas, Pará, agrojns@yahoo.com.br; 4Engenheira Agrônoma, Universidade Federal Rural da Amazônia, Parauapebas, Pará,
daiane.hidrotherm@gmail.com;
5Doutor em Agronomia, Engenheiro Agrônomo, Universidade Federal Rural da Amazônia, Belém, Pará,
jessi.galvao50@gmail.com. Revista Verde 14:1 (2019) 140-145 Revista Verde 14:1 (2019) 140-145 INTRODUÇÃO 2015; SCAPINELLI et al., 2016). Dentre fatores como
densidade, textura, matéria orgânica e umidade do solo, a
resistência do solo à penetração é considerada a propriedade
do solo adequada para expressar o grau de compactação
existente no solo. Tornando-se fundamental tanto para
recomendação de práticas de manejo do sistema de preparo
mais adequado do solo, como para a avaliação dos efeitos na
agricultura (SILVEIRA et al., 2010). Com a evolução da colheita mecanizada e o crescente
melhoramento
de
cultivares
mais
produtivas
têm-se
aumentado as áreas cultivadas (FACHIN et al., 2014). O uso
de novas áreas agricultáveis para o plantio demanda
igualmente maior número de máquinas e implementos
modernos e de elevada capacidade operacional para ser
empregado no campo, e atender a sua necessidade tecnológica
(CONAB, 2013). Existem formas para determinar a resistência do solo à
penetração, dentre elas, a penetrometria é uma técnica que
identifica
as
camadas
que
apresentam
restrição
ao
desenvolvimento radicular das plantas (CAVALCANTE et
al., 2011; MION et al., 2012). É uma técnica quantitativa
muito utilizada devido à facilidade, rapidez e a possibilidade
de se efetuar grande número de repetições para obtenção de
dados (SILVEIRA et al., 2010). No entanto, esta é variável
conforme a condição estrutural e conteúdo de água do solo no
momento de sua avaliação (VALICHESKI et al., 2012). Na atividade agrícola trabalha-se a porção mais
superficial do solo, a qual é chamada de camada arável
(ALCÂNTARA et al., 2008). Esta é preparada para oferecer
condições ideais para a semeadura, germinação, emergência e
desenvolvimento das plantas (MACEDO et al., 2009). O
preparo do solo é a primeira operação a ser realizada, pois
compreende um conjunto de práticas que, quando usadas de
forma adequada permitem alta produtividade da cultura
(EMBRAPA, 2013). O sistema de plantio convencional
(SPC) e o sistema de plantio direto (SPD) estão entre as
principais formas de manejo do solo, e a escolha varia de
acordo com a textura, estrutura e grau de compactação do
solo, bem como de acordo com a disponibilidade de
equipamentos e de recursos do produtor (NASCENTE et al.,
2011). INTRODUÇÃO Na ciência do solo muitos estudos recentes apontam
para pesquisas voltadas para a avaliação da qualidade física
dos solos em relação ao seu manejo (BROWN et al., 2018;
CAMPOS et al., 2018), visto que esse tema é constante na
literatura
mundial em
virtude das interferências
na
produtividade
das
culturas
e
sustentabilidade
dos
ecossistemas ligados ao solo (GUEDES et al., 2012). Com
isso, tornam-se de grande importância os estudos científicos
que avaliam a relação máquina-solo-planta (LIMA et al.,
2013). Nesse sentido, o presente trabalho consiste na hipótese
de que o sistema de manejo convencional do solo ocasiona
maiores valores de RP em relação ao sistema de plantio
direto. O sistema de plantio direto baseia-se nos princípios da
cobertura permanente, revolvimento mínimo do solo, rotação
de culturas e adubação verde, podendo resultar na melhor
eficiência em manter a qualidade física, química e biológica
do solo e condições favoráveis ao desenvolvimento vegetal
em decorrência da ausência de operações com máquinas para
o preparo da área (LANZANOVA et al., 2010). Diferente
disso, o sistema de plantio convencional, de maneira geral,
envolve os preparos primário e secundário que consistem no
revolvimento da camada superficial do solo com uso de
equipamentos como o arado e a grade (PEREIRA;
RODRIGUES, 2013). Com isso, o presente trabalho teve como objetivo avaliar
a resistência mecânica do solo à penetração utilizando a
técnica da penetrometria, em dois sistemas de manejo do solo,
convencional (SPC) e plantio direto (SPD). A B S T R A C T Key words:
Compression
Agricultural Machinery
Penetrometer The diference soil management systems, can result in soil physical changes, such as the
formation of compacted layers. One of the physical attributes most used as indicative of soil
compact, has been the soil mechanical resistence to penetration, because it has direct
relation with the growth of plants, and to be more efficient in identifying soil compaction
stats. In this sense, the objective was evaluate the mechanical resistence to penetration in
two soil management systens, by means of the use penetration technique. The experiment
was carried out in the area of the University Federal Rural of Amazonia, Campus
Parauapebas. The experiment design was completely randomized with two types of
management no tillage system (NTS) and conventional plating system (CPS) with 40
replicate trataments. The soil resistance to penetration (PR) was measured using the Falker-
brand Electronic Pressure Compaction Meter PenetroLOG - PLG 1020, at a depth of 0,00 to
0;40 m, which was subdivided into eight layers of 0,05 m for evaluation. The adoption of
no-tillage and conventional tillage systems caused oscillation in the soil mechanical
resistance to penetration along the soil layers. In the CPS, a lower RP was observed when
compared to the other treatment, at depth 0,00 – 0,10 m. Differently, in the NTS, the
sublayers of 0,15 – 0,25 m presented lower RP values and higher soil moisture. From the
depth of 0,20 m both systems presented indicative of compacted layers of the soil. Revista Verde
ISSN 1981-8203
Pombal, Paraíba, Brasil v. 14, n.1, jan.-mar, p.140-145, 2019
doi: 10.18378/rvads.v14i1.5892 Gislayne Farias Valente et al. Resistência mecânica à penetração em sistemas de manejo do solo Resistência mecânica à penetração em sistemas de manejo do solo marcha reduzida com velocidade média de 6,0 km h-1 a 2.000
rpm. O delineamento experimental utilizado foi inteiramente
casualizado, contendo dois sistemas de cultivo (SPD e SPC),
nos quais foram mensurados aleatoriamente, 40 pontos de RP,
em cada tratamento, totalizando 80 unidades experimentais. Os dados do penetrômetro foram tabulados no
programa de computador fornecido pela fabricante do
penetrômetro, denominado Compactação do Solo v.1.41. Os
resultados de resistência mecânica do solo à penetração foram
submetidos à análise de variância sendo as médias
comparadas pelo teste de Tukey a 5% de probabilidade. A
análise estatística foi realizada utilizando o software SISVAR
(FERREIRA, 2011). Para análise da RP, utilizou-se o Medidor Eletrônico de
compactação do solo por pressão. Para determinação de teor
de água no solo, simultaneamente à mensuração da RP, foram
coletadas amostras indeformadas de solo em pontos aleatórios
da área, para cada tratamento, utilizando o trado holandês, nas
profundidades de 0,00 - 0,20 m e 0,20 - 0,40 m. Utilizou-se o
método termogravimétrico em laboratório quantificado
segundo a norma NBR 6457/1986 – ABNT, representada pela
Equação 1. MATERIAL E MÉTODOS Todavia, as máquinas e implementos agrícolas utilizados
para as operações de preparo do solo estão cada vez maiores e
mais pesados, e com o uso crescente do maquinário nos
sistemas produtivos, o solo passou a receber maior pressão, e
quando associada ao uso inadequado de acordo com as
condições de umidade do solo, resulta no aumento da
compactação e diminuição da produtividade das culturas
(CORTEZ et al., 2017; FERRARI et al., 2018). A
compactação do solo é caracterizada pelo aumento da
densidade do solo, pelos altos valores de resistência do solo à
penetração, redução da infiltração de água, distribuição e
tamanho de poros no solo e pela diminuição de difusão dos
gases e disponibilidade de nutrientes (VALADÃO et al., O experimento foi realizado na Universidade Federal
Rural da Amazônia Campus de Parauapebas, localizada nas
coordenadas geográficas 06º 04’ 26’’ latitude Sul, 49º 49’
03’’ longitude Oeste, com altitude de 239 m e declividade
variando de 2% a 3%. O clima é classificado como Tropical
úmido, tipo Am, na classificação de Köppen, no limite de
transição para o AW, com índice pluviométrico anual em
torno de 2.000 mm. O solo foi classificado conforme a
EMBRAPA (2013), como Latossolo Vermelho-Amarelo,
textura franca, cuja composição granulométrica consta na
Tabela1. Tabela 1. Análise granulométrica do solo nas profundidades de 0,00 - 0,20 m e 0,20 - 0,40 m. Profundidade
(m)
Areia
Classe
Textural
Argila
Silte
Fina Grossa
(g kg-1)
0,00 – 0,20
120
487
206
188
Franca
0,20 – 0,40
140
479
176
205
Franca ranulométrica do solo nas profundidades de 0,00 - 0,20 m e 0,20 - 0,40 m. A i
Cl Tabela 1. Análise granulométrica do solo nas profundidades de 0,00 - 0,20 m e 0,20 - 0,40 m. f
did d
A i Os dados de resistência do solo à penetração (RP) foram
coletados no período de Junho à Setembro de 2014. A área do
experimento totalizou 800 m2 (20 x 40 m) e a vegetação
anterior à implantação dos sistemas de cultivo consistiu em Revista Verde, v.14, n.1, p.140-145, 2019 Revista Verde, v.14, n.1, p.140-145, 2019 RESULTADOS E DISCUSSÃO Na Tabela 3 observam-se os resultados da análise de
variância da RP nos sistemas de manejo, onde foi observado
maior RP superficial no SPD, e subsuperficial no SPC. Em
ambos os tratamentos observa-se comportamento crescente da
RP ao longo das camadas analisadas do SPD (0,00 – 0,40 m)
e do SPC até os 0,30 m, apresentando valores entre 0,017 e
2,58 MPa, indicando a necessidade de monitoramento e o
preparo em camadas sub superficiais. Estes resultados
corroboram com os encontrados por Pereira et al. (2002),
Bono et al. (2013), Ferrari et al. (2018) e Theodoro et al. (2018) que também constataram maiores valores de
resistência à penetração com o aumento da profundidade,
independente dos sistemas de cultivo estudados. x100 (Eq. 1) (Eq. 1) Em que: H = teor de água no solo, em %; M1 = massa do solo
úmido mais a massa do recipiente, em g; M2 = massa do solo
seco mais a massa do recipiente, em g; M3= massa do
recipiente (cápsula metálica com tampa), em g. Tabela 2. Teor de água no solo para os sistemas utilizados
nas respectivas profundidades 0,00 - 0,20 m e 0,20 - 0,40 m. Tabela 2. Teor de água no solo para os sistemas utilizados
nas respectivas profundidades 0,00 - 0,20 m e 0,20 - 0,40 m. Tratamentos
Profundidade
(m)
TAS
(%)
SPC
0,00-0,20
23,92
0,20-0,40
24,86
SPD
0,00-0,20
25,90
0,20-0,40
26,57 Tabela 2. Teor de água no solo para os sistemas utilizados
nas respectivas profundidades 0,00 - 0,20 m e 0,20 - 0,40 m. Nos sistemas avaliados, verifica-se diferença estatística
a 5% de probabilidade nas profundidades de 0,00 - 0,05 e
0,05 - 0,10 m. O SPC apresentou menores valores de RP com
médias de 0,017 e 0,366 MPa nas camadas supracitadas,
respectivamente. Tabela 3. Síntese da análise da variância para a RP (MPa) nos sistemas de manejo do solo em diferentes profundidades
Fatores
de
Variação
0,00-0,05
0,05-0,10
0,10-0,15
0,15-0,20
0,20-0,25
0,25-0,30
0,30-0,35
0,35-0,40
(m)
Sistema de Manejo
SPC
0,017 b
0,366 b
1,320
2,212 a
2,435 a
2,513
2,511
2,416
SPD
0,049 a
0,621 a
1,322
1,666 b
2,039 b
2,374
2,522
2,580
Valor de F
5,01*
10,24*
0,00NS
22,09**
9,80**
1,36 NS
0,01 NS
1,66 NS
DMS
27,94
160,92
267,55
234,80
256,15
242,37
213,85
257,11
CV (%)
37,12
18,05
24,76
26,77
25,31
21,93
18,79
22,75
Médias seguidas de mesma letra na coluna não diferem entre si pelo teste de Tukey para um nível de 5% de probabilidade. SPC: Sistema Plantio
Convencional; SPD: Sistema Plantio direto. NS: não significativo (P > 0,05); *: significativo (P ≤ 0,05); **: significativo (P ≤ 0,01); CV: coeficiente de variação
(%). álise da variância para a RP (MPa) nos sistemas de manejo do solo em diferentes profundidades
0,00-0,05
0,05-0,10
0,10-0,15
0,15-0,20
0,20-0,25
0,25-0,30
0,30-0,35
0,35-0,40
(m) CV (%)
37,12
18,05
24,76
26,77
25,31
21,93
18,79
22,7
Médias seguidas de mesma letra na coluna não diferem entre si pelo teste de Tukey para um nível de 5% de probabilidade. SPC: Sistema Pla
Convencional; SPD: Sistema Plantio direto. NS: não significativo (P > 0,05); *: significativo (P ≤ 0,05); **: significativo (P ≤ 0,01); CV: coeficiente de varia
(%). Médias seguidas de mesma letra na coluna não diferem entre si pelo teste de Tukey para um nível de 5% de prob
Convencional; SPD: Sistema Plantio direto. NS: não significativo (P > 0,05); *: significativo (P ≤ 0,05); **: significativo (P ≤ 0
(%). a letra na coluna não diferem entre si pelo teste de Tukey para um nível de 5% de probabilidade. SPC: Sistema Plantio
Plantio direto. Tabela 2. Teor de água no solo para os sistemas utilizados
nas respectivas profundidades 0,00 - 0,20 m e 0,20 - 0,40 m. (2014), em estudos sobre a resistência
mecânica do solo à penetração também em Latossolo
Vermelho sob sistema de plantio direto, obtiveram
predominância de valores na faixa de 3,0 a 5,0 MPa na
mesma profundidade do presente trabalho. Em avaliação da
RP em sistemas de cultivo, Borges et al. (2004) obtiveram
médias variando entre 2 e 3 MPa no SPC, sendo um resultado
esperado tendo-se em vista o revolvimento do solo e o tráfego
de implementos sobre a superfície do terreno. Com isso, e
pode-se inferir que os sistemas de manejo avaliados afetaram
de modo similar. diminuição das forças de coesão entre as partículas do solo e
aumento do efeito lubrificante da água, ou seja, a RP
apresenta relação inversa com o teor de umidade do solo
(SILVA et al., 2006). Esse resultado pode ser atribuído à cobertura vegetal do
solo no SPD que mantém o teor de água e a temperatura do
solo mais uniforme nas camadas mais superficiais devido ao
impedimento ou diminuição da ação direta de fatores externos
como a incidência de raios solares sobre a superfície do solo
(LOSS et al., 2011). Em ambos os tratamentos, houve
aumento no conteúdo de água ao longo das camadas do solo. Esse aumento deve-se à classe textural Franca do solo, a qual
se insere como um fator determinante para o estado estrutural
do solo e consequentemente para a sua capacidade de reter
água nos poros nas camadas mais profundas. Figura 1. Resistência mecânica do solo à penetração na
profundidade de 0,00 - 0,40 m, em dois sistemas de manejo
do solo, em Parauapebas, Pará. ,
p
, Embora a umidade do solo esteja mais elevada nessas
camadas em relação às outras profundidades, em ambos os
tratamentos, foram mensuradas RP com valores entre 2,43 e
2,58 MPa na profundidade de 0,20 – 0,40 m. Em virtude de o
solo ter atingido valores de RP superiores a 2,0 MPa nas
camadas supracitadas, esses valores podem ser um indicativo
de que, nesta profundidade, possa apresentar pé-de-grade
devido ao tráfego de trator, semeadora ou gradagens
sucessivas na mesma profundidade. LANZANOVA et al. (2007) e SILVA et al. (2002) relatam que um valor de
resistência à penetração do solo acima de 2 MPa pode estar
relacionado às condições impeditivas para o crescimento das
raízes e da parte aérea das plantas. Tabela 2. Teor de água no solo para os sistemas utilizados
nas respectivas profundidades 0,00 - 0,20 m e 0,20 - 0,40 m. NS: não significativo (P > 0,05); *: significativo (P ≤ 0,05); **: significativo (P ≤ 0,01); CV: coeficiente de variação implementos revolvem a camada superficial durante o
preparo do solo é até 0,20 m de profundidade, ocasionando
valores de RP mais baixos, e normalmente os maiores valores
encontram-se
abaixo
da
camada
trabalhada
(CORTEZ et al., 2011). Resultados semelhantes foram obtidos por Beutler et al. (2001), Ortigara et al. (2014) e Tavares Filho et al. (2001) ao
avaliarem a resistência do solo à penetração em Latossolo
Vermelho sob diferentes cultivos, verificaram maiores valores
de RP no SPD na camada mais superficial. Quando
determinada pressão externa é aplicada ao solo por máquinas,
ocorre um novo acomodamento ou disposição das partículas,
diminuindo o espaço poroso do solo, resultando em
incremento dos valores de resistência à penetração (STRECK
et al., 2004). Para as profundidades de 0,15 - 0,20 e 0,20 - 0,25 m,
observou-se comportamento da RP, nos sistemas de manejo,
contrário às camadas superficiais. No caso em questão, o SPD
apresentou menor resistência à penetração em relação ao SPC
com valores de 1,666 MPa para camada de 0,15 - 0,20 m e
2,039 MPa para 0,20 - 0,25 m. A resistência do solo à
penetração é mais afetada pela variação dos conteúdos de sua
umidade devida faixa do teor de água estar diretamente
relacionada à capacidade do solo de suportar e permitir o
trabalho do maquinário agrícola (PINTO FILHO et al., 2009;
MERCANTE et al., 2003). O SPD apresentou uma
porcentagem de umidade em torno de 8% superior ao
encontrado no SPC, com 25,90 % na camada de 0,0 – 0,20 m
e 26,57% em 0,20 – 0,40 m. Essa condição do solo ocasiona Abaixo da camada superficial, nas camadas de 0,15 -
0,20 m e 0,20 - 0,25 m houve diferença a 1% de
probabilidade entre os valores de RP. No entanto, não foi
verificada significância (P > 0,05) entre as médias de RP na
camada de 0,10 - 0,15 m, tampouco nas camadas abaixo de
0,25 m. A oscilação nos valores de RP nos diferentes sistemas
de manejo pode ser relacionado à pressão exercida no solo
durante o tráfego de maquinário específico para cada sistema
de manejo. No SPC a profundidade de trabalho em que os Revista Verde, v.14, n.1, p.140-145, 2019 Gislayne Farias Valente et al. Girardello et al. Tabela 2. Teor de água no solo para os sistemas utilizados
nas respectivas profundidades 0,00 - 0,20 m e 0,20 - 0,40 m. Contudo, é necessário
relacionar a resistência do solo à penetração com a aeração e
com o potencial matricial de água do solo (SILVA et al.,
2002). Foloni et al. (2003) ao avaliarem o efeito da
compactação sobre o desenvolvimento radicular de plantas de
milho, observaram que uma camada compactada com
resistência à penetração da ordem de 1,4 MPa na camada
subsuperficial do solo impede que o sistema radicular do
milho atravesse essa camada e se desenvolva em
profundidade. Ainda de acordo com o autor, espécies
comumente utilizadas nos sistemas de rotação de culturas,
como a soja e o milheto também mostram sensibilidade a
valores semelhantes de RP. CONCLUSÕES Os sistemas de manejo de solo causam modificações na
estrutura do solo, provocando camadas com diferentes graus
de compactação. Na figura 1 evidencia-se que o valor médio de RP para
0,10 m é similar e menor que 2 MPa em ambos os
tratamentos, sendo estes valores médios inferiores aos
observados a partir de 0,15 m, demonstrando assim que o
maior grau de compactação localiza-se nesta faixa de
profundidade. Ibiapina et al. (2014) também verificaram um
aumento abrupto do valor de RP, após a camada de 0,15 m de
profundidade, para o mesmo sistema de manejo saindo de
menos de 1 MPa para mais de 6 MPa na profundidade de 0,30
m. O sistema de plantio convencional ocasiona maiores
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solo à penetração em áreas com e sem manejo químico
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MOTA, F. D.; GABRIEL FILHO, L. R. A., TANAKA, E. M. Análise da variabilidade espacial da resistência à penetração
do solo em diferentes profundidades. Brazilian Journal of
Biosystems Engineering v. 12, n. 2, p. 164-175, 2018. MION, R. L.; NASCIMENTO, E. M. S.; SALES, F. A. L.;
SILVA, S. F.; DUARTE, J. M. L.; SOUSA, B. M. MION, R. L.; NASCIMENTO, E. M. S.; SALES, F. A. L.;
SILVA, S. F.; DUARTE, J. M. L.; SOUSA, B. M. Revista Verde, v.14, n.1, p.140-145, 2019 Gislayne Farias Valente et al. trator em plantio direto. Ciência Rural, v. 34, n. 3, p. 755-760,
2004. Variabilidade espacial da porosidade total, umidade e
resistência do solo à penetração de um Argissolo Amarelo. Revista Semina: Ciências Agrárias, v. 33, n. 6, p. 2057-2066,
2012. Variabilidade espacial da porosidade total, umidade e
resistência do solo à penetração de um Argissolo Amarelo. Revista Semina: Ciências Agrárias, v. 33, n. 6, p. 2057-2066,
2012. SILVA, V. R.; REICHERT, J. M.; REINERT, D. J. Revista Verde, v.14, n.1, p.140-145, 2019 REFERÊNCIAS Revista Verde de
Agroecologia e Desenvolvimento Sustentável, v. 4, n. 2, p. 76-84, 2009. VALICHESKI, R. R.; GROSSKLAUS, F.; STÜRMER, S. L. K.; TRAMONTIN, A. L.; BAADE, E. S. A. S. Desenvolvimento de plantas de cobertura e produtividade da
soja conforme atributos físicos em solo compactado. Revista
Brasileira de Engenharia Agrícola e Ambiental, v. 16, n. 9, p. 969-977, 2012. STRECK, C. A.; REINERT, D. J.; REICHERT, J. M.;
KAISER, D. R. Modificações em propriedades físicas com a
compactação do solo causada pelo tráfego induzido de um Revista Verde, v.14, n.1, p.140-145, 2019
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Triangles, tropes, and τὰ τοιαʋτ̃ α: a Platonic trope theory
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Triangles, tropes, and : a Platonic trope theory
Autor(es):
Buckels, Christopher
Publicado por:
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URI:http://hdl.handle.net/10316.2/47015
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DOI:https://doi.org/10.14195/2183-4105_18_1
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CHRISTOPHER BUCKELS |
Triangles, Tropes, and τὰ
τοιαʋ τ̃ α: A Platonic Trope
Theory
Christopher Buckels
University of California
cbuckels@hotmail.com
ABSTRACT
A standard interpretation of Plato’s metaphysics holds that sensible particulars are images
of Forms. Such particulars are fairly independent, like Aristotelian substances. I argue that
this is incorrect: Platonic particulars are not
Form images but aggregates of Form images,
which are property-instances (tropes). Timaeus
49e-50a focuses on “this-suches” (toiauta) and
even goes so far as to claim that they compose
other things. I argue that Form images are thissuches, which are tropes. I also examine the
geometrical account, showing that the geometrical constituents of the elements are also Form
images. Thus everything in the sensible world is
composed of tropes.
Keywords : Particulars; Tropes; Plato; Timaeus;
Substance; Metaphysics; Greek Philosophy
https://doi.org/10.14195/2183-4105_18_1
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Triangles, Tropes, and τὰ τοιαʋ τ̃ α: A Platonic Trope Theory
1. INTRODUCTION
In the Timaeus, Plato offers an account
of the world whereby macroscopic physical
objects are composed of microscopic three
‑dimensional objects, which are, in turn,
composed of two‑dimensional objects. Most,
if not all, properties of macroscopic objects are
explained by properties of microscopic objects,
which are in turn explained by the configu
ration of two‑dimensional objects. We seem
to have a completely reductionist, naturalist
account of the world; why, then, is it accompa
nied by supernatural entities such as Platonic
Forms? While I will explain why Forms are
crucial for the account of particulars in the
Timaeus, I will be focusing not on Forms, but
on sensible particulars, the three‑dimensional
objects we bump into on a day‑to‑day basis,
as well as their constituents. I will argue that
Plato does not take these particulars as basic
constituents of the physical world, but that the
ultimate constituents of Timaeus’ sensible par
ticulars are what metaphysicians call tropes,
or property‑instances; Timaeus calls them τὰ
τοιαῦτα, “this‑suches.” My argument follows:
1. Each thing that comes to be is τὸ
τοιοῦτον (this‑such).
2. All and only things that come to be
are images of Forms.
3. Therefore, τὰ τοιαῦτα (this‑suches) are
images of Forms.
4. Triangles are τὰ τοιαῦτα or are con
structed from τὰ τοιαῦτα.
5. All bodies are constructed from tri
angles.
6. Therefore, all bodies are constructed
from τὰ τοιαῦτα (images of Forms).
The second part of the paper will explain
and defend the first syllogism, the third part
will explain and defend the second syllogism
(which may be understood as laying out a con
sequence of the first syllogism), and the fourth
part will consider objections. As will become
clear in the paper, I take Timaeus’ metaphysics
of particulars to be a version of a trope theory,
and so I take τὰ τοιαῦτα to be tropes, but I will
defend this claim in the fourth part; I will also
consider the traditional account of particulars
in the Timaeus whereby Form images are bod
ies (i.e., physical objects) rather than tropes.
Let me begin by giving an initial character
ization of a Platonic trope theory. Tropes are
often used to avoid commitment to universals;
the universal or Form Whiteness, for example,
could be the set of all whiteness tropes. Most
trope theories, therefore, are nominalist theo
ries. Timaeus is not, of course, a trope nomi
nalist; his tropes coexist with transcendent
universals, Forms.1 In his terms, a trope is a
“this‑such” (τὸ τοιοῦτον) or Form image —
a trope‑theoretic interpretation of Timaeus’
ontology is thus not anachronistic, since he
himself introduces tropes, as I will show, albeit
under the name of “this‑suches” (τὰ τοιαῦτα).2
When one feels hot, one does not feel heat in
general but a particular heat, this‑heat: a heat
trope. Thus the heat in a given fire is not a
repeatable entity or “immanent universal,”
but a particular entity, distinct from each
other instance of heat. This is what I mean by
a trope: a particular, non‑repeatable instance
of a property.
While trope theorists generally take tropes
to be fundamental entities out of which uni
versals may be constructed, Timaeus’ tropes
are dependent upon transcendent universals,
as “Form image” implies. So a heat trope is an
image of Heat, and there can be many distinct
images of the same Form of Heat, each image
coming to be and then perishing as something
becomes and then ceases to be hot. I take Forms
CHRISTOPHER BUCKELS |
to be immutable, non‑spatiotemporal entities
that are universals insofar as they explain com
monality in resembling things. Heat explains
the commonality in two hot things precisely
by having an image of itself — numerically
distinct but qualitatively identical — in each
hot thing; i.e., two hot things resemble each
other because each contains a heat trope.
A trope‑bundle theorist would say that
sensible particulars are wholly constituted
by tropes. Fire is a bundle of heat, color, etc.
While I do not here defend a Platonic tropebundle theory, my arguments naturally lead
to such an interpretation. My trope‑theoretic
interpretation of the Timaeus differs from tra
ditional interpretations in that it takes tropes
rather than sensible particulars as images of
Forms. Instead of holding that sensible par
ticulars have their properties by participat
ing in Forms, a trope‑theoretic interpretation
holds that sensible particulars have properties
by having tropes as constituents. Thus Forms
and their images — in contemporary terms,
transcendent universals and tropes — play
the central role in Plato’s explanation of the
sensible world, rather than sensible particulars
playing the starring role.
2. τὰ τοιαʋ τ̃ α
2.1. EACH THING THAT COMES TO BE IS
τὰ τοιαʋ τ̃ α
Timaeus introduces τὰ τοιαῦτα at 49d5,
in the midst of a very controversial passage. 3
At the heart of the controversy is whether we
should take Timaeus to say, “fire is τὸ τοιοῦτον
(“this such” or “this sort of thing”), rather than
τοῦτο (“this” or “that”)”, or, “τὸ τοιοῦτον, not
τοῦτο, is fire.” The debate is thus over which
terms are subjects and which are predicates.
11
The context is a puzzle about the elements,
which seem to be too unstable to admit of be
ing called any one thing, since they could, at
any moment, change into different elements.
Should we say, when pointing at a bonfire, for
example, ‘fire is this sort of thing’ rather than
‘fire is this thing’, or should we say ‘this sort of
thing is fire’ instead of ‘this thing is fire’?
However one wishes to settle this dispute,
we can focus on “this‑such” and identify two
main interpretations of τὸ τοιοῦτον in the pas
sage. The first, dubbed the ‘traditional transla
tion’ by its adherents, claims that τὰ τοιαῦτα
are “temporary characteristics” rather than
“self‑subsistent” things (Zeyl 2000 lviii, n. 18).
The second, which Zeyl calls the ‘alternative
translation,’ takes τὰ τοιαῦτα to be “distinct
and self‑identical characteristics” or “recurrent,
stable, and determinate characters” (following
Cherniss 1954). I propose, for now, to follow a
middle ground, calling them “characteristics;”
we will leave aside whether they are “tempo
rary” or “recurrent, stable, and determinate.”
We are left with quite a bit of agreement. On
the one hand, these characteristics are tempo
rary in at least one sense: they may be at a cer
tain place for only a short time, and that place
may be occupied by a different characteristic
at any time. Thus the fiery characteristic may
be replaced by a watery characteristic at any
moment. On the other hand, these character
istics are stable and determinate in at least one
sense: for as long as each exists—which may be
only an instant—it is that characteristic and not
some other. It may be identified as belonging
to a certain kind. So the fiery characteristic is
fiery for as long as it exists, even if is replaced
by a watery characteristic in but a moment.
On either translation, Timaeus generalizes
his conclusions to “everything that comes to
be” (49e7). Thus every generated entity is a
characteristic, not a ‘thing;’ the characteristic
12
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Triangles, Tropes, and τὰ τοιαʋ τ̃ α: A Platonic Trope Theory
may be replaced at any time by another char
acteristic, but it remains what it is as long as
it is. If we generalize this claim, it applies to
any characteristic, e.g., that of being a garbage
truck, not just to elements. And there is no rea
son to think it does not apply completely gen
erally, since Timaeus gives “hot” and “white”
as further examples. Even if we think that the
properties of being hot and being white are
suggested from our discussion of fire, they are
widely applicable to macroscopic objects, and
we are given no reason to restrict their use to
describing elements. In fact, Timaeus cannot be
restricting his argument to elements, since he
extends it to “anything you can point to,” and
it is not, strictly speaking, possible to point to
an element, since they are microscopic particles
(we can point to some fire or water, but these
are macroscopic bodies composed of appropri
ate elements). Thus we should regard Timaeus’
proscription as perfectly general, as he insists
several times: do not call anything that comes
to be τοῦτο, but each thing that comes to be
is τὸ τοιοῦτον.4
2.2. ALL AND ONLY THINGS THAT
COME TO BE ARE IMAGES OF FORMS
Timaeus gives two accounts of the universe’s
generation, which I take to be complementary
in at least the minimal sense that the second
does not annul the first. 5 We have focused on
the second thus far. In his first account, Ti
maeus distinguishes between Being and Be
coming. He does not specify the members of
Becoming — which would, it seems, include
everything in the changeable, sensible world
— but he begins with an informative example:
the universe, taken as a whole, is in the class of
Becoming. In addition, he offers a character
ization of this exemplum of Becoming, namely
that it is an image (τόνδε τὸν κόσμον εἰκόνα
τινὸς εἶναι, 29b1‑2), viz., an image of an eter
nal, changeless model (παράδειγμα, 29a2‑b2).
So the demiurge, a divine craftsman, looks to
Being in order to make the universe, which is
an image of the Living Being that contains all
the intelligible living beings as parts just as
the universe contains us and all the other vis
ible creatures (30c2‑d1). I take the “intelligible
living beings” to be Forms of animal species,
e.g., the Form of Human Being, the Form of
Goat, etc. The Living Being that contains all
the others would be, then, the Form of Living
Being, i.e., the transcendent universal that ex
plains what all living beings have in common.6
Thus we have reason to identify Being with
Forms, an identification that is confirmed,
e.g., at 51d3‑52a4. The parts of the universe
on which Timaeus focuses, then, are the liv
ing parts (including heavenly bodies, which
are created gods), and these are all images of
animal Forms. In fact, later in the Timaeus Be
coming is explicitly identified with the class of
Form images (48e4‑49a1), so that each thing in
Becoming is an image of a Form. It is not clear,
at this point, what it means to be an “image” of
a Form, but this will become clearer when we
identify images of Forms with τὰ τοιαῦτα.
Timaeus’ second account marks a signifi
cant change in our ontology: instead of two
kinds, Being and Becoming, we now have a
third, the Receptacle. I will presuppose as little
about the Receptacle as possible, since it is a
controversial subject.7 What I will emphasize is
that everything that comes to be — everything
in the class of Becoming — is an image of a
Form.8 It is controversial to say that Becoming
is unchanged between the first and second ac
counts, as some think that the first account’s
Becoming is separated into the Receptacle
and Becoming of the second account.9 But if
we bracket the Receptacle, we can apply the
CHRISTOPHER BUCKELS |
later rule — that everything that comes to be
is an image of a Form — to the first account,
especially since Timaeus’ first and primary
exemplum of Becoming, the universe itself, is
explicitly an image of a Form. Thus Form im
ages are the things that come to be, and things
that come to be are Form images.
2.3. THEREFORE, τὰ τοιαʋ ̃τα
(THIS-SUCHES) ARE IMAGES OF FORMS
Earlier we found Timaeus extending his
conclusions about fire to “everything that
comes to be.” Thus each thing that comes to
be is τὸ τοιοῦτον. It is not a thing — at least
not as we usually conceive ‘things,’ as physi
cal objects with many characteristics — but a
characteristic. Now we have seen that Form im
ages exhaust the category of Becoming; Form
images are those things that come to be. Thus
Form images are τὰ τοιαῦτα. This makes per
fect sense, at least if we think of Forms such
as Goodness, Justice, and Beauty. Being good,
being just, and being beautiful are characteris
tics, not things; they characterize things. So an
image of Goodness should be the characteristic
of being good that some particular thing has.
An image of Justice is the just character of a
particular person or action. And the image of
Beauty a beautiful painting possesses is the
characteristic of its being beautiful.
But what about other Forms? One of the
Forms explicitly discussed in the dialogue is
Fire, which Timaeus says must exist over and
above all sensible fires (51b7‑52a4). But fire is
not, one might think, a characteristic. Fire is a
thing, at least for the ancient Greeks; it is, after
all, an element. Timaeus, however, explicitly
denies that fire is a thing at 49d5, telling us
that fire is τὸ τοιοῦτον (or that τὸ τοιοῦτον is
fire). We should think of fire as fieriness; Fire
13
Itself, mentioned at 51b8, would certainly be
understood best as Fieriness, not as a great fire
in heaven. It is distinct from each particular
fire, but it explains why each particular fire is
fire; we should understand this to mean that
Fieriness explains why each particular fire,
each with the characteristic of being fiery, is
fiery, rather than being some other quality.
There are many fiery things: each fieriness,
that is, each particular characteristic of being
fiery, is a paticular image of Fieriness itself, just
as each just character is a particular image of
Justice itself.
A word about the Receptacle: while Timaeus
denies that anything that comes to be is a this,
he turns around to argue that there is a this
that can be stably designated, although it is
not essentially characterized, i.e., in itself it
has no properties, or at least no properties that
correspond to Forms. The Receptacle evidently
has the stability that particulars lack, so that it
can be designated reliably, even though entities
are constantly coming to be in it and perishing
from it. So when we attempt to designate a bit of
flame with a demonstrative “this,” we actually
designate the bit of the Receptacle in which the
bit of flame appears. When we say “that is fire,”
speaking normally, what we really do is pick out
a location in the Receptacle and assert that it
is fiery, i.e., that there is an image of Fieriness
in that region of the Receptacle.10
A proper treatment of the Receptacle,
whether it is space, substratum, both, or neither,
would take us too far afield, but let us return to
the traditional and alternative interpretations.
Partisans of the traditional interpretation point
out that, according to the alternative, we are
using our normal, everyday terms incorrectly.11
We are wrong when we point to a bonfire and
name it “fire,” since we should only use “fire” to
name the characteristic common to it and every
other bonfire. This accusation is, of course,
14
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Triangles, Tropes, and τὰ τοιαʋ τ̃ α: A Platonic Trope Theory
true; in fact, it seems that the main point of the
passage is that we use our everyday terms in
a loose and derivative manner. Strictly speak
ing, we apply terms incorrectly. Although its
partisans do not appear to recognize it, the
traditional translation actually presupposes a
corresponding error, since we only correctly
name a bonfire “fire” if we are using the name
adjectivally, not designating the bonfire as a
self‑subsisting thing but only as a temporary
characteristic of that space. But our everyday
use of the term “fire” is intended to pick out
a real, independent thing, not a property: we
think there really is a thing there, even if it is
a thing I can pass my hand through. For ex
ample, when I get burned by some fire, I think
that there must be a thing there that burned
me. Since fire may be a strange example of a
thing to our modern ears, let us instead take
a hunk of earth. According to the traditional
translation, earth is a temporary characteristic
of a part of the Receptacle, not a self‑subsisting
thing. Calling a hunk of rock “earth,” then, is
correct only if we are applying the name not
to a this but to a characteristic. The alternative
translation insists that the name “earth” picks
out the characteristic common to this hunk and
each other hunk of earth, rather than picking
out the physical thing.
The two translations, then, have equiva
lent conclusions: our normal terms pick out
temporary characteristics, not self‑subsisting
things. The difference is that the traditional
interpretation allows us to speak of what we
see as fire, while qualifying the nature of fire
so that it is not the kind of thing to which we
think we are referring, while the alternative
prohibits us from calling what we see fire, if
we are to speak correctly, since the nature of
fire is far different from what we suppose it is.
According to both translations, fire is a charac
teristic, characterizing a bit of the Receptacle.
The Receptacle stands on its own, but fire is
parasitic.
Despite their equivalence, the traditional
translation is used to support an interpreta
tion whereby particulars in the Timaeus are
self‑subsistent substances.12 According to Zeyl,
such substances endure over time as the sub
jects and substrata of various characteristics
such as fire.13 Such a reading relies heavily on
an interpretation of the Receptacle as a sub
stratum and cannot be based simply on the
translation we have examined. In other words,
the traditional translation is not sufficient for
a substance‑interpretation of particulars; what
we have, on either translation, are character‑
istics, not things, coming to be. These charac
teristics, or τὰ τοιαῦτα, are images of Forms,
and they exhaust Becoming. Taking Forms to
exhaust Being, then, we would be left with three
ontological categories: Forms, Forms images
(τὰ τοιαῦτα), and the Receptacle.
3. TRIANGLES
We have identified all things that come to
be with images of Forms and hence with τὰ
τοιαῦτα. But there is another account of the
generation of macroscopic objects in the Ti‑
maeus, namely, that sensible particulars are
composed of more fundamental particles—
what I will call elemental triangles. In this
section of the paper I will show that these tri
angles are τὰ τοιαῦτα or are themselves com
posed of τὰ τοιαῦτα, so that our account of
sensible particulars stands. Before we discuss
these triangles, though, we need to address the
context of the passage, where Timaeus tells us
that the demiurge brings an unorganized uni
verse, full of formless “vestiges” of elements in
discordant motion, into a whole organized by
form and number. There are two readings of
CHRISTOPHER BUCKELS |
this text, a literal, which takes the divine cre
ation in time at face‑value, and a non‑literal,
which takes the cosmos to be eternal and, thus,
reads this passage as a myth.14 At stake is the
status of the elements in the pre‑cosmic state:
did “vestiges” of elements actually exist, and
thus must be accounted for, or are they simply a
relic of the mythic form of Timaeus’ discourse?
I believe my account is neutral between these
two readings, but I owe partisans of the literal
reading an explanation of how my account is
compatible with pre‑cosmic vestiges, so I will
return to this point after discussing the con
struction of elements.
Elements are, Timaeus tells us, bodies
(σώματα, 53c4‑5). Thus, he infers, they are
composed of triangles, since bodies have depth,
things with depth have planar surfaces, and
planes may be broken into triangles. Each ele
mental body of fire, for example, is a four‑sided
pyramid with faces composed of six 30‑60‑90
triangles, and these triangles, when dissolved
from their current structure, may join with
others to form molecules of water or air. Each
elemental body is defined by its number and
form, i.e., a certain number of such‑and‑such
triangles organized into such‑and‑such a shape.
So wherever triangles are combined into a tet
rahedral pyramid, there is fire. These elemental
triangles come to be in the Receptacle, thereby
forming parts of the geometrical figures that
compose elemental bodies, and elemental bod
ies, i.e., fire, earth, air, and water, in turn, form
ordinary, sensible particulars.
Although he has not said it here, Timaeus
must be speaking of the sensible, generated
elements — sensible fire, sensible water, etc.
— because he tells us earlier that bodily things
are sensible and thus also generated (28b7‑c2).
So we should not immediately conclude that
the Form of Fire is composed of elemental tri
angles, since it is not a body. The Form of Fire
15
does, however, seem to be structural, since fire
is instantiated whenever there is an instance
of a given structure — i.e., whenever elemental
triangles are arranged in a certain way. Thus we
preserve the prior reference to fire as this‑such,
i.e., such a structure as this, one that includes a
certain color, heat, and other properties. These
properties taken by themselves, scattered across
the Receptacle, do not constitute a fire, but they
do so when they are arranged correctly, i.e.,
when there is a structure that includes these
this‑suches.
Looking ahead, Timaeus explains fire’s sen
sible properties by reference to properties of its
geometrical constituents; fire is hot because
of the small, sharp nature of its body’s angles
(61d5‑62a5). Fire’s nature is not, then, qualita
tive, e.g., to be hot, since its heat is explained
by reference to its geometrical structure. Heat
is a necessary property of fire; it is included as
a necessary consequence of fire’s structure, as
we see when Timaeus describes the smallness,
sharpness, and lightness of fire (55e7‑56b2). If
fire’s other qualities can be explained likewise,
and there is no reason to think they cannot,
then fire is simply a structure that necessarily
includes certain properties. Thus, elements’
natures are their geometrical structures.15
The Form of Fire is structural.16 Notice that
we need not say that all tetrahedral pyramids
are fires, since fire need only be one of the in
finite possible kinds of tetrahedral pyramids;
in addition, we need not worry that a macro
scopic tetrahedral pyramid might burst into
flames, if it had the right proportions, since
fire is a structure of elemental triangles, not a
structure of just any triangles.17 It might help
to think of the Fieriness as a universal with
twenty‑four slots (four faces of six triangles
each), each slot being filled by the Form of the
appropriate triangle;18 when fire is instantiated,
the twenty‑four triangles are also instantiated,
16
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Triangles, Tropes, and τὰ τοιαʋ τ̃ α: A Platonic Trope Theory
since the fire trope relates the triangle tropes,
which, in turn, relate angle tropes, etc.
I can now return to the literal reading of the
demiurge’s organization of the pre‑cosmic ves
tiges. Let us assume that the vestiges had some
structure, although imperfect, so that we may
continue to assign the nature of the elements
to their geometrical structures.19 Fieriness is
structural, and there are imperfect instances
of this structure in the Receptacle even before
the demiurge sets to work. Each instance of
fire would, in these pre‑cosmic conditions, be
inexactly formed, perhaps with irregular con
stituents or no constituents at all, such that it
would be prohibited from transforming into
the other elements in the way that fire can af
ter its geometrical construction. At the time
of creation, the demiurge assigns a number
to each structure, i.e., he looks to Fieriness
and imitates its geometrical structure in the
Receptacle, crafting fiery bodies out of a cer
tain number of elemental triangles, which are
in turn formed in the Receptacle. Since this
structure has been imposed on the bodily ele
ments, they now have a certain regularity and
stability: each molecule of fire is a pyramid
with regular faces composed of triangles, and
these triangles, when dissolved from their cur
rent structure, may join with others to form
molecules of water or air. In other words, there
are now rules for an orderly transition from one
element to another, and each elemental body
is defined by its number and form. Thus my
interpretation of the elements can be reconciled
with either a literal or a non‑literal reading of
the dialogue’s creation story.
My account of the elements and their con
struction raises several concerns. First of all,
one might wonder about the consequences
of my account for the interpretation of the
Receptacle as material or nonmaterial. But
my account thus far of elemental triangles
and bodies does not lock us into a particular
interpretation of the Receptacle, since we have
nowhere assumed that the triangles are bodily
or material. In fact, the triangles cannot be
bodily, since bodies have depth (53c5-6) and
plane figures have no depth. It is also dif
ficult to hold that they are material, since
they are, again, only two‑dimensional. 20 For
the same reason, it is difficult to assign any
‘containing’ ability to the triangles, in order
that they may hold in ‘stuff ’ that then makes
particulars material: a plane can offer no kind
of resistance, let alone contain something. 21
The triangles do, however, compose elemen
tal bodies which, in turn, compose material
particulars, so there is some temptation to
call them material. “Material” will, however,
mean something peculiar. Contemporary sci
ence has acclimated us to the idea that the
most basic constituents of reality—funda
mental particles—are very different from the
objects we see and with which we interact.
Just so, Plato’s two‑dimensional triangles are
material in a stretched sense, 22 in the same
way that non‑extended simples might be said
to be material in a theory that takes them as
basic constituents of material objects.
Second, we seem to have left Form images
behind. How do we reconcile them with el
emental triangles? There are two main pos
sibilities: first, that triangles are composed of
this‑suches, even though they are not bodies,
and second, that triangles are, themselves,
simple images of Triangularity, i.e., images
with no further components. While the lat
ter suggestion would be fully compatible with
the interpretation presented above, I do not
think it correct, since elemental triangles have
multiple properties. Timaeus recognizes this
when he describes his reasoning in selecting
the ‘best’ triangles: triangles are right‑angled,
trilateral, shaped, etc. If there is a Form for
CHRISTOPHER BUCKELS |
each of these properties, elemental triangles
must be composed of Form images.
One may object, however, that I am assum
ing that triangles and their properties are Form
images, but Timaeus never explicitly calls them
such. Let us recall our treatment of fire. When
we say colloquially, “this is fire” (or, “fire is
this”), what we should really say, if we were
speaking perspicuously, is that this is a part
(region) of the Receptacle in which has come
to be this‑fieriness. We can generalize this rule
to other parts of the Timaeus, even though Ti
maeus speaks in a more colloquial manner in
other places. When he speaks of triangles, it
might seem that they abide in a way that fire
does not. But we could no more pick out “this
triangle” or “this pyramid” than we could “this
fire.” In the case of “this fire,” we pick out a por
tion of the Receptacle and a Form image, viz.,
an image of Fieriness. Similarly, when we say
“this triangle,” we are speaking loosely, picking
out a portion of the Receptacle and an image of
Triangularity. There is no more reason to think
that this‑triangle abides than to think that thisfire abides. If fire flees the use of “this,” then so
should triangles. Thus, an image of Triangular
ity is a this‑such on the same ontological level
as an image of Fieriness or Heat.
In fact, the passage immediately following
the “much misread” passage about fire tells us,
explicitly, that a triangle is a this‑such. At 50a5
‑b5, when Timaeus tells us not to call a golden
triangle “triangle” but “gold,” he goes on to say
that we should be content if the triangle (or
any other figure molded in gold) accepts the
designation of τὸ τοιοῦτον. Since the mean
ing of “triangle” has not changed between 50a
and 53c, we should continue to see triangles as
this‑suches, instances of Triangularity, which
partially constitute elemental triangles.
We have another reason to consider elemen
tal triangles as derivative objects composed of
17
Form images. Being hot is τὸ τοιοῦτον (50a2).
Since τὰ τοιαῦτα are Form images, heat is a
Form image. Fire is, of course, hot, and Ti
maeus explains this fact by appealing to its
geometrical construction. Fire is a four‑sided
pyramid with acute angles. These small, sharp
angles cut flesh, and that is what we call heat
(61d5‑62a5). And so heat, an image of Heat, is
a property of the elemental body of fire. Since
everything made out of hot etc. are τὰ τοιαῦτα
(50a3‑4), and τὰ τοιαῦτα are images of Forms,
pyramids must also be images of Forms. There
is no reason to think that the elemental tri
angles composing those pyramids — triangles
which are also made out of their properties —
are any different. So elemental triangles are
Form images or composed thereof, and the
mechanistic explanation of heat is compatible
with bodies being composed of Form images.
One may press the worry about triangles
and Form images, however, and formulate a
‘third wave’ or ‘greatest difficulty’ for my in
terpretation of the elemental triangles: such tri
angles, it seems, abide and persist in a way that
τὰ τοιαῦτα do not, and thus elemental triangles
are not subject to the worry about elements that
leads Timaeus to formulate the this/this‑such
distinction. In fact, one might say, elemental
triangles are the solution to this worry: they
must persist to underlie the geometrical ac
count of elemental transformation.
My account might lead us to think, in
contrast, that triangles are as unstable as any
other this‑such. They could, at any moment,
change into anything else. Indeed, we might
even be tempted to say that elemental trian
gles are new at every moment, i.e., that each
triangle in the universe undergoes so‑called
immaculate replacement, being replaced by a
seemingly identical triangle each instant, so
that, technically, “nothing is ever the same”
(Phaedo 78e3‑4). Although this is a tempting
18
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Triangles, Tropes, and τὰ τοιαʋ τ̃ α: A Platonic Trope Theory
way to explain Plato’s persistent calls for flux
in the sensible world, there are reasons to doubt
that elemental triangles are as unstable as the
elements they underlie. First, it is not obvious
what triangles would change into unless they
undergo immaculate replacement, and then we
are seemingly positing immaculate replace
ment just to ensure that things are “never the
same,” which is circular. Second, it seems that
triangles must persist so that the elements can
change into each other. It is clear that the same
triangles that compose a body of fire can go on
to compose a body of air (56e2‑7).
Third, Timaeus’ discussion of aging (81b5
‑e5) seems to require that elemental triangles
persist. He tells us that in newly constructed
living things, elemental triangles are “fresh”
and “straight from the stocks” (νέα μὲν οὖν
σύστασις τοῦ παντὸς ζῴου, καινὰ τὰ τρίγωνα
οἷον ἐκ δρυόχων ἔτι ἔχουσα τῶν γενῶν, 81b5
‑7). They are firmly locked together and so
they easily overcome and cut up the “older”
(παλαιότερα) incoming triangles from food.
But when a triangle’s “base” weakens (literally,
its “root,” ἡ ῥίζα τῶν τριγώνων χαλᾷ, 81c6‑7),
the living thing’s triangles can be overcome by
entering triangles, and it enters old age. Death
comes when the soul is released, which happens
when the interlocking bonds of the marrow’s
triangles no longer hold together (τῶν περὶ τὸν
μυελὸν τριγώνων οἱ συναρμοσθέντες μηκέτι
ἀντέχωσιν δεσμοὶ τῷ πόνῳ διιστάμενοι, 81d5
‑6). Marrow, which anchors soul to body, is
made of specimens of the elements with the
most precise triangles (73b5‑8).
While the information about aging and
death might lead us to believe that elemental
triangles are themselves destroyed, this is a
doubtful reading of the passage. Instead, it
seems that the bonds between triangles — ei
ther those which hold triangles together into
elements, or those that hold them together to
compose marrow, or both — are destroyed.
This is also how we should read the claim about
triangles’ bases, namely, that these refer to how
triangles are put together to form the faces of
regular solids. When their bases weaken, tri
angles are more likely to come apart from each
other, destroying marrow and releasing soul
from body. Triangles themselves re‑form with
other triangles to compose different elements,
although I do not deny that triangles could, in
principle, be destroyed; they are, after all, parts
of Becoming. Timaeus also seems to refer to
the same triangles throughout his description
of aging, rather than immaculately replaced
duplicates. 23 Thus triangles seem to persist
for some time, since they can be “fresh” and
newly constructed or “older” and more weakly
bonded together.
While it may be that some tension is un
avoidable, as Timaeus himself admits (29c4‑7),
in such an ambitious account of the universe,
we can allow some groups of τὰ τοιαῦτα to
be more stable than others; in fact, we have
independent reason for allowing such a pos
sibility, for the heavenly bodies have come to
be and yet appear to be indestructible. Timaeus
posits τὰ τοιαῦτα to distinguish Form images
and the Receptacle, since Form images have a
precarious ontological stability, due to their
complete dependence on Forms and Receptacle,
and the Receptacle is not dependent on Form
images for its existence and stability. Even if
elemental triangles or heavenly bodies have a
stability that other particulars lack, they are
still composed of images, dependent upon
originals (e.g., Triangularity) and a medium,
and so flee the designation of ‘this’ or ‘that.’
They are still destructible, derivative, and im
permanent. In these respects, they are no more
entities in their own right than elements or
other particulars, for they are constructed of
more basic entities — τὰ τοιαῦτα correspond
CHRISTOPHER BUCKELS |
ing to their properties—and these more basic
entities are fully dependent upon Forms and
the Receptacle.
4. TROPES
Timaeus’ τὰ τοιαῦτα are, I have been con
tending, tropes, which metaphysicians still call
this‑suches. 24 Consider that, for any property
Fness corresponding to predicate F, we can al
ways call an instance of Fness F. An instance
of fieriness is always fiery—a fiery trope is
fiery—whenever it exists. But predicates can
not be applied in a stable, permanent way to
any concrete subject that has come to be, since
any thing or stuff could be characterized by a
different predicate, as Timaeus shows at 49d4
‑6. Predicates can only be applied to tropes and
then derivatively to aggregates of tropes, i.e.,
the things “composed of hot and white and the
opposites.” We can concisely demarcate Plato’s
ontological division between Forms and Form
images as the division between properties (uni
versals) and instances (tropes).25
4.1. IMAGES OF FORMS ARE NOT
IMMANENT UNIVERSALS
One may object that τὰ τοιαῦτα are not
tropes but immanent universals (or ‘immanent
forms’). 26 Timaeus 49e4‑7, which gives a more
complete formula for τὸ τοιοῦτον (“this‑such
coming around always in similar fashion in
each case and all together”), seems to raise a
difficulty for taking Form images as tropes,
since τὰ τοιαῦτα here seem more like imma
nent universals; it seems that one and the same
‘sort of thing’ recurs in multiple instantiations.
This recurrence would fit well with immanent
universals, which are one and the same in all of
19
their instantiations. For example, if white were
an immanent universal, there would be only
one white, although it would be in many places
at the same time. Whiteness would ‘recur’ ev
ery time there is a white thing, but it would be
the same whiteness — numerically one and
the same — that is ‘in’ each white thing. In
contrast, there are many white tropes, each dis
tinct from the other, but each is equally white.
For a trope theorist, Whiteness recurs in white
things in the sense that each white thing has
a numerically distinct white trope ‘in’ it. So if
Form images are immanent universals, then
an image of Justice would be one and the same
in all instances of Justice, ‘recurring’ in each
instance. But if Form images are trope‑like,
then there are many images of Justice, each
one distinct and fleeting.
A trope‑theoretic reading can respond to
this difficulty by emphasizing that the Form,
not the image, recurs; the same Form is imaged,
so a characteristic “comes around always in
similar fashion” because a single Form is im
aged in many locations, not because there is
a single, multiply‑located image.27 Whiteness
recurs in every instance of white because there
is an image of one and the same Form in every
instance, even though each image is numeri
cally distinct from every other image.
The trope‑theoretic reading is, in general,
preferable to the immanent universal reading,
since Forms would be redundant if there were
immanent universals. Immanent universals
would do all the work for a theory of universals
in a way that tropes do not, since, according to
a trope‑theoretic interpretation of Plato, Forms
still serve as unitary, eternal properties and
objects of knowledge. With immanent univer
sals, however, there is already a property that
is immutable and can serve as an object for
knowledge, so we have no need of Forms in
addition. Since the Timaeus clearly makes use
20
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Triangles, Tropes, and τὰ τοιαʋ τ̃ α: A Platonic Trope Theory
of separate Forms—for instance, Timaeus ar
gues that Fieriness is distinct from sensible fires
(51b7‑c5) — and, moreover, Forms are justified
precisely because they are objects of knowledge
(51d3‑52a4), the trope‑theoretic interpretation
of τὰ τοιαῦτα is preferable.28
So, if we dismiss an immanent universal
interpretation of τὰ τοιαῦτα, which treats
them as repeatable, multiply‑located enti
ties, we are left with a trope theoretic inter
pretation, which treats each Form image as
a distinct, non‑repeatable entity located in
one spatial region. To be clear, taking Form
images to be tropes amounts to no more than
this, i.e., saying that they are non‑repeatable
instances of properties that are each located
in exactly one spatial region at a time. As
long as Form images are instances of prop
erties, then we have an exhaustive division
between interpretations that take them to
be each located at one spatial region (tropes)
and those that allow them to be multiply
‑located in space (immanent universals), as
well as reason to prefer the trope‑theoretic
interpretation.
4.2. IMAGES OF FORMS ARE NOT BODIES
If Form images are instances of proper
ties, then they are tropes. But, despite my ar
gument that all Form images are τὰ τοιαῦτα
and τὰ τοιαῦτα are Forms images, one might
find in Timaeus’ first account of the universe
a counterexample to taking them as property
‑instances. There, it seems, Timaeus discusses
things, or bodies, not properties, as images of
Forms. For example, the universe is an image of
the intelligible Living Being. But the universe is
not a property; it is a thing with properties. So
can we say it is τὸ τοιοῦτον without discarding
Timaeus’ first account?
In fact, the universe is no more an ob
stacle to a trope‑theoretic interpretation of
the Timaeus than is fire, since both are, in
fact, bodies. Just as fire is an elemental body
composed of its properties, so is the universe
a body composed of its properties. 29 Let us
review fieriness; it is a structural property,
an arrangement of other properties, includ
ing heat—which turns out to be the acuteness
of the fire molecule’s angles. Notice that the
fiery trope is structural; it does not have other
properties, but it structures them. Whenever
fire is instantiated, a certain arrangement is
also instantiated, specifically, a tetrahedral
pyramid of elemental triangles. This seems
to an observer to be an object, i.e., a fire, and
it is the apparent fire that we say is hot, not the
fiery trope. The fiery trope is not itself hot, but
always includes heat, which is hot.
Since the other elements are explained
similarly, we should explain all substantive
Forms similarly. Supposing there are Forms
of living things, since the Living Being and
‘other intelligible animals’ play an important
role as models (παραδείγματα) in creating
the universe, these Forms should be under
stood as structures parallel to the elements.
In fact, the Living Being seems simply to
be a structure of animal Forms; the Form
of any particular animal, such as Human
Being (or Humanity), would, in turn, be an
arrangement of various parts that make up
human beings. These parts include physical
parts such as hearts, which will in turn have
parts, ending with elemental triangles and
their constituent tropes; but human beings
also include non‑physical parts such as vir
tues and rationality. These latter components
are Form images, too, and components in the
structure of Humanity just as are the physi
cal parts, since all are, in the final analysis,
composed of tropes. 30
CHRISTOPHER BUCKELS |
21
5. CONCLUSION
BIBLIOGRAPHY
I have argued that the second category in
Plato’s tripartite ontology, that of Becoming,
is the category of Form images. Moreover, I
argued, Form images are tropes, i.e., proper
ty-instances, rather than bodies or immanent
universals, and particulars are composed of
tropes. The elemental triangles introduced to
constitute particulars turn out to be composed
of tropes as well, so that tropes and things
composed of tropes exhaust the category of
Becoming. There is, then, no place for sensible,
material particulars in Plato’s fundamental on
tology. Instead, such particulars are deriva
tive entities, constructed from Form images
which are, in turn, dependent on Forms and
the Receptacle. While Aristotle distinguishes
between kinds (substances), such as human be
ings and animals, and other properties, such as
qualities, quantities, and relations, Plato does
not: instances of humanity and triangularity
are just as ‘insubstantial’ as instances of white
ness and heat. I have not, however, put forth
the stronger thesis here that particulars are
only composed of tropes, i.e., that they are
bundles of tropes, since I have not directly
addressed the status of the sole member of
the third ontological category, the Receptacle.
Given, however, that particulars can success
fully be resolved into tropes, there seems little
philosophical reason to take the Receptacle to
be a substratum in which those tropes inhere.
It is more parsimonious to take particulars as
bundles of co‑located tropes in the space of
the Receptacle. Even if we do not fully accept
such a bundle theory, however, the ontology of
particulars in the Timaeus is not a substance
ontology but a trope ontology. 31
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… 2016b. “The Ontology of the Secret Doctrine in
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Gregory, Andrew. 2000. Plato’s Philosophy of Science.
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NOTES
1
Ferber 1997 also addresses the question of why
there are Forms in the Timaeus, giving an analysis of the
brief argument for Forms at 51d3‑52a7. See his 182‑4,
for an alternate account of particulars in the Timaeus as
relational entities rather than substances.
2
There are many accounts of tropes, and I
cannot here give a detailed comparison between Plato’s
tropes and contemporary trope s— this is work for
another paper. There is some similarity, however, between
Plato’s trope ontology and Lowe’s four‑category ontology
— one dissimilarity is that Plato’s ontology contains only
three categories, Forms (transcendent universals), tropes,
and the Receptacle (substantival space). For a defense
of taking the Receptacle to be substantival space, see
Buckels 2016a. For contemporary accounts of tropes, see,
e.g., Ehring 2011, Lowe 2006, Campbell 1990, Armstrong
1989, and Williams 1953.
3
All textual references are to Burnet’s OCT
Timaeus and translations are my own, unless otherwise
indicated. Note that Plato uses “τοιοῦτον” in many dif
ferent ways in the dialogues; I am not claiming that every
use of “τοιοῦτον” is a reference to something that comes
to be, only that Plato uses it thus in key passages of the
Timaeus.
4
Miller 2003, 82, argues that the proscription
generalizes only to ‘all the elements,’ not “everything that
comes to be.” This coheres with his reading of the entire
passage as restricted to elements. Harte 2002, 252, n. 162,
similarly doubts that the proscription generalizes to all
things, since the initial problem “is closely tied to the role
of earth, air, fire, and water, as candidate elements in the
traditional sense.” Broadie 2012, 202‑3, also agrees. On
this, Miller 2003, 81, writes that “Plato is not concerned
with ‘phenomenal fire’ but with the element fire that we
observe,” distinguishing between the two so as to avoid
the generalization from phenomenal fire to phenomena
in general. But it is difficult to distinguish elemental and
phenomenal fire, since, as Miller admits, we observe
elemental fire, at least when aggregated: it is visible and,
in fact, part of all visible things (31b5), so it seems likely
that Timaeus generalizes his argument regarding fire—
the visible element—to all visible particulars, which all
contain fire as a part. Cf. 50a2‑4, where the argument
concerning elements is explicitly generalized to all prop
erties; also cf. Prior 1985, 110; and Silverman 2002, 258:
“the lesson applies to the whole of the physical cosmos.”
A more problematic point Miller makes is that Timaeus
has not given us reason to think that the extension from
CHRISTOPHER BUCKELS |
the elements to all sensible phenomena is legitimate
(even if he in fact extends it), since the initial puzzle is
only about the changing of one element into another.
But the elements are the basis for all other sensible
phenomena, so if the elements are so unstable that they
might transform into each other at any moment, then the
things composed of the elements seem to be vulnerable
to the same change. Indeed, Timaeus only holds that it
is possible for one element to cease to be and another to
take its place at any time, and it seems true that any given
physical object x may cease to be what it is at any instant
and be replaced by a non‑identical, even if extremely
similar, y.
5
Timaeus ‘restarts’ his cosmogony after about
twenty Stephanus pages, so there is a ‘first’ and a ‘second’
account of the creation of the universe. I take it that the
two accounts are largely compatible, with the second
building upon the first (for one thing, the Receptacle is
added in the second, so we have a tripartite, rather than
a dualistic, ontology), but this is not uncontroversial.
Broadie 2012, 201‑2, Johansen 2004, 117, Silverman 2002,
248‑56, and Harte 2002, 213‑226, also seem to take the
two accounts as complementary, although, e.g., Silverman
sees the first account as giving us “traditional forms”
while the second gives us “geometrical forms,” and he
then joins these such that geometrical forms ground mat
ter and place while traditional forms ground qualities.
6
Cf. Broadie 2012, Ch 3, esp. 70‑74, who argues
that the “Intelligible Animal” here (my “Living Being”) is
not a “thick intelligible,” although she admits her argu
ment is not decisive.
7
It is worth pointing out that Form images be
ing τὰ τοιαῦτα, and τὰ τοιαῦτα being tropes, is compat
ible with either of the two major interpretations of the
Receptacle, namely, that it is a substratum (in which case
tropes would inhere in it) and that it is space (in which
case tropes would be located in its regions). See also note
30.
8
Silverman 2002, passim, also recognizes the
importance of Form images, which he calls Form copies.
His Form copies differ from my Form images in several
ways, some of which have to do with our readings of the
Phaedo. With respect to the Timaeus, the biggest differ
ences between our readings (which are both indebted to
Cherniss 1954 & 1957) lie in our treatment of the Recep
tacle (see Buckels 2016a) and geometrical versus “traditio
nal” Forms (see notes 15 & 19). More broadly, Silverman
argues that Form copies cannot be destroyed, whereas my
Form images come to be and perish; Silverman’s Form
copies are thus difficult to distinguish from Forms. While
Silverman and I both argue that sensible particulars (i.e.,
physical objects) are entirely composed of Form copies (in
my case, Form images), Silverman retains particulars as
a distinct category of Plato’s ontology, while I think they
reduce entirely to Form images (see note 29).
9
Cf. Silverman 2002, 261‑5, and Zeyl 2014, §6
10
Cf. Cherniss 1954, 128: “If at any time
anywhere one tries to distinguish any phase of the
phenomenal flux from any other by saying “this,” one
23
always in fact points to the permanent, unchanging, and
characterless receptacle in which are constantly occur
ring transient and indeterminate manifestations of the
determinate characteristics just mentioned [‘the such and
such, whatever the correct formula may be, that is always
identical in each and all of its occurrences’].”
11
Zeyl 2000, lviii‑lix; cf. Silverman 2002, 258‑60.
12
Cf. Silverman 2002, 260: “What is at stake
between the reconstructionist [alternative interpretation]
and the tradition is not so much these linguistic formulae
[i.e., how to address particulars] but rather the account of
the phenomena themselves.”
13
Zeyl 2000, lxi. Cf. Mertz, 83f, on Plato’s “con
crete individuals.”
14
Cf. Cornford 1937, 28‑32, Prior 1985, 95.
15
Mohr 1985, 108‑15, also argues for such a view.
For discussion of how the triangles come together to form
bodies, see, e.g., Cornford 1937, 210‑30.
16
Silverman 2002, 282, writes that there is an
“inexplicable coincidence of geometrical bodies oc
cupying space and properties entering and exiting the
place defined by those bodies.” He thinks that there is
no essential connection between a certain arrangement
of geometrical form‑copies (my Form images) and a
certain grouping of “traditional” form‑copies (hot, yel
low, fire, etc.). Thus is it a coincidence that quantitative
and qualitative form‑copies always fit together, with the
qualities entering and exiting the place of the quanti
ties. But I think Silverman (esp. 249f ) makes too much
of distinguishing geometrical from traditional Forms;
I argue that an image of Triangularity is just another
τὸ τοιοῦτον. Thus, on my account, Fieriness is a certain
species of Tetrahedral Pyramid. Harte 2002, 262‑3 and n.
189, has a position similar to mine: “particles of earth, air,
fire, and water, as constructed by the demiurge, imitate
forms through their geometrical construction; they are
structures of space, whose properties, perceptible and
other, are parasitic upon their geometrical structure.”
Harte avoids Silverman’s problem (the ‘inexplicable coin
cidence’ of geometrical Forms) but does not explain how
geometrical constructions imitate elemental Forms, since
she stops short of identifying Fire with a geometrical
structure: Harte does not suggest “that the forms of fire
and earth are themselves [the type] regular solids.”
17
Cornford 1937, 190, denies that Fire is a
certain structure, because we do not say, when we see fire,
“there are some pyramids.” Instead, Fire is a combina
tion of certain qualities. The Form is the “meaning of the
name ‘Fire,’” and the “quality is the copy” of the Form.
But we do not deny that water is H2O — even that it is
essentially H2O — even though we do not, before our
first chemistry lesson, say, “there is some H2O.” “Water”
refers to a combination of certain qualities, but this does
not prevent us from saying that it is essentially H2O. Cf.
White 1981, 331‑4.
18
Cf. Lewis 1986 for discussion of structural
universals and Gilmore 2013 for slots in universals.
19
Cornford 1937, 198‑206, instead argues that
the nature of fire is qualitative. For criticism of Cornford,
24
|
Triangles, Tropes, and τὰ τοιαʋ τ̃ α: A Platonic Trope Theory
see Mohr 1985, 108‑15. Johansen 2004, 126‑7, seems to
give a qualitative explanation, too.
20
Silverman 2002, 278‑81, holds that matter
or corporeality is an emergent property, a result of the
arrangement of geometrical form‑copies. If by this he
means that corporeality, the property of being a body,
is instantiated whenever triangular form‑copies are
arranged to form a solid, then this seems right. Cf. Silver
man 2002, 255f, and Cornford 1937, 181.
21
The position I have in mind here is that the Re
ceptacle acts as a kind of matter or stuff that is contained
by the geometrical shapes rather than the view that there
is some primitive matter in the Receptacle (and distinct
from it), as found, e.g., in Gill 1971; cf. McCabe 1994, 180.
I find this latter suggestion implausible, too, as well as
unmotivated by the text; for fairly definitive criticism of
Gill’s view, see Silverman 2002, 267‑73.
22
As an aside, holding that triangles compose
the universe hardly seems weirder than holding that the
strings of string theory, or any other posit of speculative
physics, compose the universe!
23
Gregory 2000, 203‑5, argues that the elemental
triangles undergo no intrinsic change—the only triangles
that can change are the triangular faces of the elements
air, fire, and water. It is the faces, then, that are “new or
“old,” not the elemental triangles.
24
E.g., Schaffer 2001, 247, although Schaffer
argues that tropes are better described as “here‑suches.”
Cf. Ehring 2012, 76‑91.
25
By universals, I mean that Forms are tran
scendent universals, i.e., non‑spatiotemporal entities that
explain the common features of particulars without being
“in” or located with those particulars. The traditional
relation of “participation” is replaced with a Form image
“being an image of” a Form, which we may interpret as
the relation between a trope and its transcendent uni
versal. The transcendent universal explains resemblance
among tropes, as each trope is an image (instance) of a
Form (universal). Plato does not clearly commit himself
to a range of Forms; here I assume a Form for every
meaningful predicate, but I examine this assumption
further in an unpublished manuscript.
26
See, e.g., Fine 1983 & 1986 for the view that
Forms are immanent in particulars.
27
Cf. Mohr 2005, 87: “We can tell that what
recurs is the same recurring image by referring it to the
original of which it is an image.”
28
Cf. Ferber 1997 about the justification of
Forms here. The point about the same Form being im
aged in each ‘this‑such’ is strengthened if we take ὅμοιον
in 49e4‑7 (the “similar fashion” in “this‑such coming
around always in similar fashion in each case and all
together”) as modifying the “coming around” with Miller
2003, 81, rather than as modifying τὸ τοιοῦτον (49e5). If
we take it with τὸ τοιοῦτον, we might be inclined toward
the immanent universal interpretation; the resulting
translation, following Zeyl 2000, would read: “‘what is
such,’ coming around like it was, again and again.” But
if we take it adverbially with περιφερόμενον, then ‘this
‑such’ comes around in similar fashion each time: there
need not be the same this‑such each time, as the imma
nent universalist would have it, but a this‑such need only
come to be ‘similarly’ each time the appropriate circum
stances arise. Even if we do take ὅμοιον with τὸ τοιοῦτον,
however, tropes are salvageable, since we have an account
of what it is for two tropes to be similar, namely that they
are images of the same Form.
29
Cf. Rep. 476b4‑6, where things are said, in
passing, to be made out of sounds and colors and figures
(τάς τε καλὰς φωνὰς ἀσπάζονται καὶ χρόας καὶ σχήματα
καὶ πάντα τὰ ἐκ τῶν τοιούτων δημιουργούμενα). While
I merely explain and defend a Platonic trope theory here,
in an unpublished manuscript I explore and defend a
Platonic trope bundle theory.
30
It is worthwhile to point out that, if the mem
bers of the class of Becoming are tropes, then the motiva
tion for taking the Receptacle to be a substratum has been
dealt a serious blow. Substances—particulars that are
supposed to be composed of tropes and a substratum—
feature nowhere in Plato’s tripartite ontology if not in
the category of Becoming. But they cannot be in this
category, since one of their components—tropes—makes
up this category, and another of their components—the
Receptacle, taken as a substratum—makes up a different
category. Thus substances would have to be a conglom
eration of the two categories, Form‑images and the
Receptacle, whereas, in the text, Form‑images are said to
be the product or ‘offspring’ of Forms and the Receptacle
(50d2‑4). There is plenty of room for particulars in Plato’s
ontology, but only if they are wholly composed of tropes,
as I argue in Buckels 2016a. The argument of this paper,
together with the argument of that paper, go most of the
way toward establishing Plato as a trope‑bundle theorist.
31
I would like to thank Thomas Chance, Cody
Gilmore, Peter Larsen, John Malcolm, Vasilis Politis,
Allan Silverman, and Jan Szaif for valuable discussion of
this paper and previous drafts, as well as audiences at the
Eastern and Central Division Meetings of the American
Philosophical Association, the London Ancient Science
Conference, the University of California Davis, and Trin
ity College Dublin.
|
|
https://openalex.org/W2997572800
|
http://www.journalijar.com/uploads/146_IJAR-29661.pdf
|
English
| null |
ASSESS THE KNOWLEDGE AND PERCEPTION ON HUMAN BREAST MILK BANK AMONG POSTNATAL MOTHERS
|
International journal of advanced research
| 2,019
|
cc-by
| 2,657
|
ASSESS THE KNOWLEDGE AND PERCEPTION ON HUMAN BREAST MILK BANK AMONG
POSTNATAL MOTHERS. 30
ASSESS THE KNOWLEDGE AND PERCEPTION ON HUMAN BREAST MILK BANK AMONG
POSTNATAL MOTHERS. Sharumathy V1, Dr.Vetriselvi.P2 and Dr.Adhisivam.B3. 1. B.SC[N],College of nursing,JIPMER,Puducherry. 2. Assistant professor,College of Nursing,JIPMER,Puducherry. 3. Additional Professor&HOD,Department of neonatalogy,JIPMER, Puducherry. …………………………………………………………………………………………………….... Manuscript Info
Abstract
……………………. ………………………………………………………………
Manuscript History
Received: 01 October 2019
Final Accepted: 03 November 2019
Published: December 2019
Key words:-
Human breastmilk bank, Knowledge,
Perception, Postnatal Mothers. Background:Human breast milk is considered as the best source of all
nutrients, beyond that it the only living food source for infants
especially
during
the
first
six
months
of
their
life. WHOrecommendstheexclusivebreastfeedingtill6monthsoflifeforallinfan
tsandthensupplemented breastfeedingisrecommended untilat least two
yearsofageanditisrecognized
asgloballyhighlymore
Nutritious. According to the joint statement given by the World Health
Organization and United Nations InternationalChildren’sEmergency
Fund(UNICEF)in1980. Thebestandessentialfoodforany baby whoseown
mother'smilkisnotavailable
isthebreastmilkfromanotherdonatinghealthymother. Hence this study
focuseson identifying andassessing theknowledgeand perceptionamong
postnatalmothers
aboutHumanBreastMilk
Bank
establishmentand
itsdonation.Itaimsondescribing
behaviors,beliefs,andfeelingsbehindHumanMilk Bank anditsdonation
by
identifying
donor’s
individual,socialandenvironmentalfeatures
thatcouldaffectthedonationofhumanmilk.Thefindings
willhelp
todevelophealtheducation
toolsto
promotehumanmilkdonation
by
identifying the factors that hinders the human milk donation and
rectifying it accordingly. Objectives:
1. Toassessknowledgeand
perception
on
Human
MilkBankamongpostnatal mothers. 2. Toidentifytheassociationofknowledgeand perception withselected
demographic variables. Methods:
The
design
of
the
study
is
cross
sectional. Theparticipantswho
met
the
inclusion
criteriawereselected
byconvenientsamplingtechnique. The total participants included in this
study were 100. the data was collected by using structured
questionnaire. Statistical methods like frequency and percentage, chi-
square test have been used and All statistical analysis has been carried
out at 5% level of significant and P<0.05 has considered as significant. Results:The
study
concludedthatonly
19%ofthepostnatalmothers
hadadequate knowledgeandinregardto
Corresponding Author:- Sharumathy V. Address:- B.SC[N],College of nursing,JIPMER,Puducherry. isthebreastmilkfromanotherdonatinghealthymother. Hence this study
focuseson identifying andassessing theknowledgeand perceptionamong
postnatalmothers
aboutHumanBreastMilk
Bank
establishmentand
itsdonation.Itaimsondescribing behaviors,beliefs,andfeelingsbehindHumanMilk Bank anditsdonation
by
identifying
donor’s
individual,socialandenvironmentalfeatures
thatcouldaffectthedonationofhumanmilk.Thefindings
willhelp behaviors,beliefs,andfeelingsbehindHumanMilk Bank anditsdonation
by
identifying
donor’s
individual,socialandenvironmentalfeatures
thatcouldaffectthedonationofhumanmilk.Thefindings
willhelp todevelophealtheducation
toolsto
promotehumanmilkdonation
by
identifying the factors that hinders the human milk donation and
rectifying it accordingly. y g
g y
Objectives: ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 7(12), 30-35 Journal Homepage: -www.journalijar.com
Article DOI:10.21474/IJAR01/10120
DOI URL: http://dx.doi.org/10.21474/IJAR01/10120 30
Corresponding Author:- Sharumathy V.
Address:- B.SC[N],College of nursing,JIPMER,Puducherry. Int. J. Adv. Res. 7(12), 30-35 Hence this study focuseson identifying andassessing theknowledgeand perceptionamong postnatalmothers
aboutHumanBreastMilk
Bank
establishmentand
itsdonation.Itaimsondescribing Hence this study focuseson identifying andassessing theknowledgeand perceptionamong postnatalmothers
aboutHumanBreastMilk
Bank
establishmentand
itsdonation.Itaimsondescribing
behaviors,beliefs,andfeelingsbehindHumanMilk
Bank
anditsdonation
by
identifying
donor’s
individual,socialandenvironmentalfeatures
thatcouldaffectthedonationofhumanmilk.Thefindings
willhelp behaviors,beliefs,andfeelingsbehindHumanMilk
Bank
anditsdonation
by
identify
ndividual,socialandenvironmentalfeatures
thatcouldaffectthedonationofhumanmilk.Thefinding odevelophealtheducation toolsto promotehumanmilkdonation by identifying the factors that hin
milk donation and rectifying it accordingly. y g
Objectives: 1. Toassessknowledgeand
perception
on
Human
MilkBankamongpostnatal mothers. 1. Toassessknowledgeand
perception
on
Human
MilkBankamongpostnatal mothers. MilkBankamongpostnatal mothers. 2. Toidentifytheassociationofknowledgeand perception withselected
demographic variables. g p
Methods:
The
design
of
the
study
is
cross
sectional. Theparticipantswho
met
the
inclusion
criteriawereselected
byconvenientsamplingtechnique. The total participants included in this
study were 100. the data was collected by using structured
questionnaire. Statistical methods like frequency and percentage, chi-
square test have been used and All statistical analysis has been carried
out at 5% level of significant and P<0.05 has considered as significant. Methods:
The
design
of
the
study
is
cross
sectional. Theparticipantswho
met
the
inclusion
criteriawereselected
byconvenientsamplingtechnique. The total participants included in this
study were 100. the data was collected by using structured
questionnaire. Statistical methods like frequency and percentage, chi-
square test have been used and All statistical analysis has been carried
out at 5% level of significant and P<0.05 has considered as significant. Results:The
study
concludedthatonly
19%ofthepostnatalmothers
hadadequate knowledgeandinregardto 3
Corresponding Author:- Sharumathy V. Address:- B.SC[N],College of nursing,JIPMER,Puducherry. Corresponding Author:- Sharumathy V. Address:- B.SC[N],College of nursing,JIPMER,Puducherry. 30 ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 7(12), 30-35 Int. J. Adv. Res. 7(12), 30-35 Copy Right, IJAR, 2019,. All rights reserved. WHOrecommendstheexclusivebreastfeedingtill6monthsoflifeforallinfantsandthensupplementedbreastfeedingisreco
mmended untilat least two yearsofageanditisrecognized asgloballyhighlymore Nutritious.( 2 ) It is vital because of its bio-active substances like free amino acids, nucleotides, cells, growth factors, enzymes,
hormones, pro-biotic bacteria, and prebiotic oligosaccharides that are essential for development of immature
immune system and gut micro biome of both full-term and high risk newborns like low birth weight and preterm. It
also decreases the incidences of necrotizing enterocolitis ,late onset sepsis, retinopathy, food intolerance or mal-
absorption, immunodeficiency and improvise the neuro-cognitive development. Meanwhile lower the risk of
childhood diabetes, obesity compared to those infants fed with formula milk and animal’s milk.( 1,3,4,10,12) Exclusivebreastfeedingisveryessential not only for infants but also for mothers as it prevents the risk of
breast cancer and enhances the bond between mothers and babies.(11) According to the joint statement given by the World Health Organization and United Nations
InternationalChildren’sEmergency
Fund(UNICEF)in1980. Thebestandessentialfoodforany
baby
whoseown
mother'smilkisnotavailable isthebreastmilkfromanotherdonatinghealthymother(5). BreastMilkDonation
isrecommendedandsuggestedforhospitalized
(sick)orpreterm
babiesinwhich
theirmothersdon’thavesufficientmilk production (or) underwent any breast surgery in past (or) under any
medications like chemotherapy (or) any otherconditionwheremothercan’tbeableto giveherbreastmilk. The
Human milk bank receives human milk from donor after screening, then collects, process,
pasteurize, store and then distributed to needy. ( 6 ,7 ,9 ,12) But unfortunately, most of the developing countries including our nation which has highest burden of global
neonatal mortality and morbidity rate lag in the implementation of donor breastmilk as best alternative choice for
vulnerable infants. Breast milk banks aren’t a new phenomenon in India, but they are scarce. Eventhough Asia’s
first milk bank was set up in Mumbai (Lokmanya Tilak Hospital) in 1989. Currently there are only 60 human milk
bank. Whereas Brazil, another developing country, is an international leader with 217 banks and 113 collection
centers which has helped to reduce brazil’s infant mortality rate by 73%.( 1,3,8,9) Hence this study focuseson identifying andassessing theknowledgeand perceptionamong postnatalmothers
aboutHumanBreastMilk
Bank
establishmentand
itsdonation.Itaimsondescribing
behaviors,beliefs,andfeelingsbehindHumanMilk
Bank
anditsdonation
by
identifying
donor’s
individual,socialandenvironmentalfeatures
thatcouldaffectthedonationofhumanmilk.Thefindings
willhelp
todevelophealtheducation toolsto promotehumanmilkdonation by identifying the factors that hinders the human
milk donation and rectifying it accordingly. Hence this study focuseson identifying andassessing theknowledgeand perceptionamong postnatalmothers
aboutHumanBreastMilk
Bank
establishmentand
itsdonation.Itaimsondescribing
behaviors,beliefs,andfeelingsbehindHumanMilk
Bank
anditsdonation
by
identifying
donor’s
individual,socialandenvironmentalfeatures
thatcouldaffectthedonationofhumanmilk.Thefindings
willhelp
todevelophealtheducation toolsto promotehumanmilkdonation by identifying the factors that hinders the human
milk donation and rectifying it accordingly. Toidentifytheassociationofknowledgeand perception withselected demographic variables. j
1. Toassessknowledgeand perception on Human MilkBankamongpostnatal mothers. TheQuestionnairehad 3sections.Theyare: Section – A: Socio-Demographic Data Section-B: Knowledge on human breast milk bank. Section-B: Knowledge on human breast milk bank. Section-C: Perception on human breast milk bank. Study procedure: Study procedure:
Thestudy wasconductedafterapproval fromIEC(Humanstudies), JIPMER. Theparticipantswho met the inclusion
criteriawereselected byconvenientsamplingtechnique. The total participants included in this study were 100. Study procedure:
Thestudy wasconductedafterapproval fromIEC(Humanstudies), JIPMER. Theparticipantswho
criteriawereselected byconvenientsamplingtechnique. The total participants included in this study Aftergettinginformedwrittenconsentfrom the postnatal mothers, a structuredquestionnaire was administered to them
to assess their knowledge and perception about human breast milk bank. In which it had 16 knowledge questions
[multiple choicequestions ]and 13 perception questions and instructed themto markthecorrectanswer. nknowledgequestionnairethecorrectanswerwasscoredas1andthewronganswer Inknowledgequestionnairethecorrectanswerwasscoredas1andthewronganswer
as0.Inperceptionquestionnaire,theresponseoptionsforeachquestionwasstrongly
stronglydisagree. Inknowledgequestionnairethecorrectanswerwasscoredas1andthewronganswer
as0.Inperceptionquestionnaire,theresponseoptionsforeachquestionwasstrongly
agree,agree,not
sure,disagreeand
stronglydisagree. Objectives:- j
1. Toassessknowledgeand perception on Human MilkBankamongpostnatal mothers. 1. Toassessknowledgeand perception on Human MilkBankamongpostnatal mothers. 2. Toidentifytheassociationofknowledgeand perception withselected demographic variables. 31 Int. J. Adv. Res. 7(12), 30-35 Int. J. Adv. Res. 7(12), 30-35 ISSN: 2320-5407 Sampling population:
Postnatal mothers who are admitted in Women and ChildrenHospitalJIPMER . Parameters studied: Parameters studied:
Independent variable:
1. Age
2. Education
3. Religion
4. Type of family
5. Monthly income of family
6. Domicile
Outcome variables:
Level ofKnowledge and perception on human milk bank. Confounding and interacting variables:
NIL
Statistical test used for data analysis:
Thedistributions of socio-demographic characteristics were
knowledgelevelwascategorizedintoinadequate,moderateandadequateba Independent variable: Outcome variables:
Level ofKnowledge and perception on human milk bank. Outcome variables:
Level ofKnowledge and perception on human milk bank. Confounding and interacting variables:
NIL Confounding and interacting variables:
NIL Tool description: p
structured questionnairewasused tocollectdatafrom postnatal mothers. Statistical test used for data analysis: Statistical test used for data analysis:
Thedistributions of socio-demographic characteristics were expressedasfrequency andpercentage. The
knowledgelevelwascategorizedintoinadequate,moderateandadequatebased
onthescoresanditwas
expressedin 32 Int. J. Adv. Res. 7(12), 30-35 ISSN: 2320-5407 frequency
andpercentage.Perception
levelwascategorizedinto
unfavorable,
averageand
favorablebasedonthescoresanditwasexpressedinfrequencyandpercentage.Theassociationoflevel
ofknowledge&perceptioncategorieswithdemographicvariableswascarriedoutbyusingchi-square test. All statistical
analysis has been carried out at 5% level of significant and P<0.05 has considered as significant. frequency
andpercentage.Perception
levelwascategorizedinto
un q
y
p
g
p
g
g
favorablebasedonthescoresanditwasexpressedinfrequencyandpercentage.Theassociationoflevel
ofknowledge&perceptioncategorieswithdemographicvariableswascarriedoutbyusingchi-square test. All statistical
analysis has been carried out at 5% level of significant and P<0.05 has considered as significant. Results:- Results:- Results:-
Table1:-Distribution of socio-demographicvariables of postnatal mothers N=100
Variable
Category
Frequency(N)
Percentage(%)
Agein years
20-30
90
90%
31-40
10
10%
Domicile
Rural
73
73%
Semi-urban
0
0%
Urban
27
27%
Education
Illiterate
23
23%
6th- 10
th
20
20%
11th-12
th
25
25%
Graduate
32
32%
Type of family
Nuclear
36
36%
Joint
64
64%
Extended
0
0%
Monthly
income
of family
≤2000
32
32%
2001-5000
19
19%
5001-10,000
21
21%
>10,000
28
28%
Table 2:-Knowledgelevel of postnatal mothers:N=100
Groups
Knowledge score
Frequency(NO)
Percentage(%)
Postnatal
mothers
Inadequate (0-50%)
Moderate (51-75%)
Adequate (>75%)
29
52
19
29%
52%
19%
Table 3:-Perception levelof postnatal mothers: N=100
Groups
Perceptionscore
Frequency(NO)
Percentage(%)
Postnatal
mothers
Unfavorable (0-50%)
Average (51-75%)
Favorable (75-100%)
1
29
70
1%
29%
70%
Table 4:-Association between knowledge and perception of postnatal mothers with socio demographic variables:
N=100
S
I. N
o
Variables
N
Knowledgescore
P
valu
e
Perceptionscore
P
val
ue
inadequ
ate
moder
ate
adequ
ate
unfavora
ble
avera
ge
Favora
ble
1
Agein years
20-30
31-40
9
0
1
0
26
3
45
7
19
0
0.24
6
1
0
27
2
62
8
0.7
48
2
Domicile
Rural
Semi -urban
Urban
7
3
0
2
23
0
6
36
0
16
14
0
5
0.06
17
1
0
0
19
0
10
53
0
17
0.4
83 n knowledge and perception of postnatal mothers with socio demographic variables: Table 4:-Association between knowledge and perception of postnatal mothers with socio demographic variables:
N=100
S
I. N
o
Variables
N
Knowledgescore
P
valu
e
Perceptionscore
P
val
ue
inadequ
ate
moder
ate
adequ
ate
unfavora
ble
avera
ge
Favora
ble
1
Agein years
20-30
31-40
9
0
1
0
26
3
45
7
19
0
0.24
6
1
0
27
2
62
8
0.7
48
2
Domicile
Rural
Semi -urban
Urban
7
3
0
2
23
0
6
36
0
16
14
0
5
0.06
17
1
0
0
19
0
10
53
0
17
0.4
83 33 Int. J. Adv. Res. Discussion:- Discussion:- Discussion:-
ThefirstobjectiveofthestudywastoassesstheknowledgeandperceptiononHumanMilk Bank among postnatal
mothers. Among
postnatalmothers
only
19%hadadequateknowledge,52%hadmoderateknowledgeand29%of
them
hadinadequateknowledgeandInregardtoperception70%ofthepostnatalmothershadfavorable
perception,
29%ofthemhad averageperceptionand 1%hadunfavorableperception. Theabovefindingsweresupported by thefollowingstudies:
GoodfellowHE,
Reimers
P,Ballard
K,Coutsoudis
A(2016)(13)conducted
astudy
insouthAfrica
to
determinethemothersperceptionofbreastfeedingandcommunity-basedhumanmilkbanks.Through
conveniencesamplingatotalof40motherswereinterviewed.Theresultsshowedthatonly58%of
womenwerewillingtodonatethebreastmilk,only30%werewillingtofeedtheirbabywithdonated
milkand
only57.5%consideredthe ideaofsettingupcommunitybased human milkbank. Leung
J,YauS(2015)(6)conductedastudy
inHong
Kongtoexploretheperceptionsonbreastmilk
donationandestablishmentofbreastmilkbankamongbreastfeedingmothers. Aqualitativedescriptive
studywasadopted.Throughpurposivesampling10participantswereselected. Thedatawerecollected
throughin-depth
face-to-face
andsemi-structuredinterviews. The
resultsshowedthatmostof
the
breastfeedingmotherswelcomedtheideaofbreastmilkdonationandtheestablishmentofbreastmilk bank. Alencar
L,seidlE(2009)(14)
conductedanexploratory,descriptiveandcross-sectionalstudyamong36
womenintheagegroupof
14to33yearswho
were
breastmilk
donorsinBrazil. Theobjectivewas
to
describethecharacteristicsofdonationbehaviorandtoidentifythereasons,beliefsandfeelingsrelative
tobreastmilk
donationpractice. Throughconvenientsampling
thesampleswereselectedanddatawere
collectedthroughinterviewmethod.Theresultsshowedthat91.7%ofthemothersthoughtthatbreast
milkdonationexperience
ispositive.Themostfrequently
reportedreasonsfordonatingbreastmilkwere
altruismand
excessmilkproduction. Discussion:-
ThefirstobjectiveofthestudywastoassesstheknowledgeandperceptiononHumanMilk Bank among postnatal
mothers. Among
postnatalmothers
only
19%hadadequateknowledge,52%hadmoderateknowledgeand29%of
them
hadinadequateknowledgeandInregardtoperception70%ofthepostnatalmothershadfavorable
perception,
29%ofthemhad averageperceptionand 1%hadunfavorableperception. Theabovefindingsweresupported by thefollowingstudies:
GoodfellowHE,
Reimers
P,Ballard
K,Coutsoudis
A(2016)(13)conducted
astudy
insouthAfrica
to
determinethemothersperceptionofbreastfeedingandcommunity-basedhumanmilkbanks.Through
conveniencesamplingatotalof40motherswereinterviewed.Theresultsshowedthatonly58%of
womenwerewillingtodonatethebreastmilk,only30%werewillingtofeedtheirbabywithdonated
milkand
only57.5%consideredthe ideaofsettingupcommunitybased human milkbank. Leung
J,YauS(2015)(6)conductedastudy
inHong
Kongtoexploretheperceptionsonbreastmilk
donationandestablishmentofbreastmilkbankamongbreastfeedingmothers. Aqualitativedescriptive
studywasadopted.Throughpurposivesampling10participantswereselected. Thedatawerecollected
throughin-depth
face-to-face
andsemi-structuredinterviews. The
resultsshowedthatmostof
the
breastfeedingmotherswelcomedtheideaofbreastmilkdonationandtheestablishmentofbreastmilk bank. Alencar
L,seidlE(2009)(14)
conductedanexploratory,descriptiveandcross-sectionalstudyamong36
womenintheagegroupof
14to33yearswho
were
breastmilk
donorsinBrazil. Theobjectivewas
to
describethecharacteristicsofdonationbehaviorandtoidentifythereasons,beliefsandfeelingsrelative
tobreastmilk
donationpractice. Throughconvenientsampling
thesampleswereselectedanddatawere
collectedthroughinterviewmethod.Theresultsshowedthat91.7%ofthemothersthoughtthatbreast
milkdonationexperience
ispositive.Themostfrequently
reportedreasonsfordonatingbreastmilkwere
altruismand
excessmilkproduction. ThefirstobjectiveofthestudywastoassesstheknowledgeandperceptiononHumanMilk Bank among postnatal
mothers Among
postnatalmothers
only
19%hadadequateknowledge,52%hadmoderateknowledgeand29%of
them
hadinadequateknowledgeandInregardtoperception70%ofthepostnatalmothershadfavorable
perception,
29%ofthemhad averageperceptionand 1%hadunfavorableperception. Results:- 7(12), 30-35 ISSN: 2320-5407 7
3
Education
Illiterate
6th-10th
11th-12th
Graduate
2
3
2
0
2
5
3
2
9
6
8
6
9
11
13
19
5
3
4
7
0.72
0
1
0
0
0
7
3
9
10
15
17
16
22
0.41
8
4
Typeoffamily
Nucl
ear
Joint
Exte
nded
3
6
6
4
0
9
20
0
20
32
0
7
12
0
0.79
7
1
0
0
6
23
0
29
41
0
0.06
0
5
Monthlyinco
me
offamily
≤2000
2001-5000
5000-10,000
>10,000
3
2
1
9
2
1
2
8
12
5
6
6
15
9
10
18
5
5
5
4
0.69
8
0
0
1
0
10
4
7
8
22
15
13
20
0.57
0 Theabovefindingsweresupported by thefollowingstudies: GoodfellowHE,
Reimers
P,Ballard
K,Coutsoudis
A(2016)(13)conducted
astudy
insouthAfrica
to
determinethemothersperceptionofbreastfeedingandcommunity-basedhumanmilkbanks.Through
conveniencesamplingatotalof40motherswereinterviewed.Theresultsshowedthatonly58%of
womenwerewillingtodonatethebreastmilk,only30%werewillingtofeedtheirbabywithdonated
milkand
only57.5%consideredthe ideaofsettingupcommunitybased human milkbank. Leung
J,YauS(2015)(6)conductedastudy
inHong
Kongtoexploretheperceptionsonbreastmilk
donationandestablishmentofbreastmilkbankamongbreastfeedingmothers. Aqualitativedescriptive
studywasadopted.Throughpurposivesampling10participantswereselected. Thedatawerecollected
throughin-depth
face-to-face
andsemi-structuredinterviews. The
resultsshowedthatmostof
the
breastfeedingmotherswelcomedtheideaofbreastmilkdonationandtheestablishmentofbreastmilk bank. Alencar
L,seidlE(2009)(14)
conductedanexploratory,descriptiveandcross-sectionalstudyamong36
womenintheagegroupof
14to33yearswho
were
breastmilk
donorsinBrazil. Theobjectivewas
to
describethecharacteristicsofdonationbehaviorandtoidentifythereasons,beliefsandfeelingsrelative
tobreastmilk
donationpractice. Throughconvenientsampling
thesampleswereselectedanddatawere
collectedthroughinterviewmethod.Theresultsshowedthat91.7%ofthemothersthoughtthatbreast
milkdonationexperience
ispositive.Themostfrequently
reportedreasonsfordonatingbreastmilkwere
altruismand
excessmilkproduction. Iloh K, Osuorah C, Ndu I, et.all. (2018)(1) conducted a study in south-east Nigeria to determine the perception and
acceptability of donor breast milk among mothers. It is a cross- sectional multi-center study in which the data was
collected using pretested questionnaire among 1235 selected mothers by using purposive and convenient sampling 34 ISSN: 2320-5407 Int. J. Adv. Res. 7(12), 30-35 method. Results showed that 39% (480/1225) of them heard about it, whereas only 10% had adequate knowledge
about the concept and policy of donor milk and 60% of them were willing to donate and use the donor breast milk. Summary: Summary:
The
study
concludedthatonly19%ofthepostnatal
mothershadadequate
knowledgeandinregardto
perception70%ofpostnatal
mothershadfavorable
perception. Therewasno
significantassociationofknowledgeandperception with any of thesocio- demographic variables. The above results stress the importance of organizing health education topostnatalmothers,broadcasting
abouthumanmilk
bank
inantenatalOPD, The above results stress the importance of organizing health education topostnatalmothers,broadcasting
abouthumanmilk
bank
inantenatalOPD, postnatalward&NICUwillenhancetheirknowledgeandincreasethenumberofdonors.Handouts
stressingtheimportanceofhumanmilkdonation should beprovidedto allpostnatalmother postnatalward&NICUwillenhancetheirknowledgeandincreasethenumberofdonors.Handouts
t
i
th i
t
fh
ilkd
ti
h
ld b
id dt
ll
t
t l
th postnatalward&NICUwillenhancetheirknowledgeandincreasethenumberofdonors.Handouts with g
p
Thecurrentstudy
findingsrevealedthattherewasnosignificantassociationofknowledgeandperception
with
anyof
theselecteddemographicvariablesamong postnatalmothers. Theabovefindingsweresupported by thefollowingstudies: Ghuge S, Aghamkar J, Salvi R (2018)(7) conducted a study in Maharastra to determine the knowledge and attitude
towards human milk bank among postnatal mothers. The research design was one group pretest posttest quasi
experimental design. Descriptive research approach has been carried out. By using non-probability convenience
sampling technique a total of 60 postnatal mothers were included in this study and the data has been collected
through structured knowledge and attitude questionnaires. The results concluded that there was no significant
association between the baseline variables with knowledge, attitude scores and the demographic variables. 14. YauSY,LeungJCY.PerceptionsofbreastfeedingMothersonBreastMilkDonationandEstablishmentofHuman
BreastMilkBankinHong Kong. IntJ NursPract .2015; 2:72-80. References:- 1. AlencarLC,SeidlEM.Breastmilkdonation:women’sdonorexperience.Rev.SaudePublica.2009 2. Arnold LDW.Humanmilkbanking in North America. JHumLact.1997;13:159–62. 3. Barin J, Lotscher M. Contextualizing Human Milk Banking and Milk Sharing Practices and Perception in
Switzerland. THE MILK GAP report. 2018 (August) 3. Barin J, Lotscher M. Contextualizing Human Milk Banking and Milk Sharing Practices and Perception in
Switzerland. THE MILK GAP report. 2018 (August) 4. DennisCL. Breastfeeding Initiation andDuration.J ObstetGynecol Neonatal Nurs.2001;31:12-32 5. Donor breast milk banks: the operation of donor milk bank services. NICE clinical guideline 93. 2018 (July). 6. Ghuge S, Aghamkar J, Salvi R. A study to assess the knowledge and attitude regarding donating milk to the
human milk bank among postnatal mothers of selected hospital. International Journal of Applied Research. 2018; 4(9): 94-98. 6. Ghuge S, Aghamkar J, Salvi R. A study to assess the knowledge and attitude regarding donating milk to the
human milk bank among postnatal mothers of selected hospital. International Journal of Applied Research. 2018; 4(9): 94-98. ( )
7. GoodfellowHE,ReimersP,BallardK,CoutsoudisA.Perceptionsofcommunity-basedhumanmilkbanks
beforeand
aftertraining ina resource-limited SouthAfrican setting. S Afr J ChildHealth.2016;10(1):83-86. 8. Iloh K, Osuorah C, Ndu I, Asinobi I, Anyim I,et al. Perception of donor breast milk and determinants of its
acceptability among mothers in a developing community: a cross-sectional multi-center study in south-east
Nigeria. International Breastfeeing Journal . 2018; 13:47 : 1-2. 8. Iloh K, Osuorah C, Ndu I, Asinobi I, Anyim I,et al. Perception of donor breast milk and determinants of its
acceptability among mothers in a developing community: a cross-sectional multi-center study in south-east
Nigeria. International Breastfeeing Journal . 2018; 13:47 : 1-2. g
g
9. Kim JH, Unger S. Human Milk Banking. Canadian Pediatric society Nutrition and Gastroenterology
Committee.Paediatr ChildHealth .2010;15:595-98. 9. Kim JH, Unger S. Human Milk Banking. Canadian Pediatric society Nutrition and Gastroenterology
Committee.Paediatr ChildHealth .2010;15:595-98. 10. Mackenzie C, Hons B, Javanparast S, Newman L,Hons B. Mother’s knowledge of and attitudes toward human
milk banking in south Australia: A qualitative study. Journal of Human Lactation. 2013; 29(2):222-29. 11. McGuireW,AnthonyMY.Donorhumanmilkversusinfantformulaforpreventingnecrotizingenter
inpreterminfants:systematicreview. ArchDisChild Fetal NeonatalEd.2003;88:11-14. 12. WilliamsAF,FisherC,GreasleyV,TraylerH,WoofrideMW.Humanmilkbanking.JTropPediatr.1985; 31:185
13. Wong’sTextbookofEssential pediatricNursing .7thed.Philadelphia:Elsevier publications;2005. y
y
g
p
13. Wong’sTextbookofEssential pediatricNursing .7thed.Philadelphia:Elsevier publications;2005. 14. YauSY,LeungJCY.PerceptionsofbreastfeedingMothersonBreastMilkDonationandEstablishmentofHuman
BreastMilkBankinHong Kong. IntJ NursPract .2015; 2:72-80. 35
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Spectral Domain Optical Coherence Tomography Assessment of Macular and Optic Nerve Alterations in Patients with Glaucoma and Correlation with Visual Field Index
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Hindawi
Journal of Ophthalmology
Volume 2018, Article ID 6581846, 9 pages
https://doi.org/10.1155/2018/6581846
Clinical Study
Spectral Domain Optical Coherence Tomography Assessment of
Macular and Optic Nerve Alterations in Patients with Glaucoma
and Correlation with Visual Field Index
Alessio Martucci ,1 Nicola Toschi ,2,3,4 Massimo Cesareo,1 Clarissa Giannini,1
Giulio Pocobelli,1 Francesco Garaci ,2 Raffaele Mancino,1 and Carlo Nucci 1
1
Ophthalmology Unit, Department of Experimental Medicine, University of Rome Tor Vergata, Rome, Italy
Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
3
Department of Radiology, Athinoula A. Martinos Center for Biomedical Imaging, Boston, MA, USA
4
Harvard Medical School, Boston, MA, USA
2
Correspondence should be addressed to Carlo Nucci; nucci@med.uniroma2.it
Received 4 July 2018; Accepted 3 September 2018; Published 8 October 2018
Academic Editor: Angelo Balestrazzi
Copyright © 2018 Alessio Martucci et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Introduction. To evaluate the sectorial thickness of single retinal layers and optic nerve using spectral domain optic coherence
tomography (SD-OCT) and highlight the parameters with the best diagnostic accuracy in distinguishing between normal and
glaucoma subjects at different stages of the disease. Material and Methods. For this cross-sectional study, 25 glaucomatous (49 eyes)
and 18 age-matched healthy subjects (35 eyes) underwent a complete ophthalmologic examination including visual field testing.
Sectorial thickness values of each retinal layer and of the optic nerve were measured using SD-OCT Glaucoma Module Premium
Edition (GMPE) software. Each parameter was compared between the groups, and the layers and sectors with the best area under the
receiver operating characteristic curve (AUC) were identified. Correlation of visual field index with the most relevant structural
parameters was also evaluated. Results and Discussion. All subjects were grouped according to stage as follows: Controls (CTRL);
Early Stage Group (EG) (Stage 1 + Stage 2); Advanced Stage Group (AG) (Stage 3 + Stage 4 + Stage 5). mGCL TI, mGCL TO, mIPL
TO, mean mGCL, cpRNFLt NS, and cpRNFLt TI showed the best results in terms of AUC according classification proposed by Swets
(0.9 < AUC < 1.0). These parameters also showed significantly different values among group when CTRL vs EG, CTRL vs AG, and EG
vs AG were compared. SD-OCT examination showed significant sectorial thickness differences in most of the macular layers when
glaucomatous patients at different stages of the disease were compared each other and to the controls.
1. Introduction
Primary open-angle glaucoma, a leading cause of blindness
in the world, is an optic neuropathy characterized by the
death of ganglion cells of the retina, which is associated
with the loss of axons that make up the optic nerve. These
ultrastructural alterations gradually progress becoming
clinically evident as an increased excavation of the optic
disc and the presence of specific visual field (VF) defects [1].
Diagnosing and monitoring disease progression is therefore essential for the management of patients with glaucoma. Given that a significant structural loss usually
precedes detectable function loss [2], technologies and
strategies able to quantify glaucomatous changes at an
early stage have the potential to impact prognosis and
hence influence quality of life [3]. In this context, spectral
domain-optical coherence tomography (SD-OCT) provides a tool for macular segmentation and thickness
evaluation of individual retinal layers as well as retinal
nerve fiber layer thickness (RNFLt) and Bruch’s membrane
opening (BMO)-minimum rim width (MRW) assessment.
The patented Anatomic Position System (APS) creates an
anatomic map of each patient’s eye using the center of the
fovea and the center of BMO as landmarks. In turn, this
allows accurate localization and hence highly sensitive
assessment of structural changes.
2
In this study, sectorial thickness values of each retinal
layer at macular level, circumpapillary RNFLt of the optic
nerve, and BMO-MRW were measured using SD-OCT
Glaucoma Module Premium Edition (GMPE) software
(Heidelberg Engineering, Germany) to assess the putative
thickness differences between controls and initial glaucoma,
controls and advanced glaucoma, and initial and advanced
glaucoma.
2. Methods
In this cross-sectional study, 49 eyes of 25 glaucomatous
patients and 35 age-matched healthy eyes of 18 subjects were
recruited from the Glaucoma Clinic and the General Outpatients clinic (respectively) at the University Hospital
“Policlinico Tor Vergata” (Rome, Italy). Patients and controls
were aged 61.86 ± 6.79 and 60.58 ± 9.22 years, respectively.
The study protocol was approved by the local institutional
review board and adhered to the tenets of the Declaration of
Helsinki. All subjects provided written informed consent.
All subjects underwent a complete ophthalmologic examination including the administration of a medical history
questionnaire focused on local and systemic treatments and
family history of glaucoma, determination of best-corrected
visual acuity with logarithmic Early Treatment Diabetic
Retinopathy Study visual acuity charts (Precision Vision, la
Salle USA), slit-lamp examination of the anterior segment,
intraocular pressure (IOP) evaluation using Goldmann
applanation tonometry, pachymetry using an ultrasound
pachymeter (Pachette DGH500; DGH Technology, Inc.,
Philadelphia, PA), gonioscopy, and 24-2 Swedish Interactive
Threshold Algorithm (SITA) standard visual field (VF)
testing. After pupillary dilation, slit-lamp fundus examination and SD-OCT were performed.
All participants met the following inclusion criteria: bestcorrected visual acuity >0.1 logMAR, refractive error < ±5
spherical diopters or < ±3 cylindrical diopters, transparent
ocular media, and open anterior chamber (Shaffer classification >20°).
The exclusion criteria comprehended previous or active
optic neuropathies, retinal vascular diseases, preproliferative
or proliferative diabetic retinopathy, macular degeneration,
hereditary retinal dystrophy, use of medication that could
affect VF, and previous or active neurological, cerebrovascular, or neurodegenerative diseases. Normal tension
glaucoma (NTG) patients were also excluded.
A glaucoma diagnosis was defined, following the European Glaucoma Society criteria [4], as the presence of an
elevated IOP (>21 mmHg), marked excavation of the optic
nerve head with thinning of the neural rim, notching, focal
or diffuse atrophy of neural rim, cup/disc ratio (CDr) in the
vertical meridian >0.6, CDr asymmetry between the eyes
>0.2, optic disc haemorrhages, denuded circumlinear vessels, and the presence of typical VF defects.
2.1. Visual Field Examination. VF examination was performed using Humphrey Swedish Interactive Threshold Algorithm (SITA) standard visual fields with 24-2 test point
Journal of Ophthalmology
pattern (Carl Zeiss Meditec Inc., Dublin, CA). As reported in
the literature [5], Standard Automated Perimetry (SAP) examinations were considered unreliable and discarded if fixation losses were >20%, false-positive errors >15%, and falsenegative errors were >33%. The minimal glaucomatous abnormality was defined as the presence of pattern deviation
probability plots with <5%, more than three of which contiguous and one of which <1%, corrected pattern standard
deviation or pattern standard deviation significant at p < 0.05,
or glaucoma hemifield test outside normal limits [5]. VFs were
confirmed in at least 3 subsequent VF examinations. For this
study, VFs were classified according to the glaucoma staging
system based on the visual field index (VFI) [5]. VFI was found
to be in excellent correlation with MD across the spectrum of
glaucomatous visual loss [5]. The VFI expresses the amount of
visual field loss as a percentage relative to the sensitivity of
a reference group of healthy observers. To reduce the potentially confounding effects of cataract, the VFI disregards reductions in sensitivity unless they are associated with a pattern
deviation probability outside normal limits. Locations at which
the pattern deviations are within the 95th percentile of healthy
observers are treated as normal and assigned a value of 100%.
In addition, locations in the center of the visual field are more
heavily weighted and therefore make a greater contribution to
the VFI than do those in the periphery. This classification has
been deemed easy to use, accurate, and its staging performance
has been reported to be either equal or superior to other
existing glaucoma staging systems [5].
All subjects were subsequently grouped according to
stage as follows: Controls (CTRL); Initial Stages Group
(IG) (Stage 1 + Stage 2); Advanced Stages Group (AG)
(Stage 3 + Stage 4 + Stage 5).
2.2. Optical Coherence Tomography Examination. After
pupil dilation, all subjects underwent SD-OCT examination
with GMPE software (Heidelberg Retinal Engineering,
Dossenheim, Germany).
During the initial Anatomic Positioning System (APS)
scan, the scanner performs automatic detection of landmarks and automatic alignment of scans relative to the
patient’s individual fovea-to-BMO center axis, hence improving accuracy and reproducibility measurements and
overcoming measurement errors due to head tilt and eye
rotation. Moreover, custom TruTrack technology actively
tracks the eye during imaging with simultaneous dual-beam
imaging minimizing motion artifacts. The GMPE, unlike the
previous software versions, offers multi-layer segmentation
for assessment of the isolated retinal layers providing
a thorough assessment of the macular region via single layer
thickness maps, APS, and BMO-MRW-based optic nerve
head (ONH) evaluation.
To obtain perifoveal volumetric retinal scans, both eyes
of all subjects were examined using the Spectralis OCT
posterior pole vertical-oriented scan lines (PPoleV scan)
protocols. PPoleV scan includes 19 single vertical axial scans
(30° × 15° OCT volume scan), aligned to the individual foveato-BMO center axis with 240 microns distance between
sections (Figure 1). Segmentation of the retinal layers in each
™
Journal of Ophthalmology
3
Figure 1: Sample Spectralis OCT posterior pole vertical-oriented scan lines (PPoleV scan) protocols.
vertical foveal scan was performed automatically using
GMPE software for Spectralis OCT. For each layer provided
by the new segmentation software (macular total retina
(RETINA), Retinal Nerve Fiber Layer (RNFL), Ganglion Cell
Layer (GCL), Inner Plexiform Layer (IPL), Inner Nuclear
Layer (INL), Outer Plexiform Layer (OPL), Outer Nuclear
Layer (ONL), Retinal Pigmented Epithelium (RPE), Inner
Retinal Layers (IRL), and Outer Retinal Layers (ORL)),
thickness measurements of all sectors, as defined by the Early
Treatment Diabetic Retinopathy Study scheme (temporal
inner, superior inner, nasal inner, inferior inner, temporal
outer, superior outer, nasal outer, and inferior outer), were
considered (Figure 2).
To perform optic nerve head (ONH) analysis, using BMO
as the anatomical border of the rim, within 24 scan lines, the
GMPE software automatically detects 48 BMO positions
along the ONH determining the BMO-based disc margin.
BMO-MRW is calculated from the BMO to the nearest point
on the internal limiting membrane (ILM). In this study, for
each scan, the following BMO-MRW measurements were
considered: global, temporal superior, nasal superior, nasal,
nasal inferior, temporal inferior, and temporal (Figure 3).
Circumpapillary RNFLt (cpRNFLt) analysis is performed acquiring three circle scans automatically centred on
the individual fovea-to-BMO center axis ensuring the accurate definition of each single sector independent of head
position. For each scan, the central circle has been analyzed
and the following cpRNFLt measurements were considered:
global, temporal superior, nasal superior, nasal, nasal inferior, temporal inferior, and temporal (Figure 4).
No manual corrections were necessary and only good
quality OCT scans, with an OCT score >25, were included in
the study.
2.3. Statistical Analysis. All data were initially entered into
an EXCEL database (Microsoft, Redmond, Washington,
United States). Statistical analysis was performed in Statistical Package for the Social Sciences (SPSS) version 23.
Descriptive statistics consisted of the mean ± SD for parameter with Gaussian distributions (after confirmation using
the Kolmogorov–Smirnov test), median and interquartile
range for variables with nonGaussian distributions.
Comparisons among groups were performed using
ANOVA/ANCOVA, for continuous Gaussianly distributed
variables, and Mann–Whitney U test, for non-Gaussianly
distributed variables. p < 0.05 was considered statistically
significant.
Figure 2: Sample of segmentation of retinal layers.
Age comparisons among groups were performed using
Friedman ANOVA. A chi-square test was used to test independence among categorical variables.
For all the parameters which showed a statistically significant difference comparing controls vs. initial stages
glaucoma, controls vs. advanced stages glaucoma, and initial
stages glaucoma vs. advanced stages glaucoma, the diagnostic
accuracy was evaluated by fitting a binary logistic regression
model and examining the area under the generated receiver
operating characteristics (ROC) curve (AUC).
For the interpretation of the AUC values, we referred to
the classification proposed by Swets and only retained parameters with high diagnostic accuracy [6]:
(a) AUC � 0.5, the test is not informative
(b) 0.5 < AUC < 0.7, the test is not accurate
(c) 0.7 < AUC < 0.9, the test is moderately accurate
4
Journal of Ophthalmology
Figure 3: Bruch’s membrane opening minimum rim width analysis.
Figure 4: Circumpapillary retinal nerve fiber layer thickness analysis.
(d) 0.9 < AUC < 1.0, the test is highly accurate
The correlation between functional, VFI, and structural
loss, layers, and sector thicknesses was determined using
Spearman’s r correlation coefficient. Spearman’s r correlation coefficient was classified accordingly [7]:
(a) 0.9 to 1.0 (−0.9 to −1.0): very high positive (negative)
correlation
(b) 0.7 to 0.9 (−0.7 to −0.9): high positive (negative)
correlation
(c) 0.5 to 0.7 (−0.5 to −0.7): moderate positive (negative)
correlation
(d) 0.3 to 0.5 (−0.3 to −0.5): low positive (negative)
correlation
(e) 0.0 to 0.3 (−0.0 to −0.3): negligible correlation
Journal of Ophthalmology
3. Results
A total of 84 eyes were included in the study. Control group
was constituted of 35 eyes (age 60.83 ± 1.53), while initial
and advanced stages glaucoma groups were constituted of
22 eyes (age 63.23 ± 1.34) and 27 eyes (age 60.52 ± 1.37),
respectively.
There were no statistically significant differences in terms
of age (p � 0.58) and gender (p � 0.087) among groups.
At macular level, the mean thicknesses of GCL superior,
inferior, temporal, and nasal in the inner (GCL mean inner)
and outer (GCL mean outer) sectors showed a highly accurate diagnostic ability in all the comparisons considered
(CTRL vs. IG, AUC � 0.9; CTRL vs. AG, AUC � 1.0; IG vs.
AG, AUC � 0.9). The same result was obtained when
macular GCL temporal inner and outer and IPL temporal
inner thicknesses were evaluated (CTRL vs. IG, AUC � 0.9;
CTRL vs. AG, AUC � 1.0; IG vs. AG, AUC � 0.9). The mean
of macular RNFL superior, inferior, temporal, and nasal
outer sectors thickness (RNFL mean outer) also showed
a highly accurate diagnostic ability in discriminating among
all the groups considered for the study (CTRL vs. IG,
AUC � 0.9; CTRL vs. AG, AUC � 1.0; IG vs. AG, AUC � 0.9).
Moreover, at macular level, both the mean GCL and mean
RNFL thickness values, given by the respective mean of
superior, inferior, temporal, and nasal inner and outer
sectors, resulted highly accurate in discriminating among
all the groups (CTRL vs. IG, AUC � 0.9; CTRL vs. AG,
AUC � 1.0; IG vs. AG, AUC � 0.9) (Table 1).
At ONH level, circumpapillary RNFL global and circumpapillary RNFL temporal superior sector thicknesses
resulted highly accurate in discriminating among all the
groups considered for the study (CTRL vs. IG, AUC � 0.9;
CTRL vs. AG, AUC � 1.0; IG vs. AG, AUC � 0.9) (Table 1).
Table 2 reports descriptive statistics of the parameters
that showed the highest diagnostic ability in all the following
comparison: CTRL vs. IG, CTRL vs. AG, and IG vs. AG.
The correlation between functional, VFI, and structural
loss thickness of the OCT parameters has been determined
using Spearman’s r correlation coefficient. Table 3 reports
the OCT parameters that showed high and very high correlation coefficient with VFI (see Supplementary Material
for the other parameters). Interestingly, all the parameters
that showed the highest AUC also showed a positive correlation with VFI. In particular, if we consider the parameters that showed the best AUC values, at macular level, GCL
mean inner (r � 0.81), GCL mean outer (r � 0.75), GCL
temporal inner (r � 0.79), GCL temporal outer (r � 0.72), IPL
temporal inner (r � 0.78), RNFL mean outer (r � 0.71), mean
GCL (r � 0.76), and mean RNFL (r � 0.73) were highly
positively correlated with VFI. On the contrary, circumpapillary RNFLt global (r � 0.45) and circumpapillary RNFLt
temporal superior (r � 0.52) showed a low positive correlation with VFI.
4. Discussion
OCT technology has been proposed more than 20 years ago
for noninvasive cross-sectional imaging in biological
5
systems [8], and the first generation of time domain OCT has
been superseded by the newest spectral domain instruments.
Given that SD-OCT provides an accurate reconstruction of
ONH and of the macular area, hence highlighting the presence of possible structural damage, it is increasingly becoming
part of common clinical practice. Moreover, thanks to the
faster scanning speed and the increased axial resolution, the
latest OCT generation offers high resolution images which are
less affected by eye movement artifacts and are more reproducible [9–11]. In this regard, local intrasession and intersession variability in OCT have been previously described
as very low and uniform across eyes and layers [12, 13].
Before Zeimer et al. [14] suggested the use of macular
imaging for glaucoma evaluation, the most important clinical
parameter was the circumpapillary RNFL thickness [15–20].
However, due to the presence of blood vessels and the high
variability of the ONH structure (even among healthy subjects), circumpapillary RNFLt measurements may not always
be completely reliable in glaucoma diagnosis [21]. Instead, the
macula is a relatively simple structure, constituted by multiple
layers whose structure is not influenced by the presence of
blood vessels. The shape of the macula, and more specifically
the RGC layer, is generally less variable among healthy individuals as compared to other ocular structures such as the
RNFL and ONH. Thus, in conjunction with the fact that it
contains 50% of the total retinal ganglion cells (RGCs) and
35% of RNFL, macula appears to be a promising area for
glaucoma evaluation [14, 21].
While previous studies with TD-OCT reported a good
diagnostic ability of macular thickness, sensitivity and
specificity were lower than those provided by circumpapillary RNFLt [22–28].
Using the fast-macular cube scan of a prototype software,
Martinez-de-la-Martinez-de-la-Casa et al. [29] observed that
with the macular segmentation software provided the Spectralis
OCT, macular RNFLt measurements performed better than
other algorithms in discriminating healthy subjects from
glaucoma suspects. A similar result was also found considering
RNFL + GCL + IPL thickness and total macular thickness [30].
Another study by Mathers et al. [31] also highlighted the
usefulness of macular thickness as measured by SD-OCT in
confirming the existence and extent of VF defects.
In a previous study using 3D-OCT 2000 (Topcon Corp.,
Tokyo, Japan), a thickness reduction of macular layers with
progression of glaucoma was reported. This study showed
a significantly lower ganglion cell-inner plexiform layer thickness
(GCIPLT), as well as lower macular ganglion cell complex
thickness (GCCT: a combination of macular RNFL thickness
and GCIPLT) in POAG patients, compared to normal controls,
progressing with the severity of glaucomatous damage [32].
In this study, we evaluated the ability of the parameters
assessed using the GMPE software at macular and optic
nerve level to discriminate among the following groups:
controls versus initial glaucoma, controls versus advanced
glaucoma, and initial versus advanced glaucoma. GMPE is
a specific software for glaucoma diagnosis that, unlike other
software, allows a layer by layer and sector by sector macular
assessment. Thanks to this software, we are able to obtain, at
macular level, macular total retina, retinal nerve fiber layer,
6
Journal of Ophthalmology
Table 1: Mann–Whitney and AUC of the parameters with the highest diagnostic ability in the CTRL vs. IG, CTRL vs. AG, and IG vs. AG
comparisons.
CTRL vs. IG
Parameter
Macula GCL mean
inner
Macula GCL mean
outer
Macula GCL
temporal inner
Macula GCL
temporal outer
Macula IPL
temporal inner
Macula RNFL
mean outer
Macula mean GCL
Macula mean
RNFL
cpRNFL global
cpRNFL temporal
superior
VFI
AUC
p value
0.9
4.29449E − 06
0.9
2.3797E − 06
0.9
6.40501E − 07
0.9
2.26545E− 07
0.9
4.81767E − 06
0.9
1.6614E − 05
0.9
9.86195E − 07
0.9
2.51857E − 06
0.9
3.19772E − 06
0.9
2.83685E − 06
0.9
2.14775E − 06
CTRL
Parameter
Macula GCL mean
inner
Macula GCL mean
outer
Macula GCL
temporal inner
Macula GCL
temporal outer
Macula IPL
temporal inner
Macula RNFL
mean outer
Macula mean GCL
Macula mean
RNFL
cpRNFL global
cpRNFL temporal
superior
VFI
vs. AG
AUC
p value
1.0
3.94592E − 10
1.0
3.11259E − 10
1.0
3.59673E − 10
1.0
1.6719E − 10
1.0
1.67747E − 10
1.0
4.46656E − 10
1.0
1.78465E − 10
1.0
3.14624E − 10
1.0
2.68425E − 10
1.0
2.70908E − 10
1.0
3.81295E − 11
IG vs. AG
Parameter
AUC
Macula GCL mean
0.9
inner
Macula GCL mean
0.9
outer
Macula GCL
0.9
temporal inner
Macula GCL
0.9
temporal outer
Macula IPL
0.9
temporal inner
Macula RNFL
0.9
mean outer
Macula mean GCL
0.9
Macula mean
0.9
RNFL
cpRNFL global
0.9
cpRNFL temporal
0.9
superior
VFI
1.0
p value
9.29422E − 06
4.87372E − 07
3.5386E − 06
4.12819E − 06
1.42929E − 05
3.41429E − 06
1.04181E − 06
9.886E − 07
7.41664E − 07
4.33798E − 06
6.5955E − 09
GCL: ganglion cell layer; IPL: inner plexiform layer; cpRNFL: circumpapillary retinal nerve fiber layer thickness; VFI: visual field index; CTRL: control group;
IG: Early Glaucoma Group; AG: Advanced Glaucoma Group; p value Mann–Whitney <0.05.
Table 2: Descriptive statistics of the parameters with the highest diagnostic ability in the CTRL vs. IG, CTRL vs. AG, and IG vs. AG
comparisons.
Parameter
CTRL
Interquartile
Median
range low
Macula GCL
52.00
mean inner
Macula GCL
35.50
mean outer
Macula GCL
51.00
temporal inner
Macula GCL
39.00
temporal outer
Macula IPL
43.00
temporal inner
Macula RNFL
34.75
mean outer
Macula mean
31.59
GCL
Macula mean
40.94
RNFL
cpRNFL global
90.00
cpRNFL temporal
113.00
superior
VFI
99.00
IG
AG
Interquartile
Interquartile Interquartile
Interquartile
Median
Median
range low
range low
range high
range high
Interquartile
range high
50.25
54.75
43.13
39.25
45.75
27.75
22.50
36.50
32.75
38.50
30.13
27.75
32.25
22.50
20.75
25.75
47.00
53.00
35.00
29.00
42.00
20.00
18.00
27.00
35.00
40.00
26.50
24.00
31.00
19.00
17.00
22.00
40.00
45.00
34.00
32.00
37.00
25.00
22.00
27.00
33.50
37.00
28.88
25.25
31.50
21.00
19.00
24.50
29.27
33.20
27.34
26.06
27.88
21.41
19.80
24.14
38.61
42.73
32.03
26.06
35.78
20.66
19.52
25.16
79.00
97.00
61.50
58.00
72.00
47.00
39.00
51.00
101.00
138.00
79.50
66.00
96.00
39.00
31.00
57.00
98.00
99.00
92.00
86.00
96.00
49.00
13.00
66.00
GCL: ganglion cell layer; IPL: inner plexiform layer; cpRNFL: circumpapillary retinal nerve fiber layer thickness; VFI: visual field index; CTRL: control group;
IG: Early Glaucoma Group; AG: Advanced Glaucoma Group.
ganglion cell layer, inner plexiform layer, inner nuclear layer,
outer plexiform layer, outer nuclear layer, retinal pigmented
epithelium, inner retinal layers, and outer retinal layer
thickness measurements in all sectors as defined by the Early
Treatment Diabetic Retinopathy Study scheme (temporal
inner, superior inner, nasal inner, inferior inner, temporal
outer, superior outer, nasal outer, and inferior outer). In
addition, for the purpose of the study, we calculated and
evaluated the mean thickness value for each layer.
ONH was also assessed using the GMPE software and
retinal nerve fiber layer thickness, and Bruch’s membrane
opening minimum rim width was obtained.
Journal of Ophthalmology
7
Table 3: Pearson correlation coefficient between VFI and OCT
parameters.
Parameter
Macula GCL mean inner
Macula GCL temporal inner
Macula GCL superior inner
Macula IPL temporal inner
Macula IPL mean inner
Macula GCL inferior inner
Mean GCL
Macula GCL mean outer
Macula RNFL nasal outer
Mean RNFL
Macula GCL temporal outer
Macula GCL nasal outer
Macula GCL nasal inner
Macula RNFL mean outer
Macula IPL inferior inner
Correlation with VFI (r)
0.81
0.79
0.78
0.78
0.77
0.76
0.76
0.75
0.75
0.73
0.72
0.71
0.71
0.71
0.70
GCL: ganglion cell layer; IPL: inner plexiform layer; VFI: visual field index;
CTRL: control group; IG: Early Glaucoma Group; AG: Advanced Glaucoma
Group; r: Pearson correlation coefficient.
Of all the parameters, the presence of statistically
significant differences among the groups was evaluated.
Subsequently the diagnostic ability of those parameters to
discriminate controls versus initial glaucoma, controls
versus advanced glaucoma, and initial versus advanced
glaucoma was tested using AUC. Subsequently, only the
parameters with good ability to discriminate among
groups in all of the three comparisons were considered. At
macular level, the parameters that best performed were
mean GCL, GCL mean inner and outer, GCL temporal
inner and outer, IPL temporal inner, mean RNFL, and
RNFL mean outer.
At ONH level, the parameters that best performed were
circumpapillary RNFL global and temporal superior. None
of the Bruch’s membrane opening parameters had very high
diagnostic accuracy in discriminating among all the groups.
Interestingly, the median of all the parameters discussed
above showed reducing thickness values trend with progression of the disease.
Although many efforts have been made in the development
of new technologies to diagnose and evaluate the glaucoma
progression, clinicians still base their decisions on standard
“white-on-white” automated VF testing, which remains the
best-studied way to assess disease progression. Nevertheless,
many confounding factors, such as media opacity, may affect
the results. Most importantly, distraction, or other factors
involving the patient’s participation make VF tests unreliable.
This leads to a high level of disagreement among clinicians
whether or not glaucoma is progressing in their patients.
The VFI is a new global metric that represents the entire VF
as a single percentage of normal. Based on an aggregate percentage of visual function with 100% being a perfect ageadjusted visual field, it assigns a number between 1% and
100%. Central VF points are more heavily weighted, and the
percentage of VF loss is calculated based on pattern or total
deviations depending on the depth of loss. Interestingly, the
progression rates calculated by VFI are much less affected by
cataract development and cataract surgery than the traditional
mean deviation index or the pattern standard deviation [33, 34].
Due to the clinical relevance of VFI, its possible correlation with the OCT parameters has been assessed in this
study. Interestingly, most of the OCT parameters showed
a correlation with VFI. In particular, macular parameters
such as GCL mean inner, GCL temporal inner, GCL superior
inner, IPL temporal inner, IPL mean inner, GCL inferior
inner, mean GCL, GCL mean outer, RNFL nasal outer, mean
RNFL, GCL temporal outer, GCL nasal outer, GCL nasal
inner, RNFL mean outer, and IPL inferior inner showed
from high to very high correlation with VFI. Thus, suggesting their possible usefulness in the objective evaluation
of the progression of the disease.
Interestingly, our data go beyond those of a previous
study that showed a good diagnostic ability of macular GCL
temporal inner thicknesses in discriminating controls vs.
initial glaucoma [35]. In fact, our results suggest that this
parameter had, not only a good ability in discriminating
controls from initial glaucoma, but also controls from advanced glaucoma and initial glaucoma from advanced glaucoma. Moreover, this parameter showed a very high positive
correlation with VFI, showing a strong structure-function
correlation, thus further supporting a possible usefulness of
OCT parameters in glaucoma diagnosis and follow-up.
There is much interest in the world of scientific research
regarding the use of OCT in the diagnosis and follow-up of
glaucoma, which is now considered a real neurodegenerative
disease [36, 37]. Many studies have focused on the analysis of
the diagnostic capacity of this tool in discriminating healthy
subjects from subjects with suspected glaucoma or at the initial
stage of the disease. Our work has been aimed at pushing
beyond searching parameters that would allow, not only an
early diagnosis of the disease but also a patient evaluation
during lifetime, allowing to discriminate among the patients at
the initial stage of the disease and those at an advanced stage.
There are several limitations to this prospective study. The
cohort included a small number of patients, and this may have
affected our analysis. Moreover, while we aimed to include
patients with a broad range of glaucoma severity, the group of
patients affected by advanced stages of disease was smaller
than all other groups, possibly affecting statistical power.
In conclusion, this study suggests a possible usefulness of
macular segmentation and ONH analysis with GMPE in the
evaluation of glaucoma patients. Our data suggest that OCT
may be a useful tool in detecting macular microstructural
changes related to the progression of glaucoma and that this
tracking is possible since the early stages of the disease. Our
initial results warrant further prospective longitudinal
studies on a larger cohort to confirm the ability of OCT
parameters, to track disease progression in glaucoma, and
eventually test new neuroprotective agents in the management of glaucoma [38, 39].
Data Availability
The data used to support the findings of this study are included within the text and the supplementary information
file.
8
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Supplementary Materials
In the supplementary materials, AUC and Mann–Whitney p
values for each comparison, complete descriptive statistics,
and correlation of each parameter with VFI are reported.
(Supplementary Materials)
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A background correction method to compensate illumination variation in hyperspectral imaging
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PLOS ONE RESEARCH ARTICLE A background correction method to
compensate illumination variation in
hyperspectral imaging Jonghee Yoon1,2*, Alexandru Grigoroiu1,2, Sarah E. BohndiekID1,2*
1 Department of Physics, University of Cambridge, Cambridge, England, United Kingdom, 2 Li Ka Shing Centre,
Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, England, United Kingdom * jonghee.yoon@cruk.cam.ac.uk (JY); seb53@cam.ac.uk (SEB) a1111111111
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a1111111111 Editor: Ireneusz Grulkowski, Nicolaus Copernicus
University, POLAND Editor: Ireneusz Grulkowski, Nicolaus Copernicus
University, POLAND Editor: Ireneusz Grulkowski, Nicolaus Copernicus
University, POLAND Received: October 9, 2019
Accepted: February 9, 2020
Published: March 13, 2020 Received: October 9, 2019
Accepted: February 9, 2020
Published: March 13, 2020 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.0229502 Abstract Hyperspectral imaging (HSI) can measure both spatial (morphological) and spectral (bio-
chemical) information from biological tissues. While HSI appears promising for biomedical
applications, interpretation of hyperspectral images can be challenging when data is
acquired in complex biological environments. Variations in surface topology or optical power
distribution at the sample, encountered for example during endoscopy, can lead to errors in
post-processing of the HSI data, compromising disease diagnostic capabilities. Here, we
propose a background correction method to compensate for such variations, which esti-
mates the optical properties of illumination at the target based on the normalised spectral
profile of the light source and the measured HSI intensity values at a fixed wavelength
where the absorption characteristics of the sample are relatively low (in this case, 800 nm). We demonstrate the feasibility of the proposed method by imaging blood samples, tissue-
mimicking phantoms, and ex vivo chicken tissue. Moreover, using synthetic HSI data com-
posed from experimentally measured spectra, we show the proposed method would
improve statistical analysis of HSI data. The proposed method could help the implementa-
tion of HSI techniques in practical clinical applications, where controlling the illumination pat-
tern and power is difficult. OPEN ACCESS Citation: Yoon J, Grigoroiu A, Bohndiek SE (2020)
A background correction method to compensate
illumination variation in hyperspectral imaging. PLoS ONE 15(3): e0229502. https://doi.org/
10.1371/journal.pone.0229502 Editor: Ireneusz Grulkowski, Nicolaus Copernicus
University, POLAND
Received: October 9, 2019
Accepted: February 9, 2020
Published: March 13, 2020 PLOS ONE PLOS ONE Introduction I, I0, and Idark are the intensities of the spectral signals mea-
sured from the sample, the background spectral signals recorded without the sample in place
(also referred to as the “white” signals) and the dark signals recorded without any illumination,
respectively. Reflectance and absorbance metrics thus indicate the true spectral features of a
sample as these calculations correct for variations in illumination conditions and errors intro-
duced by the optical components. These processed reflectance or absorbance signals can then
be further subjected to statistical analyses such as principal component analysis (PCA)[22,23],
spectral angle mapping (SAM)[24], or machine learning methods[5,25–27], to extract signifi-
cant spectral features that can discriminate or classify the samples of interest. ,where R(x,y,λ) and A(x,y,λ) are the reflectance and absorbance at a given spatial position (x, y)
and wavelength (λ), respectively. I, I0, and Idark are the intensities of the spectral signals mea-
sured from the sample, the background spectral signals recorded without the sample in place
(also referred to as the “white” signals) and the dark signals recorded without any illumination,
respectively. Reflectance and absorbance metrics thus indicate the true spectral features of a
sample as these calculations correct for variations in illumination conditions and errors intro-
duced by the optical components. These processed reflectance or absorbance signals can then
be further subjected to statistical analyses such as principal component analysis (PCA)[22,23],
spectral angle mapping (SAM)[24], or machine learning methods[5,25–27], to extract signifi-
cant spectral features that can discriminate or classify the samples of interest. Competing interests: The authors have declared
that no competing interests exist. A key limitation of these reflectance and absorbance calculations is the assumption of uni-
form sample illumination. Several methods are used in hardware to ensure that this assump-
tion remains valid, including: uniform illumination instrumentation[28,29]; 3D shape
measurement[30]; reference intrinsic / fluorescence imaging[31–34]; and ratiometric mea-
surements[35–37]. Uniform illumination instrumentation can be achieved with specialised
devices such as ring illuminators or diffuse domes, however, there are difficulties in applying
these for in vivo imaging, such as during endoscopy, due to the need for bulky illuminating
units. In addition, uniform illumination instrumentation does not guarantee intensity homo-
geneity along the axial direction, which means illumination issues can still occur ex vivo when
measuring most biological tissues due to their uneven surfaces. Introduction Hyperspectral imaging, originating from remote sensing applications[1], enables a combined
simultaneous measurement of both spatial and spectral information from biological tissues. Analysis of the resulting 3D data set, or ‘hypercube’, enables spatial discrimination of healthy
and abnormal tissues based on the rich morphological and biochemical information contained
within the spatial and spectral features[2,3]. HSI has shown potential in a range of biomedical
applications, from label-free tumour diagnoses[4–6] and detection of tumour margins during
surgical operations[7–9], to quantification of blood oxygenation levels[10–12], and multi-col-
our fluorescent imaging[12,13]. HSI methods have thus been developed for the fast and accu-
rate analysis of biological samples ex vivo[14–17] as well as for diagnostic and intraoperative
applications in vivo[16,18]. Copyright: © 2020 Yoon 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: Data will be uploaded
into the University of Cambridge Open data
repository Apollo, more information can be found
at: www.data.cam.ac.uk. The DOI for this data set
is https://doi.org/10.17863/CAM.42338 1 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 PLOS ONE Background correction in hyperspectral imaging Funding: This work was funded by CRUK (C47594/
A16267, C14303/A17197, C9545/A29580,
C47594/A21102; www.cancerresearchuk.org),
EPSRC (EP/R003599/1, EP/N014588/1, EP/
L015889/1; www.epsrc.ac.uk) and the EU FP7
agreement FP7-PEOPLE-2013-CIG-630729 (ec. europa.eu). These grants were awarded to SEB and
support the work of JY and AG, with the exception
of EP/L015889/1, which is a Centre for Doctoral
Training award that supports AG. The funders had
no role in study design, data collection and
analysis, decision to publish, or preparation of the
manuscript. The high complexity of handling the 3D hypercube requires careful consideration of appro-
priate analysis methods[3,19–21]. HSI data are commonly subjected to a normalisation proce-
dure to calculate reflectance and/or absorbance of the sample using the following Eq[3]: R x; y; l
ð
Þ ¼ Iðx; y; lÞ Idark
I0ðx; y; lÞ Idark
ð1Þ ð1Þ A x; y; l
ð
Þ ¼ log10
Iðx; y; lÞ Idark
I0ðx; y; lÞ Idark
ð2Þ ð2Þ ,where R(x,y,λ) and A(x,y,λ) are the reflectance and absorbance at a given spatial position (x, y)
and wavelength (λ), respectively. PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 Image acquisition Spectral image acquisition was performed after allowing 15 minutes for temperature stabilisa-
tion of the equipment. The image acquisition process consisted of three steps: (1) dark imag-
ing; (2) white reflectance imaging; and (3) sample measurement. Dark imaging was performed
under closed camera shutter conditions. White reflectance imaging was performed using a
standard white reflectance target (Spectralon diffuse reflectance target, Labsphere) to obtain
information of the spectral profile and intensity of the light source. All image acquisition pro-
cesses were performed under the same experimental conditions, including exposure time, gain
and light source power. Hyperspectral imaging system The hyperspectral imaging endoscopy system, reported previously[18], exploits a flexible CE-
marked endoscope (Polyscope, PolyDiagnostics) and a line-scanning (pushbroom) method. Briefly, the endoscope consists of a reusable imaging fibre bundle with 10,000 individual fibre-
lets and a disposable sterile catheter that contains an imaging channel, an illumination fibre
and an accessory channel. The proximal end of the imaging fibre bundle was imaged and mag-
nified using an infinity corrected objective lens (40×, 0.6NA, Nikon) and a tube lens (L1, f = 75
mm), with the image being measured by an electron multiplying CCD camera (sCam, ProEM
512, Princeton Instruments) combined with a spectrograph (IsoPlane 160, Princeton Instru-
ment) to obtain hyperspectral information. The spectrograph consisted of a mechanical
entrance slit of manually adjustable width (10 μm– 3 mm) and a grating (150 lines/mm with
500 nm blaze, Princeton Instruments); thus a spectral image with a spectral bandwidth of 250
nm can be measured in a single image acquisition. The spectrograph and camera were con-
trolled by LightField software v6.7 (Princeton Instrument). In order to obtain a wide-area
hyperspectral image, the line-scanning was performed using a motorized translational stage
(MTS50/M-Z8, Thorlabs). All equipment was synchronously controlled in Labview 2017
(National Instruments) environment. A broadband light source (OSL2, Thorlabs) with a Halogen light bulb (OSL2bIR, Thorlabs)
whose emission spectrum spanned across the visible to NIR (1050 nm) region was used to illu-
minate a sample either internally or externally, depending on experimental purposes. For
internal illumination, the light source was directly coupled to the illumination fibre of the
endoscope by using a collimating lens (L2, f = 150 mm) and an objective lens (60×, NA 0.9,
Olympus). For external illumination, the light source was coupled to a large core fibre and the
distal end of the fibre was placed 2 cm away from the sample at a tilted angle. Olympus). For external illumination, the light source was coupled to a large core fibre and the
distal end of the fibre was placed 2 cm away from the sample at a tilted angle. Introduction Estimating optical illumination
power is possible using 3D shape measurement techniques and optical model-based analysis
but predicting illumination conditions within shadowed regions is challenging. Intrinsic
image or reference fluorescence signals measured by multimodal imaging systems have been
used to provide a reference background (BG) that enables estimation of the optical power dis-
tribution of the light source and correction of sample signals but again, a complex optical sys-
tem is required to measure reference BGs and additional errors are introduced by variations in
tissue absorption or the concentration of fluorescence agents. Ratiometric measurements,
such as narrow-band imaging, record spectral information from only a few spectral bands, dis-
playing physiological information based on a (weighted) sum of the images. Although ratio-
metric imaging is usually insensitive to illumination conditions and sample morphology, only
limited spectral information is recorded. Here, we introduce a BG correction method that estimates the optical power of illumination
at a sample by exploiting the normalised spectral profile of the light source and the hyperspec-
tral signal of the sample. We experimentally demonstrate the proof-of-concept of the method
using HSI data acquired via a hyperspectral endoscopy system from blood samples, tissue-
mimicking phantoms, and ex vivo chicken tissue. Moreover, the importance and applicability
of the proposed method to hyperspectral image analysis (PCA and SAM) and machine learn-
ing classification of hyperspectral data were tested using synthetic reflection and absorption
hypercubes based on these experimentally measured spectra. The proposed BG correction PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 2 / 21 PLOS ONE Background correction in hyperspectral imaging method, referred to as retrieved background (RB), enables the estimation of optical character-
istics of illumination at the sample, avoiding the need for additional complex hardware, and
results in accurate hyperspectral data analysis and classification. Hypercube reconstruction The recorded 2D line-scan image contains one spatial coordinate and the spectral coordinate,
because the grating inside the spectrograph disperses the image horizontally; hypercube recon-
struction is required to retrieve the other spatial coordinate, obtained during the motorized
translation. Before commencing hypercube reconstruction, the dark image was subtracted
from the white reflectance and sample images. A single column of the corrected image, which
contains information from a single wavelength, was selected and duplicated horizontally to 3 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 PLOS ONE Background correction in hyperspectral imaging match its image size to the physical slit width. For example, in hyperspectral imaging of the
chicken tissue, line-scan hyperspectral images of the sample and white-reflectance target were
measured with a step scanning size of 250 μm, which corresponds to 5 pixels. Thus, each pro-
cessed image was placed 5 pixels apart from the previous image. By repeating this process for
all column images, a slice of the hypercube at a single wavelength was created. The 3D hyper-
cube was then reconstructed by repeating the process to create a wide-area spatial image at all
wavelengths. Hypercubes of the sample and white-reflectance target were reconstructed sepa-
rately, enabling the calculation of normalised reflectance and absorbance values by dividing
the sample and white-reflectance hypercubes. Preparation of chicken tissue A food-grade chicken drumstick purchased from a local grocery market was horizontally
dissected using a knife. Local handling of the tissue was approved by our Biological Safety
Committee. The test sample was then placed on a petri dish and measured by using the hyper-
spectral endoscope. To obtain background signals, a white-diffuse-reflectance target was mea-
sured under the same experimental conditions as the sample measurement. The hyperspectral
imaging was performed at a working distance of 7 cm with a step size of 250 μm on the motor-
ized stage. A total of 150 spectral images were measured, resulting in a total scanning area of
31.56 mm × 37.50 mm, with an exposure time of 1s. The experiments were conducted within a
3 hour timeframe to ensure sample freshness. Generation of a synthetic RGB image from the hypercube For visualization purposes, the hypercube can be converted to a synthetic RGB (colour) image
using an artificially generated RGB filter based on a previously published method[18]. The
spectrum of the RGB filter employed Rayleigh probability density functions (raylpdf function
in Matlab R2018b), with centre wavelengths of each colour being set to 442, 518, and 579 nm,
respectively. Amplitudes of each filter were determined such that saturation of the synthetic
RGB image was avoided. The hyperspectral signal from the hypercube was multiplied by the
artificially generated RGB filters, with the R, G and B values of the synthetic RGB image being
determined by calculating the area-under-curve values of the filtered signals. Synthesized RGB
images were displayed using imshow function in Matlab R2018b. Preparation of tissue-mimicking phantom and blood samples For blood oxygenation measurements, fresh heparinized mouse blood was collected from
deceased mice provided by the Biological Resources Unit of the Cancer Research UK Cam-
bridge Institute (mice were not sacrificed for the purpose of this study). 1 mL of mouse blood
was divided between two 1 mL Eppendorf tubes. To make a fully oxygenated blood sample,
1 μL of 30% hydrogen peroxide (Sigma-Aldrich) was added and the sample was gently mixed
by inversion. 1.5 mg of sodium hydrosulphite (Sigma-Aldrich) was added to the other tube to
make a completely deoxygenated blood sample, again mixing by inversion. The tubes were
kept at room temperature for 10 mins and 20 μL of the oxygenated and deoxygenated blood
samples were transferred to a petri dish and covered by a cover slip. As a reference target,
20 μL of distilled water was put on the petri dish and covered by the cover slip. To test the effects of scattering, absorption and fluorescence on the suggested method, tis-
sue mimicking phantoms with defined optical properties that closely mimic biological tissue
were fabricated using agar, intralipid, nigrosin and methylene blue[38]. All chemicals were
purchased from Sigma-Aldrich. Before fabricating the tissue phantoms, two different concen-
trations of absorbance and fluorescence dyes were prepared. Nigrosin (0.1 and 0.05 g/mL) and PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 4 / 21 PLOS ONE Background correction in hyperspectral imaging methylene blue (0.1 and 0.05%) were prepared by diluting dyes using distilled water. 0.75 g of
agarose was dissolved in 48.5 mL of distilled water and then heated to the boiling point using a
microwave oven. The solution was left to cool to ~40˚C, with 1 mL of 20% intralipid being
added to the solution and gently mixed to induce optical scattering. 500 μL of the solution was
transferred to 6 wells of an 8 well dish (μ-Slide 8 Well, ibidi GmbH) using a pipette and then
100 μL of the four prepared dyes were added to 4 of the cells. The dish was covered by plastic
wrap and kept inside a refrigerator to set. Creation of synthetic absorption and reflection hypercubes In order to test machine-learning methods, synthetic hypercubes that mimic experimental
conditions were used, composed of spectral signals from experimentally measured BG and
samples (pork muscle tissue, oxygenated blood, methylene blue and nigrosin dyes). All sam-
ples were measured four times under different experimental conditions to include noise gener-
ated by the optical systems and environment to synthetic hypercubes. To generate
uncorrelated training and test hypercubes, three of the four measured data sets were used for
training data and the other data set was used for test data. Synthetic reflection and absorption hypercubes were created by following four steps: (1)
generation of a random illumination pattern; (2) creation of a GT reflectance hypercube based
on four experimentally measured signals with an uncorrelated noise; (3) creation of SB and RB
reflectance hypercubes by combining the GT hypercube with the random illumination pattern;
and (4) applying a log-transformation of the produced reflectance hypercubes to generate
absorbance hypercubes Step (1): 2D random Gaussian distributions, M, were used as ground-truth optical power dis-
tributions, with values were ranging between 0 and 1. Gaussian distribution was created
using ‘mvnpdf’ function in Matlab, and its central location was randomly assigned using
‘rand’ function in Matlab. Optical characteristics of the illumination conditions were
decided by the following equation: BGðx; y; lÞ ¼ Mðx; yÞ SlightðlÞ where BG(x,y,λ) is light intensity at the wavelength of λ at the point x,y in the image, M(x,y) is
the optical power at the point x,y, and Slight(λ) is the experimentally measured spectral intensity
of the light source at the wavelength of λ, respectively. Step (2): The GT reflectance hypercube was created by assigning experimentally measured
hyperspectral signals of samples (pork muscle tissue, oxygenated blood, methylene blue,
and nigrosin dye) with an uncorrelated noise obtained from independent experimental
measurements of spectral signals from a colour chart (ColorChecker Classic Mini, x-rite) to
each of the corresponding clusters in the spatial regions of the image (either target or back-
ground regions). Step (2): The GT reflectance hypercube was created by assigning experimentally measured
hyperspectral signals of samples (pork muscle tissue, oxygenated blood, methylene blue,
and nigrosin dye) with an uncorrelated noise obtained from independent experimental
measurements of spectral signals from a colour chart (ColorChecker Classic Mini, x-rite) to
each of the corresponding clusters in the spatial regions of the image (either target or back-
ground regions). PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 Specular angle mapping For SAM analysis, the average hyperspectral signal of cluster i of each hypercube was used as a
reference hyperspectral signal. Then, the spectral angles, α, between the hyperspectral signal of
each pixel of a hypercube and the reference spectral signal were calculated using the following
equation[24]: a ¼ cos 1ð
Pn
l¼1 tlrl
ðPn
l¼1 tl
2Þ
0:5ðPn
l¼1 rl
2Þ
0:5Þ
ð3Þ ð3Þ , where tλ and rλ are values of the target and reference spectral profiles at wavelength λ, respec-
tively and n indicates the total number of spectral channels. Creation of synthetic absorption and reflection hypercubes GTðx; y; lÞ ¼ SsampleðlÞ þ a NðlÞ where Ssample(λ) is the experimentally measured spectral intensity among the samples at the
wavelength of λ, α is randomly generated weighting factor between 0 to 0.1 (‘rand’ function in
Matlab), and N(λ) is an experimentally measured spectral intensity of the colour chart at the
wavelength of λ, respectively. The noise, α×N(λ) has a scale less than 10% of sample signals, PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 5 / 21 PLOS ONE Background correction in hyperspectral imaging Ssample(λ), and the range of the weighting factor, α, was determined to make the noise scale
consistent with the scale of average experimental noise. The uncorrelated noise makes the
training process more robust and reduces generalization error. Step (3): SB and RB reflectance hypercubes were created based on the GT hypercube from Step
(2) and single and retrieved illumination conditions by following equations, respectively: SB x; y; l
ð
Þ ¼ BGðx; y; lÞ GTðx; y; lÞ
SlightðlÞ
RB x; y; l
ð
Þ ¼ BGðx; y; lÞ GTðx; y; lÞ
RMðx; y; lÞ where RM(x,y,λ) is retrieved optical power at the wavelength of λ at the point x,y obtained via
the BG retrieval method. Step (4): Absorbance hypercubes were calculated by performing logarithmic transformation of
the GT, SB and RB reflectance hypercubes. Principal component analysis A pixel-wise approach and singular value decomposition (SVD) were exploited to perform
PCA of the hypercube[39]. Three pre-processing steps were required before calculating the
SVD of the hypercube. First, the 3D hypercube was vectorised into a 2D matrix, consisting of
pixels (vertical axis) and hyperspectral signals (horizontal axis). Then, hyperspectral data was
centred by subtracting mean values of the hyperspectral signal of each pixel from its corre-
sponding signal. Finally, the covariance matrix of the pre-processed hyperspectral data was cal-
culated, which was used as an input of SVD. SVD was performed using svd function in Matlab
R2018b. An NVIDA GeForce GTX 1080 graphical processing unit was exploited for fast SVD
calculation. Software Matlab R2017b and Python were used for image processing. Lightfield v6.7 (Princeton Instru-
ment) was used to control the spectrograph and EMCCD. Labview 2017 (National Instru-
ments) was used for synchronized control of the wide-field camera, spectrograph and
EMCCD, and motorized stage. Code availability All custom data analysis code will be made available online at: https://doi.org/10.17863/CAM. 42338 Machine-learning based classification of emulated hypercube Learning algorithms were implemented in Python, with K-means clustering and SVM algo-
rithms being implemented via the sklearn library and CNNs being implemented via Lasagne, a
Theano supplementary library. Learning was performed on a machine with access to 16 GB
RAM and a NVDIA GeForce GTX 1050Ti graphical processing unit. To validate the lack of
overfitting of the classifiers, the synthetic data was split into training and testing datasets, with PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 6 / 21 PLOS ONE Background correction in hyperspectral imaging base spectral measurements for the simulations being independent of one-another. All results
presented are based on the performance of the classifiers on the test data. A pixel-wise approach was used for learning and classification processes. A 3D hypercube,
consisting of 256 × 512 spatial points and 300 spectral channels, was converted to a 2D image
with sizes of 131072 × 300. Each row of the converted image with 300 spectral channels was
then used as an input to the learning and classification processes. We found that twenty-five
training datasets were sufficient to achieve 100% classification accuracy in the ground-truth
data. For a better supervised learning process, fifty hypercubes were exploited to train super-
vised learning models (SVMs and CNNs). Due to the large data sizes and memory limitations,
learning was performed incrementally in batches of one hypercube (131072 × 300). Thus, for
the K-mean algorithm, the MiniBatchKMeans function was employed, with a 21 epochs early
stopping decision and a target of 4 clusters. An incremental SVM algorithm has been imple-
mented by employing the SGDClassifier function with a hinge loss function and l2 regulari-
sers. For the CNN a six layered network was implemented with three convolutional layers, two
fully connected layers and an output layer. Unlike in the previous two methods (K-means and
SVM algorithms), a subset of a hypercube (200 × 300) was used as a batch size of CNN to facili-
tate a more effective learning process. 5-fold cross-validation was performed to test the accu-
racy of CNNs. PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 Background correction using the normalised source profile and target
hyperspectral signals The influence of varying illumination power on the calculation of reflectance and absorbance
spectra along with the proposed correction method is demonstrated in Fig 1. Experimentally
measured hyperspectral signals were acquired from absorbing nigrosin black dye as the sample
(Fig 1A) and a standard diffuse reflectance target as the background (Fig 1B, ω = 1). To emu-
late varying illumination intensities, two weighting factors were multiplied with the ground-
truth BG signal (Fig 1B, ω = 0.8, 1.2) and the resulting reflectance (Fig 1C) and absorbance
(Fig 1D) spectra were calculated according to the following equations (see Methods for com-
plete definition of all variables): R l
ð Þ ¼
IðlÞ Idark
o ðI0ðlÞ IdarkÞ
ð4Þ ð4Þ A l
ð Þ ¼ log10
IðlÞ Idark
o ðI0ðlÞ IdarkÞ
¼ log10
IðlÞ Idark
ðI0ðlÞ IdarkÞ
þ log10w
ð5Þ ð5Þ Varying the intensity in this way resulted in the expected change in the scale of the calculated PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 7 / 21 PLOS ONE Background correction in hyperspectral imaging Fig 1. The effect of illumination power on absorbance and reflectance spectra. Raw spectra of 0.05 g/mL nigrosin
dye (a) and the background halogen light source (b, ω = 1) were measured. To simulate the effects of the low and high
illumination power, weighting factors (ω = 0.8 and 1.2) were multiplied with the background spectrum. (c, d)
Reflectance and absorbance were obtained for the three different weighting factors. Illumination power can be
observed to change the scaling of reflectance spectra and the offset of absorbance spectra. (e, f) The proposed
background retrieval method estimates the optical spectral power of the illumination at the sample by exploiting a
normalised spectral profile of the light source (e) and the intensity ratio between the normalised spectral profile of the
light source and sample data (Cs/Cb) at a wavelength displaying low absorbance in the sample of interest, here selected
as 800 nm (f). Fig 1. The effect of illumination power on absorbance and reflectance spectra. Raw spectra of 0.05 g/mL nigrosin Fig 1. The effect of illumination power on absorbance and reflectance spectra. Raw spectra of 0.05 g/mL nigrosin
dye (a) and the background halogen light source (b, ω = 1) were measured. Proof-of-concept using a standard reflectance target and phantoms In order to test the proposed method, hyperspectral imaging data were acquired via a hyper-
spectral endoscope (HySE) that consists of a line-scanning spectrograph and multi-core optical
fibre endoscope (S1 Fig, see Methods)[18]. The endoscopy system can image the sample using
light from an external fibre-coupled light source (referred to as ‘external illumination’) or
using light delivered through an internal illumination fibre (referred to as ‘internal illumina-
tion’). We introduce external illumination here to provide light with an easily adjustable dis-
tance and angle relative to the sample. During clinical endoscopy, internal illumination is
used, and the changing working distance and angle of endoscope lead to additional heteroge-
neities in sample illumination. Data were first acquired from a standard white reflectance target that reflects 99% of illumi-
nating light using external illumination (Fig 2A), where a fibre coupled to a broadband light
source was tilted to create a variation in the optical power distribution across the sample. Line-
scanning HSI was performed at three different positions (indicated in Fig 2A with coloured
rectangles) and the resulting line-scan HySE image containing 1D spatial (vertical axis) and
spectral (horizontal axis) information (Fig 2B) was then processed at each position to retrieve
the average spectral profiles (Fig 2C). Min-max normalisation led to complete overlap of the
spectra (Fig 2D), indicating that the light source illuminates each position with the same spec-
tral profile but with different optical powers. Applying the proposed BG correction method,
ratios of the intensity values of three hyperspectral signals (Fig 2C; 2C1, 2C2 and 2C3), and the
normalised signal (Fig 2D and 2Cb) at 800 nm were taken (c1/cb, c2/cb, and c3/cb) and multi-
plied by the normalised spectrum (Fig 2D) to successfully retrieve the original signal (Fig 2E). Repeating the same process via internal illumination (Fig 2F) also showed appropriate back-
ground retrieval (Fig 2G–2J). To compare our results to other BG correction methods when imaging a range of samples,
we then defined three different BG conditions: ground-truth BG (GT); single BG (SB); and our
retrieved BG method (RB). GT was obtained by measuring HSI data from the standard white
reflectance target under precisely the same conditions as the sample imaging (S2A Fig). For
example, GT data was acquired at every working distance used. Background correction using the normalised source profile and target
hyperspectral signals To simulate the effects of the low and high
illumination power, weighting factors (ω = 0.8 and 1.2) were multiplied with the background spectrum. (c, d)
Reflectance and absorbance were obtained for the three different weighting factors. Illumination power can be
observed to change the scaling of reflectance spectra and the offset of absorbance spectra. (e, f) The proposed
background retrieval method estimates the optical spectral power of the illumination at the sample by exploiting a
normalised spectral profile of the light source (e) and the intensity ratio between the normalised spectral profile of the
light source and sample data (Cs/Cb) at a wavelength displaying low absorbance in the sample of interest, here selected
as 800 nm (f). https://doi.org/10.1371/journal.pone.0229502.g001 reflectance, since ω is a multiplicative factor in Eq (4), and a change in the offset of the calcu-
lated absorbance, because ω becomes an additive constant in Eq (5) due to logarithm calcula-
tions. This simple illustration highlights how image processing with incorrect BG data would
cause errors in the interpretation of hyperspectral data. We propose instead to multiply the
normalised spectrum of the light source (Fig 1E) with the intensity ratio between the normal-
ised spectral profiles of the light source (Cb) and the sample (Cs) at a wavelength of low absor-
bance in the sample to estimate the actual spectrum of the light source at the target (Fig 1F). Here, we select 800 nm as the wavelength for comparison, since this is central in the near-
infrared (NIR) tissue ‘optical window’ of low absorbance in biological tissues[40,41]. More-
over, there is an isosbestic absorption of haemoglobin at 800 nm meaning that any change in
absorption due to haemoglobin oxygenation status would not affect the calculation and the
loss of information at the normalisation is minimal since there are several other isosbestic
points for haemoglobin within the visible spectral region. PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 8 / 21 PLOS ONE Background correction in hyperspectral imaging The proposed method calculates reflectance and/or absorbance of the sample using the fol-
lowing equations: R x; y; l
ð
Þ ¼ Iðx; y; lÞ Idark
Csðx;yÞ
Cbðx;yÞ NSðlÞ
ð6Þ ð6Þ A x; y; l
ð
Þ ¼ log10
Iðx; y; lÞ Idark
Csðx;yÞ
Cbðx;yÞ NSðlÞ
! Background correction using the normalised source profile and target
hyperspectral signals ð7Þ ð7Þ , where NS(λ) is the normalised spectrum of the light source, Cs(x,y) and Cb(x,y) are intensity
values of I(800) at the point x,y, and NS(800), respectively. PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 Proof-of-concept using a standard reflectance target and phantoms (b) Representative spectral image from the
hyperspectral endoscope during external illumination. (c) Average spectral signals of the three hyperspectral images measured at the different locations shown in (a)
obtained from the white-dashed region in (b). (d) Min-max normalisation of spectral signals in (c) show complete overlap. (e) Spectra obtained (black dashed lines)
using the normalised spectral profile in (d) and ratio of values at 800 nm (c1, c2, c3, and cb). (f-j) As above but for the internal illumination method. https://doi.org/10.1371/journal.pone.0229502.g002 illumination power and working distance. In the present study, an arbitrary choice of a single
GT HSI data sets was taken as SB. RB is calculated as described above (S2B and S2C Fig). We then analysed blood samples, since blood has distinct absorption profiles depending on
the level of oxygenation and provides relatively low absorbance at 800 nm[42]. Fully oxygen-
ated and deoxygenated blood (see Methods) and distilled water (20 μL) were pipetted onto a
plate, covered by a coverslip (Fig 3A) and imaged immediately. HySE was applied using inter-
nal illumination at 3 working distances (Fig 3B) and the spectral profile of distilled water was
used as GT. The measured absorption spectra of the deoxygenated (Fig 3C) and oxygenated
blood (Fig 3D) clearly show one (550 nm) and two (540 and 560 nm) peaks respectively, con-
sistent with known blood absorption spectra[42]. The slightly different absorption values of
three measurements might be originating from the varying imaging areas, which are depen-
dent on the working distance of the endoscope. The absorbance spectra calculated using the
retrieved BG are consistent with the GT results, albeit slightly lower in magnitude
(8.73 ± 1.56% lower at 550 nm peak and 6.84 ± 1.22% lower at 560 nm peak of the absorption
spectra of the deoxygenated and oxygenated blood, respectively), however, the conventional
single BG method produces substantial differences. (8.73 ± 1.56% lower at 550 nm peak and 6.84 ± 1.22% lower at 560 nm peak of the absorption
spectra of the deoxygenated and oxygenated blood, respectively), however, the conventional
single BG method produces substantial differences. As the proposed method only uses a single normalisation wavelength, the accuracy of the
method may be affected by noise. The influence of noise levels to the retrieved signals was
assessed via simulation (S3 Fig). Proof-of-concept using a standard reflectance target and phantoms This is rarely feasible in practi-
cal clinical applications, such as during endoscopy, as a reference target cannot be introduced
into the lumen being imaged nor are working distance variations normally accounted for. SB
is the conventional background correction method commonly used in HSI and obtained by
measuring HSI data from the standard white reflectance target prior to sample measurements
under arbitrary illumination conditions and assuming this spectral profile to be representative
of the illumination conditions during the sample imaging[3]. SB does not allow compensation
of any variations that are introduced during the imaging condition, such as variations PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 9 / 21 PLOS ONE Background correction in hyperspectral imaging Fig 2. Spectral profiles of different illumination conditions can be accurately retrieved through hyperspectral endoscopy by different illumination methods. (a)
Schematic of the external illumination methods. Red, green, and blue lines indicate the hyperspectral imaging regions. (b) Representative spectral image from the
hyperspectral endoscope during external illumination. (c) Average spectral signals of the three hyperspectral images measured at the different locations shown in (a)
obtained from the white-dashed region in (b). (d) Min-max normalisation of spectral signals in (c) show complete overlap. (e) Spectra obtained (black dashed lines)
using the normalised spectral profile in (d) and ratio of values at 800 nm (c1, c2, c3, and cb). (f-j) As above but for the internal illumination method. htt
//d i
/10 1371/j
l
0229502 002 Fig 2. Spectral profiles of different illumination conditions can be accurately retrieved through hyperspectral endoscopy by different illumination methods. (a)
Schematic of the external illumination methods. Red, green, and blue lines indicate the hyperspectral imaging regions. (b) Representative spectral image from the
hyperspectral endoscope during external illumination. (c) Average spectral signals of the three hyperspectral images measured at the different locations shown in (a)
obtained from the white-dashed region in (b). (d) Min-max normalisation of spectral signals in (c) show complete overlap. (e) Spectra obtained (black dashed lines)
using the normalised spectral profile in (d) and ratio of values at 800 nm (c1, c2, c3, and cb). (f-j) As above but for the internal illumination method. Fig 2. Spectral profiles of different illumination conditions can be accurately retrieved through hyperspectral endoscopy by different illumination methods. (a)
Schematic of the external illumination methods. Red, green, and blue lines indicate the hyperspectral imaging regions. PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 Proof-of-concept using a standard reflectance target and phantoms Four different noise levels (1%, 5%, 10%, and 20%) were
added to the spectral profile of oxygenated blood (S3A Fig) and absorbance was calculated
based on simulated spectral signals with different noise levels and the proposed method (S3B
Fig). S3C Fig shows that the error levels are 3.62 ± 0.31%, 4.35 ± 1.64, 4.98 ± 2.26%, and
7.81 ± 2.90% with increasing a noise level from 1% to 20%, respectively. This indicates that
high noise levels could compromise the accuracy of the proposed method and care should be
taken when applying the approach to noisy spectra. To demonstrate that the BG correction method remains accurate under scattering, absorp-
tion and fluorescence conditions, a tissue-mimicking phantom was exploited. The phantom
was made of agarose and intralipid with high and low concentrations of nigrosin and 10 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 PLOS ONE Background correction in hyperspectral imaging Fig 3. Retrieved background (RB) signals enable accurate measurements of absorbance of deoxygenated and oxygenated blood compared to ground truth (GT). (a) Photograph of the experiment setup. Water (control) and blood (deoxygenation and oxygenation) were covered by a cover glass to prevent the sample from drying
during the measurement. Hyperspectral imaging was performed using the internal illumination method at three working distances. (b) Experimentally measured
reflectance signals of a control target (water) were used GT, with different optical illumination power according to working distance. Absorbance of deoxygenated (c)
and oxygenated (d) blood measured at three working distances were then calculated using GT, SB and RB methods, showing good agreement between GT and RB, but
substantial deviation for SB method. https://doi org/10 1371/journal pone 0229502 g003 Fig 3. Retrieved background (RB) signals enable accurate measurements of absorbance of deoxygenated and oxygenated blood compared to ground truth (GT). (a) Photograph of the experiment setup. Water (control) and blood (deoxygenation and oxygenation) were covered by a cover glass to prevent the sample from drying
during the measurement. Hyperspectral imaging was performed using the internal illumination method at three working distances. (b) Experimentally measured
reflectance signals of a control target (water) were used GT, with different optical illumination power according to working distance. Absorbance of deoxygenated (c)
and oxygenated (d) blood measured at three working distances were then calculated using GT, SB and RB methods, showing good agreement between GT and RB, but
substantial deviation for SB method. Proof-of-concept using a standard reflectance target and phantoms https://doi.org/10.1371/journal.pone.0229502.g003 methylene blue dyes added to test the effects of absorption and fluorescence, respectively (Fig
4A, see Methods). HySE was applied using internal illumination at 2 working distances; the
spectral profile of agarose containing intralipid alone was used as GT (Fig 4B). Absorbance
spectra of nigrosin and methylene blue calculated using the GT, SB and RB methods (Fig 4C
and 4D) again show that GT and RB provide consistent spectral shapes, whereas SB has a sub-
stantial deviation in the profiles. The absorbance of nigrosin obtained using the retrieved BG is
slightly lower in magnitude compared to GT, however, methylene blue is indistinguishable
(0.83 ± 0.67% at peak 550 nm). This suggests that the underestimation observed in the blood
and phantom experiments occurs because both haemoglobin and nigrosin have a small but
finite absorption of light at 800 nm causing a slight inaccuracy in the BG estimation, whereas
methylene blue has truly negligible absorption around 800 nm. Our RB method therefore
leads to a slight underestimation of the actual absorbance values if light absorption around the
chosen background wavelength is not negligible, though it does not change the absorbance
spectrum itself. Application of the background correction method to biological tissue and
endoscopic imaging conditions To examine the practical application of the method, dissected chicken bone tissue, consisting
of compact bone and bone marrow, was first measured (see Methods). A total of 150 spectral
images of dissected chicken bone tissue were measured using the external illumination method
to cast shadows across the topology of the sample, which can be seen in the synthetic RGB
images (Fig 5A), created by the convolution of emulated RGB filters and measured hyperspec-
tral signals (see Methods). Before examining the proposed method, the raw spectral signals in PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 11 / 21 PLOS ONE Background correction in hyperspectral imaging Fig 4. Background correction using the retrieved background method performs favourably in measurement of the absorbance spectra of nigrosin and methylene blue
(a) Photograph of tissue-mimicking phantoms with intralipid (control), nigrosin (absorbing dye), and methylene blue (fluorescent dye). (b) Experimentally measured
reflectance signals of the control phantom (intralipid only) at two different working distances gave the ground-truth background. (c) Absorbance of low and high
concentrations of nigrosin dye calculated using the ground truth background (GT), single background (SB) and our retrieved background (RB) method respectively. (d)
Absorbance of low and high concentrations of methylene blue calculated using GT, SB and RB methods respectively. Fig 4. Background correction using the retrieved background method performs favourably in measurement of the absorbance spectra of nigrosin and methylene blue
(a) Photograph of tissue-mimicking phantoms with intralipid (control), nigrosin (absorbing dye), and methylene blue (fluorescent dye). (b) Experimentally measured
reflectance signals of the control phantom (intralipid only) at two different working distances gave the ground-truth background. (c) Absorbance of low and high
concentrations of nigrosin dye calculated using the ground truth background (GT), single background (SB) and our retrieved background (RB) method respectively. (d)
Absorbance of low and high concentrations of methylene blue calculated using GT, SB and RB methods respectively. https://doi.org/10.1371/journal.pone.0229502.g004 two square areas (4 by 4 pixels) within each of bone marrow, compact bone, and shadowed
regions were assessed (S4A and S4B Fig). The reflected signal at the normalisation wavelength
under the same illumination conditions should be similar in order to use the proposed
method. The two nearby small squares in each tissue type were selected because illumination
conditions in these small areas could be considered as homogenous. S4C Fig shows that raw
reflected intensities at 800 nm of each tissue type are similar. PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 Application of the background correction method to biological tissue and
endoscopic imaging conditions HSI data from a chicken tissue sample obtained using the RB method agrees well with the GT (a) Synthetic RGB images of the sample were created from
hypercubes obtained by exploiting ground truth, single, and retrieved backgrounds. (b) Representative slice images from each hypercube (GT, SB and RB) were
illustrated at three wavelengths (456 1 531 4 and 612 9 nm) Solid and dashed arrows indicate anatomically similar structures in the sample (c) Average absorbance of Fig 5. HSI data from a chicken tissue sample obtained using the RB method agrees well with the GT (a) Synthetic RGB images of the sample were created from
hypercubes obtained by exploiting ground truth, single, and retrieved backgrounds. (b) Representative slice images from each hypercube (GT, SB and RB) were
illustrated at three wavelengths (456.1, 531.4 and 612.9 nm). Solid and dashed arrows indicate anatomically similar structures in the sample. (c) Average absorbance of
the hypercube reconstructed by using the GT, SB and RB methods within solid and dashed squares shown in (a) were obtained. Gray shaded area indicates the standard
deviation. Scale bars: 1 cm. https://doi.org/10.1371/journal.pone.0229502.g005 horizontally into the tube with a motorised stage, which leads to a gradual decrease in the
working distance of the endoscope. While the SB spectra show an offset as a function of work-
ing distance (S5C Fig), the RB spectra show the consistent measurement of absorbance regard-
less of the working distance (S5D Fig). horizontally into the tube with a motorised stage, which leads to a gradual decrease in the
working distance of the endoscope. While the SB spectra show an offset as a function of work-
ing distance (S5C Fig), the RB spectra show the consistent measurement of absorbance regard-
less of the working distance (S5D Fig). Application of the background correction method to biological tissue and
endoscopic imaging conditions There is no significant difference
in the data recorded from the same tissue type. The synthetic RGB images of GT and RB methods clearly show the structure of the tissue
with uniform brightness, but the SB image shows bright and dark regions arising due to the
uneven illumination (Fig 5A). Moreover, the shadowed region resulting from the sample mor-
phology was restored to its original white colour only in the RB method. Representative absor-
bance images at three different wavelengths (456.1, 531.4 and 612.9 nm; Fig 5B) allow
structures of the dissected chicken bone tissue to be visualised, showing qualitative similarity
between GT and RB at all wavelengths, while the single BG reconstructions show different
absorbance even in the same anatomical structures (solid and dashed white lines in Fig 5B). In order to investigate spectral fidelity of the BG retrieval method, the average and standard
deviation of the absorbance spectra in 6 different regions (red: bone marrow, orange: compact
bone, and blue: white reflectance; indicated in Fig 5A) were quantified (Fig 5C). Spectral pro-
files of bone marrow and compact bone, in GT and RB show similar values and trends. In
addition, our RB method brings the absorbance values on the left and right side of the white
reflectance target closer compared to the result obtained by GT. The SB result, however, shows
very different values and trends compared to the results obtained by the ground-truth and
retrieved BG. HySE was then applied in a tubular tissue-mimicking phantom with homogeneous methy-
lene blue concentration (S5A Fig) placed on a tilted surface (S5B Fig). HySE was advanced 12 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 PLOS ONE Background correction in hyperspectral imaging Fig 5. HSI data from a chicken tissue sample obtained using the RB method agrees well with the GT (a) Synthetic RGB images of the sample were created from
hypercubes obtained by exploiting ground truth, single, and retrieved backgrounds. (b) Representative slice images from each hypercube (GT, SB and RB) were
illustrated at three wavelengths (456.1, 531.4 and 612.9 nm). Solid and dashed arrows indicate anatomically similar structures in the sample. (c) Average absorbance of
the hypercube reconstructed by using the GT, SB and RB methods within solid and dashed squares shown in (a) were obtained. Gray shaded area indicates the standard
deviation. Scale bars: 1 cm. Fig 5. Investigating the influence of background correction on hyperspectral data
classification To understand the extent to which incorrect background compensation influences HSI data
classification, experimentally measured data obtained in the previous sections were composed
into a set of 53 synthetic hypercubes in four steps: (1) generation of a random illumination pat-
tern; (2) creation of a GT reflectance hypercube based on four experimentally measured signals PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 13 / 21 PLOS ONE Background correction in hyperspectral imaging Fig 6. Synthetic absorbance and reflectance hypercubes created based on experimentally measured hyperspectral signals and randomly generated illumination
conditions. (a) Representative image of Gaussian illumination power. (b) Representative projection images of synthetic GT, SB, and RB hypercubes. (i)-(iv) indicate the
areas with spectral profiles of corresponding signals shown in (c, d). Three circles in the same horizontal position had the same spectral profile and were defined as a
cluster. (c, d) Four experimentally measured absorbance and reflectance spectra (muscle tissue, oxygenated blood, methylene blue, and nigrosin samples) were exploited
to create synthetic absorbance and reflectance hypercubes. Scale bar: 100 pixels. Fig 6. Synthetic absorbance and reflectance hypercubes created based on experimentally measured hyperspectral signals and randomly generated illumination Fig 6. Synthetic absorbance and reflectance hypercubes created based on experimentally measured hyperspectral signals and randomly generated illumination
conditions. (a) Representative image of Gaussian illumination power. (b) Representative projection images of synthetic GT, SB, and RB hypercubes. (i)-(iv) indicate the
areas with spectral profiles of corresponding signals shown in (c, d). Three circles in the same horizontal position had the same spectral profile and were defined as a
cluster. (c, d) Four experimentally measured absorbance and reflectance spectra (muscle tissue, oxygenated blood, methylene blue, and nigrosin samples) were exploited
to create synthetic absorbance and reflectance hypercubes. Scale bar: 100 pixels. https://doi.org/10.1371/journal.pone.0229502.g006 https://doi.org/10.1371/journal.pone.0229502.g006 (muscle tissue, oxygenated blood, methylene blue and nigrosine dyes) with an uncorrelated
noise; (3) creation of SB and RB reflectance hypercubes by combining the GT hypercube with
the random illumination pattern; and (4) applying a log-transformation of the produced
reflectance hypercubes to generate absorbance hypercubes (Fig 6; see Methods). The synthetic hypercubes were then subjected to PCA, SAM and machine learning classifi-
cation. PCA is commonly used in HSI analysis for dimensionality reduction by finding a small
number of orthonormal PCs that explain most of the variance of hyperspectral data, thus
enabling simpler interpretation and classification. PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 Investigating the influence of background correction on hyperspectral data
classification PCA was performed pixel-wise with singu-
lar value decomposition (SVD): hyperspectral data were centred by subtracting the mean val-
ues of each pixel from its corresponding signal, while the scaling (variance) was preserved due
to the synthetic hypercubes being created under the same scale and unit conditions; the covari-
ance matrix of the centred data was used as the SVD input. As the first and second PCs capture
over 99% of the original variance, they were used to compare the influence of background cor-
rection methods. Scatter plots of PC2 against PC1 for GT, SB and RB in absorbance show no
differences and 2D image of the scores on PC1 are also identical (S6A Fig). For reflectance,
however, the scatter plots for SB show a dramatic elongation compared to the GT and RB
methods and the 2D image of the SB PC1 scores clearly shows the power distribution of PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 14 / 21 PLOS ONE Background correction in hyperspectral imaging illumination (S6B Fig), indicating an incomplete correction of the BG. Such behaviour arises
because the incorrect BG causes scaling and shifting of the ground-truth reflectance and absor-
bance signals, respectively. Scaling changes the variance of the hyperspectral data, which pro-
duces an incorrect PCA result for the reflectance hypercube, whereas shifting of absorption
data does not change PCA results as the variance is preserved. SAM is widely used to evaluate the similarity between measured hyperspectral signals by
calculating angles between them. Substantial differences in SAM analysis of the absorbance
hypercube using the SB method were found compared to GT and RB and the SAM image
again shows the power distribution of the illumination indicating an incomplete correction of
the BG (S6C Fig). The reflectance data are identical regardless of the BG correction used (S6D
Fig). This is because the scaling factor of the reflectance signal is eliminated through the calcu-
lation of the spectral angle in Eq (3) so it does not affect the SAM results, but shifting the
absorption signal changes the calculated spectral angle values. Investigating the influence of background correction on hyperspectral data
classification Finally, the effect of BG correction on machine learning-based data classification was evalu-
ated through: classification based on the distance between the data and the centroid of each
cluster by k-means clustering (K-Means, k = 4); maximising the distance between a decision
boundary and members of different classes by support vector machines (SVMs); and training
convolutional neural networks (CNNs). To enhance the learning process, min-max normalisa-
tion was employed with all three algorithms, to constrain the data between -1 and 1. For SVMs
and CNNs, the supervised learning approach was employed with ground-truth data of 50
training hypercubes produced for each of the three BG correction methods, whereas K-Means
was performed in an unsupervised learning manner without using data reduction methods
such as PCA or SAM. The test dataset was composed of three GT, SB, and RB hypercubes and the accuracy of all
established classifiers was tested on all datasets (9 total comparisons). 100% classification accu-
racy is theoretically achievable due to the use of synthetic hypercubes, consisting of only four
distinct spectral signals, for training and test. Using k-means clustering, the SB method showed
accuracies of only 47.1% and 48.7%, respectively for the absorbance and reflectance hyper-
cubes (Fig 7A and 7B) when clustered using the SB method classifier, compared to over 97.0% Fig 7. Investigating the effect of background correction on the accuracy of machine learning-based hyperspectral imaging classification. Classification accuracy of
three machine learning methods (k-means clustering, support vector machine and convolutional neural network) in absorbance (a) and reflectance (b) hypercubes
obtained by GT, SB and RB methods. (c—f) Representative images of classification results indicated by c–f in (a) and (b). Scale bar: 100 pixels. https://doi.org/10.1371/journal.pone.0229502.g007 Fig 7. Investigating the effect of background correction on the accuracy of machine learning-based hyperspectral imaging classification. Classification accuracy of
three machine learning methods (k-means clustering, support vector machine and convolutional neural network) in absorbance (a) and reflectance (b) hypercubes
obtained by GT, SB and RB methods. (c—f) Representative images of classification results indicated by c–f in (a) and (b). Scale bar: 100 pixels. https://doi.org/10.1371/journal.pone.0229502.g007 PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 15 / 21 PLOS ONE Background correction in hyperspectral imaging for GT and RB method classifiers. The resulting segmented images again indicate incomplete
background correction (Fig 7C and 7D) for the SB method. Discussion Applications of HSI in biomedicine frequently calculate optical reflectance and absorbance
spectra for tissue classification. The data processing procedures assume that the samples are
uniformly illuminated and while several methods can be employed to ensure that this assump-
tion holds, applications that encounter variations in surface topology or optical power distri-
bution, such as endoscopy, may result in classification errors. Here, we demonstrated a simple
background correction method that enables estimation of the spectral profile and optical
power distribution of illumination across a sample by exploiting the normalised spectra of the
light source and intensity values of the measured hyperspectral signals at a fixed wavelength
with negligible absorbance. The advantage of the method is that it is applied in software, so it
does not require any specialised equipment or application of contrast agents and can be
applied to any HSI data where a wavelength of negligible absorbance is available. It is therefore
practical for application in biomedical imaging, for example, during hyperspectral endoscopy
as demonstrated here using the HySE system. It could also be easily applied to snapshot multi-
spectral biomedical imaging applications, if one of the wavelength bands is located in the NIR
or other minimally absorbing wavelength range, which could enable the fast acquisition and
online post-processing of the data. We selected 800 nm as the wavelength for normalisation in these studies. The results sug-
gest that in samples that are not absorbing at the selected normalisation wavelength, our
retrieved background (RB) method accurately recovers the ground truth (GT) HSI data com-
pared to the standard approach of using a single background (SB). The feasibility and applica-
bility of the proposed RB method were demonstrated by measuring oxygenated and
deoxygenated blood samples, a tissue-mimicking phantom with scattering, absorption, and
fluorescence agents and ex vivo chicken tissue. These experiments indicated the importance of
a complete background correction for analysis and interpretation of HSI data, with variations
in optical power distribution causing rescaling of reflectance data and introducing offsets in
absorbance data. Moreover, the importance of precisely retrieved and corrected background
was assessed using HSI analysis methods and machine-learning based image classification
techniques. In particular, the standard SB method led to erroneous findings for reflectance
data in PCA and absorbance data in SAM. Investigating the influence of background correction on hyperspectral data
classification SVMs successfully segmented the
four clusters in absorption hypercubes with 100% accuracy under all BG corrections, however,
the classification accuracy of SB reflectance hypercubes segmented by using the SVMs trained
via GT and RB hypercubes dropped to 69.1% and 89.1%, respectively again with incomplete
background correction (Fig 7E and 7F). Lastly, CNNs were implemented via a six-layered net-
work, including three convolutional layers, two fully connected layers and a softmax layer (S7
Fig). Trained CNNs classified the hypercubes with 100% accuracy regardless of hypercube
types and BG conditions. Discussion It also led to misclassification in both data types for
k-means clustering and in reflectance data for SVMs, compromising their accuracy, however,
well-trained CNNs were not vulnerable to changes in BG corrections or data types. Nonetheless, there remain some limitations to the present study. The proposed method
assumes that absorption at the normalisation wavelength is negligible. Should there arise some
non-negligible or spatially inhomogeneous absorption at the normalisation wavelength, the
calculated reflectance and absorbance may still introduce errors. For example, in our case
using 800 nm as the normalisation wavelength, we saw that the blood samples and nigrosin
dye samples had some non-negligible absorption at 800 nm, which meant that the magnitude PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 16 / 21 PLOS ONE Background correction in hyperspectral imaging of the corrected spectrum could be up to 29.6% lower than the GT, but importantly, the shapes
of the spectral profiles of the calculated reflectance and absorbance remained unaffected. Therefore, prior information about the absorbance of a given sample at the normalisation
wavelength is necessary when making comparisons between the magnitude of the recorded
spectra. While many biological tissues have little absorption at 800 nm [40,41] choosing this
wavelength may produce problems for experiments that introduce NIR dyes for molecular
imaging. Selecting a wavelength further into NIR tissue optical window could overcome this,
though would require illumination of the tissue with further NIR/IR optical power and the
associated thermal deposition characteristics should be carefully considered from a safety
perspective. In addition, we examined the influence of noise on the study and found that the accuracy of
the normalisation method decreases with increasing noise in the spectra. Care should therefore
be taken when applying the method to a noisy spectral data set. Another consideration is the
need for spectrally uniform illumination across the target, which is an important precondition
for many experiments in HSI and also affects the proposed method. If multiple incoherent
light sources are used, then spectral homogeneity should be checked before using the proposed
method. A further consideration is that the effects of BG correction on HSI classification using
machine learning algorithms were tested here using simple synthetic hypercubes composed of
experimentally measured data from only four spectra components. Discussion While these serve to illus-
trate the potential of the method in cases where known ground truth is available, further exper-
iments would be needed to establish the bounds of operation of the method in another chosen
application. Finally, we focused on the influence of background correction on reflectance and
absorbance hypercubes. Further work would be needed to understand how well the method
could perform for other HSI applications, such as multiplexing of fluorescence contrast agents
[12]. Despite these limitations, the proposed background correction method allows for accurate
and consistent measurement of HSI data, regardless of illumination method and optical power
distribution. Application of the method could facilitate further exploitation of multi- and
hyperspectral imaging techniques in practical clinical applications, where controlling the illu-
mination pattern and power are non-trivial. PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 Acknowledgments We thank Dr Laura Bollepalli for technical assistance in the completion of this study. Supporting information Scale bars: 1 cm (b)
Average measured spectral profiles of the bone marrow (BM), compact bone (C), and shade
(S) areas within solid squares shown in (a) were obtained. Gray shaded area indicates the stan-
dard deviation. (c). Bar graphs show average intensities of six regions shown in (a) were calcu-
lated. Error bar indicates the standard deviation. Statistical analysis was performed using
Student t-test. (PNG) S5 Fig. The RB method enables the accurate measurement of absorbance in endoscopy
conditions. (a) Photograph of the tubular tissue-mimicking phantom with homogeneous
methylene blue concentration. (b) Schematic of the experiment. Absorbance of the tissue-
mimicking phantom at three working distances was obtained using SB (c) and RB methods
(d). The solid line and the gray shaded area indicate average absorbance and standard devia-
tion, respectively. (PNG) S6 Fig. Assessment of background effects on hyperspectral image analysis via principal
component analysis (PCA) and spectral angle mapping (SAM). (a, b) Scatter plots of 2nd
principal component (PC) versus 1st PC (top) and representative images of 1st PC scores (bot-
tom) of absorbance and reflectance hypercubes, respectively. Scale bar: 100 pixels. (c, d) Bar
graphs indicate mean and standard deviation (error bars) of angle values for each cluster
shown in the bottom image (top) and 2D images of spectral angle values (bottom) of absor-
bance and reflectance hypercubes, respectively. SAM was performed using the average spectral
profile of the cluster i of each hypercube. Scale bar is 100 pixels. (PNG) Supporting information S1 Fig. Optical design of the line-scanning hyperspectral endoscope. The system is assem-
bled using a CE-marked endoscope with an imaging fibre bundle and an integrated illumina-
tion fibre. A sample is illuminated either by coupling a halogen light source to the illumination
fibre (internal illumination method) or by directly illuminating via the fibre-coupled halogen
light source (external illumination method). Hyperspectral data is acquired using a CCD cou-
pled to the spectrograph. For line-scanning hyperspectral imaging, a motorized translational
stage is exploited to control imaging position in these studies. Abbreviations: CCD, charge
coupled device; L1–2, lens; Obj1–2, objective lens. (PNG) S2 Fig. Schematic for obtaining the ground truth background (GT), single background
(SB) and retrieved background (RB). (a) GT was obtained by measuring a white reflectance
target under the same position and illumination conditions as the sample measurement. From
the GT, the normalised spectral profile of the background was calculated by averaging across
all spatial locations within the hyperspectral image frame. One of GTs was used as SB. (b) To
obtain RB, the intensity ratio (Cs/Cb) at 800 nm and the normalised spectral profile of the
background was calculated. The intensity ratio of each vertical pixel was calculated by dividing PLOS ONE | https://doi.org/10.1371/journal.pone.0229502
March 13, 2020 17 / 21 PLOS ONE Background correction in hyperspectral imaging intensity values of a sample spectral image (Cs, red dashed line) at 800 nm by the intensity
value of normalised background signal at 800 nm (Cb). (c) The spectrum of the RB used for
correction at a specific vertical pixel was determined by multiplying the normalised back-
ground to the intensity ratio value corresponding to the pixel. (PNG) S3 Fig. Influence of noise-to-signal ratio to the retrieved BG method (a) Simulation of raw spec-
tral profiles of oxygenated blood with different signal-to-noise ratios (1%, 5%, 10%, and 20%). (b)
Absorbance obtained using spectral signals in (a) and the retrieved BG method. Gray shaded area
indicates the standard deviation. (c). Bar graphs show the average error percentages of absorbance
at four different signal-to-noise ratios. Error bar indicates the standard deviation. (PNG) S4 Fig. Investigation of intensity variation at 800 nm (a) Left: Synthetic RGB image of chicken
tissue. Right: Magnified image of the dashed square shown in left figure. Data curation: Jonghee Yoon. References 1. Van der Meer FD, Van der Werff HM, Van Ruitenbeek FJ, Hecker CA, Bakker WH, et al. (2012) Multi-
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https://openalex.org/W3209782791
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https://www.scielo.br/j/ac/a/4LZ8qtKr468XLzxyzgPgVVB/?lang=en&format=pdf
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English
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Effects of termal treatment on the physical properties of Buchenavia sp. (branquilho) wood
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Ambiente Construído/Ambiente construído
| 2,022
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cc-by
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Palavras-chave: Densidade da madeira. Higroscopicidade da madeira. Madeira tropical.
Retratibilidade da madeira. VOLLBRECHT, L. T.; MASCARENHAS, A. R. P.; MELO, R. R. de; LIMA, M. F. de M.; SOTEIL, R. P.; PIMENTA, A. S.
Effects of termal treatment on the physical properties of Buchenavia sp. (branquilho) wood. Ambiente Construído,
Porto Alegre, v. 22, n. 1, p. 95-103, jan./mar. 2022.
ISSN 1678-8621 Associação Nacional de Tecnologia do Ambiente Construído.
http://dx.doi.org/10.1590/s1678-86212022000100580
9 Effects of termal treatment on the physical
properties of Buchenavia sp. (branquilho) wood Efeitos do tratamento térmico nas propriedades físicas da
madeira de Buchenavia sp. (branquilho) Laysa Teles Vollbrecht
Adriano Reis Prazeres Mascarenhas
Rafael Rodolfo de Melo
Maúcha Fernanda de Mota Lima
Ricardo Pereira Soteil
Alexandre Santos Pimenta Abstract his work aimed to evaluate the effect of heat treatment on the physical
properties of wood from Buchenavia sp. The heat treatments were
carried out at temperatures of 180 °C and 200 °C for 2 h. Apparent
density (AD), basic density (BD), porosity (Ф), mass loss, longitudinal
(LS), radial (RS), tangential (TS) and volumetric (VS) shrinkages and anisotropic
factor (AF) were determined. The lowest values of basic density (0.67 g cm-3),
apparent density (0.77 g cm-3), and porosity (43.3%) were observed for the wood
treated at a temperature of 200 °C. Mass losses increased with increasing
temperature and the highest values were observed under the condition of 200 °C
(9.3%). The LS and AF was not affected by heat treatments. The mean values for
RS (3.1%), TS (5.1%), and VS (9.1%) were reduced after the performance of heat
treatments at temperatures of 180 °C and 200 °C, which did not differ from each
other. The thermal treatments were able to reduce the dimensional instability of
Buchenavia sp. Thermal treatments enhance the use of less prestigious Amazonian
woods in the civil construction market. T T 2Adriano Reis Prazeres
Mascarenhas
2Universidade Federal de Rondônia
Rolim de Moura – RO – Brasil Keywords: Hygroscopicity of wood. Tropical wood. Wood density. Wood shrinkage. 1Laysa Teles Vollbrecht
1Universidade Federal de Rondônia
Rolim de Moura – RO – Brasil Effects of termal treatment on the physical
properties of Buchenavia sp. (branquilho) wood
Efeitos do tratamento térmico nas propriedades físicas da
madeira de Buchenavia sp. (branquilho) Effects of termal treatment on the physical
properties of Buchenavia sp. (branquilho) wood Resumo Objetivou-se avaliar o efeito do tratamento térmico nas propriedades físicas
da madeira de Buchenavia sp. Os tratamentos térmicos foram feitos em
temperaturas de 180 °C e 200 °C durante 2 h. Avaliou-se a densidade
aparente (AD), densidade básica (BD), porosidade (Ф), perda de massa,
contração longitudinal (LS), radial (RS), tangencial (TS) e volumétrica (VS) e
fator anisotrópico (AF). Os menores valores de densidade básica (0,67 g cm-
3), densidade aparente (0,77 g cm-3) e porosidade (43,3%) foram observados
para a madeira tratada em temperatura de 200 °C. As perdas de massa foram
crescentes com o aumento da temperatura e os maiores valores foram
observados na condição de 200 °C (9,3%). A LS e o AF não foram afetados
pelos tratamentos térmicos. Os valores médios para RS (3,1%), TS (5,1%) e
VS (9,1%) foram reduzidos após a realização dos tratamentos térmicos nas
temperaturas de 180 °C e 200 °C, que não diferiram entre si. Os tratamentos
térmicos foram capazes de diminuir a instabilidade dimensional da madeira
de Buchenavia sp. Os tratamentos térmicos potencializam o uso de madeiras
da Amazônia de menor prestígio no mercado no contexto da construção civil. Introduction Wood is by far one of the materials more used by mankind from pre-historical times to the present day,
mainly due to its excellent and versatile technological properties, which permit its application in several
forms, such as: civil construction, flooring, furniture uses, watercraft, musical instruments, pulp and paper,
source of chemicals and firewood, among other uses (FIGUEROA; MORAES, 2009; RAMAGE et al.,
2017). The anisotropic character of wood can raise some problems along variations in the moisture content,
for instance, reflected negatively as twisting, cracking, splitting, bowing and also changes in shape and size
of sawn pieces from the green to dry condition (SARGENT, 2019; MASCARENHAS; GHILARDI; MELO,
2020). Therefore, in order to prevent or even eliminate the occurrence of defects during wood processing and
drying, and also to avoid decay during its utilization, some techniques have been successfully developed
with more or less efficiency on reducing the hygroscopicity of wood and the gain and loss of moisture with
ambient conditions. Among the processes employed on wood modification, the thermal treatment stands out since it can improve
wood properties such as dimensional stability, water resistance and also durability, especially without
addition of any type of chemicals (HILL, 2007; JIROUŠ-RAJKOVIĆ;; MIKLEČIĆ, 2019). According to
Silva et al. (2015), the benefits of heat treatments are majorly associated to the degradation of
hemicelluloses, which are responsible for a significant role on sorption and desorption of water in the wood
cell wall. This way, temperatures for heat treatment are usually in the range of 160 a 260 °C and the higher
the temperature is greater will be the losses in mechanical strength of the wood, what can sometimes
severely restrain its use for structural applications (ESTEVES; PEREIRA, 2009; SILVA et al., 2015;
PERCIN; PEKER; ATILGAN, 2016; SANDBERG; KUTNAR, 2016). The contributions of heat-treated wood are also decisive to a low-carbon economy as presented by kutnar,
Sandberg and Haller (2015), who highlighted that new modification processes of the material, such as
thermal treatment, are important strategies to offer to the wood some technological applications without
changing its eco-friendly characteristics and additionally broadening its fields of use. Other characteristics to
be considered when thermal treatment is applied to the wood is the improvement of the color of the
heartwood in lighter-colored woods, turning them into a higher-value-added species (FREITAS;
GONÇALVEZ; DEL MENEZZI, 2016). 3Rafael Rodolfo de Melo
3Universidade Federal Rural do Semi-
Árido
Mossoró – RN – Brasil 3Rafael Rodolfo de Melo
3Universidade Federal Rural do Semi-
Árido
Mossoró – RN – Brasil 4Maúcha Fernanda de Mota Lima
4Universidade Federal de Mato Grosso
Cuiabá – MT – Brasil 5Ricardo Pereira Soteil
5Universidade Federal de Rondônia
Rolim de Moura – RO – Brasil 6Alexandre Santos Pimenta
6Universidade Federal do Rio Grande do
Norte
Macaíba – RN – Brasil Recebido em 02/07/20
Aceito em 07/09/21 Recebido em 02/07/20
Aceito em 07/09/21 95 Ambiente Construído, Porto Alegre, v. 22, n. 1, p. 95-103, jan./mar. 2022. Vollbrecht, L. T.; Mascarenhas, A. R. P.; Melo, R. R. de; Lima, M. F. de M.; Soteil, R. P.; Pimenta, A. S.
96 Introduction However, most of the information on the application of thermal
treatment to improve wood quality and value is available for non-Brazilian species. Within the rationale exposed above, there is a lack in the literature of research works dealing with the use of
thermal treatment to improve the quality of low-rated Amazonian wood species. There are so many useful
and noble species in the Amazon rainforest that several of them, although having medium or high density
and good characteristics for woodworking, only because of one or another physical property such as
dimensional stability, for example, or yet for not having good aesthetical characteristics, are left behind at
the time of harvesting in sustainable management plans. Within this context, the species Buchenavia sp. (Combretaceae), commonly known as “branquilho” (INSTITUTO BRASILEIRO…, 2021; RONDÔNIA,
2021) may be included. Its wood does not have any consolidated market compared to other Amazonian
species, despite presenting acceptable physical-mechanical properties to be employed in civil construction,
tools handles, and veneering (REIS et al., 2019; INSTITUTO DE PESQUISAS…, 2021). The main
limitation of wood of this species is its dimensional instability, which promotes the occurrence of warping
and cracking during drying and its use in external applications Moreover, its light color, within the Brazilian
market, is associated to low quality woods. Because of this, is does not have prestige for more noble uses as
wood sheeting and furniture, the interior finishing of homes, and frames and sheets for coating engineered
wood panels.” The presented context meets the aspects associated with the technology of the built environment, such as
sustainability, the need for technological development in civil construction, and rationalization of costs. The
understanding of techniques, such as thermo-rectification, to enhance the use of little-known Amazonian
woods, whose aesthetic and technological characteristics are undesirable for the market, can contribute to
reducethe overexploitation of species and reduce costs with wood, allowing its application in buildings for
uses for which it would not be considered previously. Thus, the objective of the present work was to assess
the effects of thermal treatment on the physical properties of the Buchenavia sp. wood. Ambiente Construído, Porto Alegre, v. 22, n. 1, p. 95-103, jan./mar. 2022. Calculation of analyzed variables The basic density of the wood was determined by following the procedures described in the standard NBR
7190 (ABNT, 1997), and is defined as the ratio between the wood dry mass and the saturated volume of the
sample. Saturated volume was determined from the radial, tangential, and longitudinal linear measures. For
all samples, the basic density was determined, and for those submitted to thermal treatment that property was
evaluated after the experimental treatment. Following the aforementioned standard, the apparent density of the wood was obtained by the ratio between
the mass and the volume of the wood specimen with moisture content equal to 12%. To reach this target
moisture content, the wood specimens were conditioned at 65% of relative humidity and temperature of 22
°C in a climatic chamber until reaching equilibrium. The dry masses of the samples were recorded before
and after the heat treatments to allow the calculation of the mass loss caused by the heat treatment, as
pointed out above. For radial and tangential faces, in each face, three measures were taken, one in the center and two others in
each far end of the specimen with a precision of 0.01 mm, obtaining, this way, the average measures for
each face. For the longitudinal face, the average measure of the four faces along the length direction was
also taken with a precision of 0.01 mm. Material and methods
Obtaining the samples In order to obtain the test specimens, in a sawmill located at the municipality of Rolim de Moura (Rondônia
State, Brazil), two 0.6 m diameter logs were quarter-sawn by using a band saw and from each log, three
radial boards with final dimensions of 2 m length x 0.2 m width x 0.025 m thickness were obtained. Next,
the boards underwent resawing to remove sapwood and pith, and as well wood defects such as knots, cracks,
and grain slopes. From each board, 60 test specimens with final dimensions of 0.1 m length x 0.025 m width x 0.025 m
thickness were cut, according to procedures described in the standard D143-14 (AMERICAN…, 2014). The
test specimens were accurately cut to expose the longitudinal, tangential and radial wood sections. Conducting thermal treatment The samples were conditioned at 20 °C and 65% relative humidity until reaching the hygroscopic moisture
content (~12%). This initial condition was established before the heat treatment. After, the test specimens
were randomly divided into three groups. Two of them were thermally treated at 180 °C (T1) and 200 °C
(T2) for 2 h. The third group of wood samples was maintained untreated and used as a reference. Before
starting the thermal treatment, all test specimens were oven-dried for 24 h at 105 ± 2 °C. At this time, the
dry mass was measured to calculate the mass loss after thermal treatment. Treatments T1 and T2 were applied to the test specimens right after the oven-drying. To reach the final
temperatures of the respective thermal treatments, a heating rate of 20 °C/5 min was carried out. After 2 h in
the respective final temperatures, the oven was switched off and left to cool until reaching 30 °C. After this
step, the thermal-treated test specimens were taken to conditioning until reaching constant weight. A
microprocessor-controlled oven with electric heating was employed both for drying and heat treatment. All
tests were carried out without any gas injection, relying only on atmospheric air inside the equipment,
according to the methodology reported by Delucis et al. (2014) and Modes, Santini and Haselein (2017). Effects of termal treatment on the physical properties of Buchenavia sp. (branquilho) wood Results and discussion Such
sort of result is expected since in the longitudinal direction the dimensional variations are negligible around
0.1 and 0.3%, depending on the type of wood, basic density, and either amount and type of extractives. As observed, a reduction in the radial and tangential shrinkage occurred when compared to the control. Korkut and Guller (2008) pointed out that thermal treatment brings about a decrease in the water sorption
capacity of the wood cell walls as a result of the decrease in the number of hydroxyl groups and
consequently both radial and tangential shrinkage decrease as well. Table 1 - Means of basic (BD) and apparent (AD) densities and porosity (Ф) for the of wood Buchenavia
sp. (branquilho) before and after thermal treatment at 180 °C and 200 °C
Treatments
BD
(g cm-3)
AD
(g cm-3)
Ф
(%)
Control
0.70 (± 0.04) a
0.82 (± 0.05) a
45.26 (± 2.84) a
180 °C
0.72 (± 0.05) a
0.83 (± 0.06) a
47.00 (± 3.23) a
200 °C
0.67 (± 0.05) b
0.77 (± 0.06) b
43.31 (± 3.24) b
Note: numbers between brackets are standard deviations; and means followed by different letters are statistically
dissimilar by the Scott-Knott test at 95% of probability. le 1 - Means of basic (BD) and apparent (AD) densities and porosity (Ф) for the of wood Buchenavia
(branquilho) before and after thermal treatment at 180 °C and 200 °C Note: numbers between brackets are standard deviations; and means followed by different letters are statistically
dissimilar by the Scott-Knott test at 95% of probability. Table 2 - Means of mass loss for the of wood Buchenavia sp. (branquilho) before and after thermal
treatment at 180 °C and 200 °C
Treatments
Mass Loss (%)
Control
0.0 a
180 °C
2.56 (± 0.99) a
200 °C
9.32 (± 3.95) b
Note: numbers between brackets are standard deviations; and means followed by different letters are statistically
dissimilar by the Scott-Knott test at 95% of probability. Table 2 - Means of mass loss for the of wood Buchenavia sp. (branquilho) before and after thermal
treatment at 180 °C and 200 °C
Treatments
Mass Loss (%)
Control
0.0 a
180 °C
2.56 (± 0.99) a
200 °C
9.32 (± 3.95) b
Note: numbers between brackets are standard deviations; and means followed by different letters are statistically
dissimilar by the Scott-Knott test at 95% of probability. Results and discussion As experimentally observed, there were no statistical differences between the values of basic and apparent
densities and, also, between the porosity for the wood treated at 180 °C and the control treatment (Table 1). However, the standard deviation for the porosity of the thermally-treated wood was slightly higher when
compared to the control. As pointed out by Percin, Peker and Atilgan (2016), when the wood is heated at a high temperature, it
becomes more brittle and its mechanical strength decreases depending on the level and duration of the
thermal treatment which is followed by a concomitant decrease in the wood density. So, more likely it
occurred in the assessed material, despite no differences in density was observed after the thermal treatment,
even though the heat promotes degradation of the wood cell leaving it susceptible to mechanical fatigue
(FIGUEROA; MORAES, 2009). In Table 2, the experimental results for wood mass loss were brought about by the thermal treatment. As
observed, there was no statistical difference of mass loss for the wood treated at 180 °C compared to the
control. Regarding the mass loss, Delucis et al. (2014) discussed that usually, temperatures up to 180 °C are
not enough to promote significant mass losses and as well volume decrease of the wood as determined in the
present work. On the other hand, thermal treatment at 200 °C resulted in a concomitant decrease in basic and apparent
densities, porosity and mass loss. Within this context, Poubel et al. (2013) and Cademartori et al. (2015)
reported that such a pattern of decrease in these physical properties of the wood is mainly associated with the
degradation of the hemicelluloses and extractives, which is followed by a significant decrease in the wood
hygroscopicity. Those components are the most sensitive to temperature and thermal degradation and their both structural
and chemical modification are irreversible since the hydroxyl groups are removed by volatilization, and this
is the reason why the wood hygroscopicity decreases (BORREGA; KÄRENLAMPI, 2008). Moreover, as
temperatures of thermal treatment rise higher is the mass loss, as found by Conte et al. (2014) who worked
with Pinus elliottii wood and determined a mass loss of 6.67% at 210 °C of temperature. The mass losses
observed for these species were 4.5 times when compared to their original wood. As shown in Table 3, the longitudinal shrinkage was not significantly influenced by thermal treatment. Statistical analysis The experiment was set as in an entirely randomized design with three treatments, two temperatures of
thermal treatment, and a control witness treatment, respectively, with 60 replicates (test specimens) per
experimental treatment. Experimental data were submitted to analysis of variance and when the significant
statistical difference was detected, the Scott-Knott test at 95% of probability was applied. 97 Ambiente Construído, Porto Alegre, v. 22, n. 1, p. 95-103, jan./mar. 2022. Vollbrecht, L. T.; Mascarenhas, A. R. P.; Melo, R. R. de; Lima, M. F. de M.; Soteil, R. P.; Pimenta, A. S.
98 Note: numbers between brackets are standard deviations; and means followed by different letters are statistically
dissimilar by the Scott-Knott test at 95% of probability. Results and discussion Table 2 - Means of mass loss for the of wood Buchenavia sp. (branquilho) before and after thermal
treatment at 180 °C and 200 °C Note: numbers between brackets are standard deviations; and means followed by different letters are statistically
dissimilar by the Scott-Knott test at 95% of probability. Ambiente Construído, Porto Alegre, v. 22, n. 1, p. 95-103, jan./mar. 2022. Table 3 - Means of longitudinal (LS), radial (RS), and tangential shrinkage (TS) Means of mass loss for
the of wood Buchenavia sp. (branquilho) before and after thermal treatment at 180 and 200 °C before
and after thermal treatment at 180 °C and 200 °C
Treatments
LS
RS
TS
(%)
Control
0.28 (± 0.25) a
4.60 (± 1.76) a
7.54 (± 1.68) a
180 °C
0.22 (± 0.16) a
3.30 (± 1.04) b
5.59 (± 1.50) b
200 °C
0.25 (± 0.14) a
2.90 (± 0.95) b
4.61 (± 2.37) b
Note: numbers between brackets are standard deviations; and means followed by different letters are statistically
dissimilar by the Scott-Knott test at 95% of probability. le 3 - Means of longitudinal (LS), radial (RS), and tangential shrinkage (TS) Means of mass loss for
of wood Buchenavia sp. (branquilho) before and after thermal treatment at 180 and 200 °C before
after thermal treatment at 180 °C and 200 °C Note: numbers between brackets are standard deviations; and means followed by different letters are statistically
dissimilar by the Scott-Knott test at 95% of probability. Usually, the tangential shrinkage is the most affected property when the wood is submitted to thermal
treatment and such behavior was observed in the present work where the tangential wood movement
decreased in 71% and 63% at temperatures of 180 °C and 200 °C, respectively, when compared to the
original wood. The wood radial is commonly twice as less than the tangential one. In this research study,
thermal treatment was able to reduce the radial movement by about 74% and 61% for the temperatures of
180 °C and 200 °C. Since, for the pristine wood of the forest species assessed here, the mean radial and tangential shrinkage are
higher than 4% and 7%, respectively, the stability can be classified as medium. Results and discussion After thermal treatment
application, the values were reduced to the range of 2.90% to 3.30% for the radial movement and 4.61% to
5.59% for the tangential shrinkage, which led the stability of the wood to be classified as high dimensional
(RUFFINATTO; CRIVELLARO; WIEDENHOEFT, 2015). Bearing in mind the patterns discussed above, for a practical application, it is preferable to employ the
thermal treatment at 180 °C because, besides improving dimensional stability of the Buchenavia sp. wood, it
also caused a smaller mass loss and no alteration on the wood density. On the other hand, as shown in Figure
1, the thermal treatment at 200 °C promoted a higher darkening of the wood. Such a darker color can make
some sorts of wood more attractive to the final consumers, depending on the market preferences, as pointed
out by Freitas, Gonçalvez and Del Menezzi (2016). In Table 4, the results obtained for the volumetric shrinkage and the anisotropy factor are presented. The
values of volumetric shrinkage are statistically different when compared to the control with the decrease in
this parameter. However, there were no differences between the two thermal treatments. Once again, as
mentioned before concerning the linear shrinkages, the thermal treatment at 180 °C is recommended since it
brings about low mass loss. Additionally, as volumetric shrinkage (VS) is an unwelcome characteristic for many kinds of wood uses, the
results accomplished by applying the thermal treatments on the Buchenavia sp. wood turned it from medium
dimensional stability with VS of 13.81% in the control to high stability with 8.32% and 9.81% of VS for
thermal treatment at 180 °C and 200 °C, respectively, results explained by the significant reduction in the
combined tangential and radial shrinkages. Nevertheless, no statistical differences were observed in the
anisotropy factor (AF) between the control and the thermal treatments, which probably can be due to a
proportional decrease in both tangential and radial shrinkages, whose ratio was kept constant. This way, by comparing the values, the AFs can be considered normal-wood-like since they are in the range
of 1.50 and 2.00. As discussed by Melo et al. (2013) and Mascarenhas et al. Effects of termal treatment on the physical properties of Buchenavia sp. (branquilho) wood Results and discussion (2021a, 2021b), the AF cannot
be solely considered as a parameter to define the dimensional stability of wood, because it is not exactly a
limiting parameter for its use, since by using proper conditions of drying and woodworking woods with high
AF can be normally used without any problems. Within a general context, the experimental results found in this work showed that 2 h duration thermal
treatments at 180 °C and 200 °C can satisfactorily increase the dimensional stability of the Buchenavia sp. wood without significant mass losses and alteration on its basic density. The improvement in dimensional
stability acquired by thermal treatment enables the wood of the species to be indicated to nobler end uses
such as fine furniture, window and door frames, wood sheeting for walls, which typically require more
expensive and less widely available woods. 99 Ambiente Construído, Porto Alegre, v. 22, n. 1, p. 95-103, jan./mar. 2022. Figure 1 - Color differences among the of wood Buchenavia sp. (branquilho) of control (a), before and
after thermal treatment at 180 °C (b) and 200 °C (c)
(a) (b) (c)
Table 4 - Means of volumetric shrinkage (VS) and anisotropy factor (AF) for the of wood Buchenavia sp. (branquilho) before and after thermal treatment at 180 °C and 200 °C Figure 1 - Color differences among the of wood Buchenavia sp. (branquilho) of control (a), before an
after thermal treatment at 180 °C (b) and 200 °C (c) (c) (c) (b) (a) Table 4 - Means of volumetric shrinkage (VS) and anisotropy factor (AF) for the of wood Buchenavia sp. (branquilho) before and after thermal treatment at 180 °C and 200 °C Treatments
VS (%)
AF
Control
13.81 (± 3.55) a
1.86 (± 0.78) a
180 °C
9.81 (± 2.23) b
1.87 (± 0.87) a
200 °C
8.32 (± 3.24) b
1.68 (± 0.85) a
Note: numbers between brackets are standard deviations; and means followed by different letters are statistically
dissimilar by the Scott-Knott test at 95% of probability. Note: numbers between brackets are standard deviations; and means followed by different letters are statistically
dissimilar by the Scott-Knott test at 95% of probability. As observed, the heat treatment contributed to the increase in the dimensional stability of the Buchenavia sp. wood to ranges similar to those reported for high-dimensional-stability woods, especially the values
presented by Pereira (2013) for the radial, tangential, and volumetric shrinkages. Conclusions Thermal treatments with 2 h of duration at 180 °C and 200 °C increased significantly the dimensional
stability of Buchenavia sp. wood with the decrease of basic density and concomitant mass loss as
temperature increased. The longitudinal shrinkage was not significantly influenced by thermal treatment. The application of thermal treatment at 180 °C is recommended when the objective is to obtain
improvements in dimensional stability with negligible mass loss and minimum alterations in the basic
density. Results and discussion Moreoverl, there was a
color alteration of the wood becoming darker, which can make it more attractive for uses such as siding or
fine finishing. Therefore, woods commonly employed for those uses such as cumaru (Dipteryx odorata) and
ipê (Tabebuia impetiginosa) may be substituted by the forest species assessed after thermal treatment in this
work. More likely, these new possibilities of use can contribute to decrease the overexploitation of small
groups of tropical forest species (REIS et al., 2019; MASCARENHAS et al., 2021c), since now an
unconventional species, when properly heat-treated, can present similar characteristics to those traditionally
marketed. These aspects are in context with the technology from the built environment, given that they are
based on sustainability, the need for technological development in the production of civil construction, and
the rationalization of costs. AMERICAN SOCIETY FOR TESTING AND MATERIALS. D143-14: standard methods testing: small
clear specimens of timber. Philadelphia, 2014. References AMERICAN SOCIETY FOR TESTING AND MATERIALS. D143-14: standard methods testing: small
clear specimens of timber. Philadelphia, 2014. Vollbrecht, L. T.; Mascarenhas, A. R. P.; Melo, R. R. de; Lima, M. F. de M.; Soteil, R. P.; Pimenta, A. S. 100 Ambiente Construído, Porto Alegre, v. 22, n. 1, p. 95-103, jan./mar. 2022. ASSOCIAÇÃO BRASILEIRA DE NORMAS TÉCNICAS. NBR 7190: projeto de estruturas de madeira. Rio de Janeiro, 1997. BORREGA, M.; KÄRENLAMPI, P. P. Mechanical behavior of heat-treated spruce (Picea abies) wood at
constant moisture content and ambient humidity. Holz als Roh-und Werkstoff, v. 66, n. 1, p. 63-69, 2008. CADEMARTORI, P. H. G. D. et al. Effect of thermal treatments on technological properties of wood from
two Eucalyptus species. Anais da Academia Brasileira de Ciências, v. 87, n. 1, p. 471-481, 2015. CONTE, B. et al. Physical and colorimetric properties of Pinus elliottii var. elliottii thermally treated wood. Scientia Forestalis, v. 42, n. 104, p. 555-563, 2014. DELUCIS, R. A. et al. Physical properties of four thermally treated hardwoods. Floresta e Ambiente, v. 21,
n. 1, p. 99-107, 2014. ESTEVES, B. M.; PEREIRA, H.M. Wood modification by heat treatment: a review. BioResources, v. 4, n. 1, p. 370-404, 2009. FIGUEROA, M. J. M.; MORAES, P. D. Wood behavior at high temperatures. Ambiente Construído, Porto
Alegre, v. 9, n. 4, p. 157-174, out./dez. 2009. FREITAS, A. S.; GONÇALEZ, J. C.; DEL MENEZZI, C. H. Thermomechanical treatment and the effects
on the properties of Simarouba amara (Aubl.). Floresta e Ambiente, v. 23, n. 4, p. 565-572, 2016. HILL, C. A. Wood modification: chemical, thermal and other processes. Hoboken: John Wiley & Sons,
2007. INSTITUTO BRASILEIRO DO MEIO AMBIENTE E DOS RECURSOS NATURAIS RENOVÁVEIS. Sistema Nacional de Controle da Origem dos Produtos Florestais (Sinaflor): tabela de espécies do
Sistaxon. Brasília, 2021. Available: https://www.ibama.gov.br/sinaflor. Access: 26 feb. 2021. INSTITUTO DE PESQUISAS TECNOLÓGICAS. Informações sobre madeiras. Nome científico:
Buchenavia sp. São Paulo, 2021. Available at: https://www.ipt.br/informacoes_madeiras3.php?madeira=76. Access on: 26 fev. 2021. JIROUŠ-RAJKOVIĆ, V.;; MIKLEČIĆ, J. Heat-treated wood as a substrate for coatings, weathering of heat-
treated wood, and coating performance on heat-treated wood. Advances in Materials Science and
Engineering, v. 2019, p. 1-9, 2019. KORKUT, D. S.; GULLER, B. The effects of heat treatment on physical properties and surface roughness of
red-bud maple (Acer trautvetteri Medw.) wood. Bioresource technology, v. 99, n. 8, p. 2846-2851, 2008. KUTNAR, A.; SANDBERG, D.; HALLER, P. References Compressed and moulded wood from processing to products:
COST action FP0904 2010–2014: thermo-hydro-mechanical wood behaviour and processing. Holzforschung, v. 69, n. 7, p. 885-897, 2015. MASCARENHAS, A. R. P. et al. Characterization of wood from Schizolobium parahyba var. amazonicum
Huber × Ducke trees from a multi-stratified agroforestry system established in the Amazon rainforest. Agroforestry Systems, v. 95, p. 475-486, 2021a. MASCARENHAS, A. R. P. et al. Ultrasound to estimate the physical-mechanical properties of tropical
wood species grown in an agroforestry system. Holzforschung, ahead of print, p. 1-13, 2021c. MASCARENHAS, A. R. P. et al. Wood quality of Khaya senegalensis trees from a multi-stratified
agroforestry system established in an open ombrophilous forest zone. Wood Material Science and
Engineering, p. 1-10, 2021b. MASCARENHAS, A. R. P.; GHILARDI, D. S.; MELO, R. R. Geotecnologias aplicadas ao zoneamento
sazonal da umidade de equilíbrio da madeira em Rondônia, Brasil. Anuário do Instituto de Geociências, v. 43, n. 2, p. 119-127, 2020. MELO, R. R. et al. Evaluation of physical, chemical, mechanical and surface properties of the Schizolobium
amazonicum Veneer. Floresta e Ambiente, v. 20, n. 2, p. 238-249, 2013. MODES, K. S.; SANTINI, E. J.; HASELEIN, C. R. Effect of heat treatment on mechanical properties of
Pinus taeda and Eucalyptus grandis woods. Ciência Florestal, v. 27, n. 1, p. 291-302, 2017. PERCIN, O.; PEKER, H.; ATILGAN, A. The effect of heat treatment on some physical and mechanical
properties of beech (Fagus orientalis Lipsky) wood. Wood Research, v. 61, n. 3, p. 443-456, 2016. 101 Effects of termal treatment on the physical properties of Buchenavia sp. (branquilho) wood Ambiente Construído, Porto Alegre, v. 22, n. 1, p. 95-103, jan./mar. 2022. PEREIRA, A. F. Madeiras Brasileiras. São Paulo: Blucher, 2013. PEREIRA, A. F. Madeiras Brasileiras. São Paulo: Blucher, 2013. PEREIRA, A. F. Madeiras Brasileiras. São Paulo: Blucher, 2013. POUBEL, D. S. et al. Effect of the heat treatment on physical and chemical properties of Pinus caribaea
wood. Cerne, v. 19, n. 3, p. 391-398, 2013. RAMAGE, M. H. et al. The wood from the trees: the use of timber in construction. Renewable and
Sustainable Energy Reviews, v. 68, p. 333-359, 2017. REIS, P. C. M. R. et al. Clustering of Amazon wood species based on physical and mechanical properties
Ciência Florestal, v. 29, n. 1, p. 336-346, 2019. RONDÔNIA. Pauta Fiscal de mercadorias e produtos (Nome científico de madeiras comercializadas
em Rondônia). Instrução Normativa n° 8/2021/SEFIN-GETRI de 01 de fevereiro de 2021. Porto Velho,
2021. RUFFINATTO, F.; CRIVELLARO, A.; WIEDENHOEFT, A. C. Review of macroscopic features for
hardwood and softwood identification and a proposal for a new character list. IAWA Journal, v. 36, n. 2, p. 208-241, 2015. SANDBERG, D.; KUTNAR, A. Thermally modified timber: recent developments in Europe and North
America. Wood and Fiber Science, v. 48, n. 1, p. 28-39, 2016 SARGENT, R. Evaluating dimensional stability in solid wood: a review of current practice. Journal of
Wood Science, v. 65, n. 1, p. 1-11, 2019 SILVA, M. R. et al. Chemical and mechanical properties changes in Corymbia citriodora wood submitted to
heat treatment. International Journal of Materials Engineering, v. 5, n. 4, p. 98-104, 2015. Laysa Teles Vollbrecht
Departamento Acadêmico de Engenharia Florestal | Universidade Federal de Rondônia | Av. Norte Sul, 7300, Nova Morada | Rolim de
Moura – RO – Brasil | CEP 76940-000 | Tel.: (69) 3449-3802 | E-mail: laysateles@hotmail.com Adriano Reis Prazeres Mascarenhas
Departamento Acadêmico de Engenharia Florestal | Universidade Federal de Rondônia | E-mail: adriano.mascarenhas@unir.br Adriano Reis Prazeres Mascarenhas
Departamento Acadêmico de Engenharia Florestal | Universidade Federal de Rondônia | E-mail: adriano.mascarenhas@unir.br
Rafael Rodolfo de Melo
Á Departamento Acadêmico de Engenharia Florestal | Universidade Federal de Rondônia | E-mail: adriano.mascarenhas@unir.br
Rafael Rodolfo de Melo
Departamento de Ciências Agronômicas e Florestais | Universidade Federal Rural do Semi-Árido | Rua Francisco Mota Bairro, 572, Rafael Rodolfo de Melo
Departamento de Ciências Agronômicas e Florestais | Universidade Federal Rural do Semi-Árido | Rua Francisco Mota Bairro, 572,
Presidente Costa e Silva | Mossoró – RN – Brasil | CEP 59625-900 | Tel.: (69) 98179-0123 | E-mail: rafael.melo@ufersa.edu.br Maúcha Fernanda de Mota Lima Maúcha Fernanda de Mota Lima
Faculdade de Engenharia Florestal | Universidade Federal de Mato Grosso | Rua Fernando Corrêa da Costa, 2367, Boa Esperança | Cuiabá
– MT – Brasil | CEP 78068-600 | Tel.: (65) 3615-8632 | E-mail: mauchaf@gmail.com Maúcha Fernanda de Mota Lima
Faculdade de Engenharia Florestal | Universidade Federal de Mato Grosso | Rua Fernando Corrêa da Costa, 2367, Boa Esperança | Cuiabá
– MT – Brasil | CEP 78068-600 | Tel.: (65) 3615-8632 | E-mail: mauchaf@gmail.com Ricardo Pereira Soteil
Departamento Acadêmico de Engenharia Florestal | Universidade Federal de Rondônia | E-mail: ricardo.soteli@unir.br Alexandre Santos Pimenta
Engenharia Florestal, Escola Agrícola de Jundiaí | Universidade Federal do Rio Grande do Norte | Rodovia RN 160, Km 03 s/n, Distrito de
Jundiaí | Macaíba – RN – Brasil | CEP 59280-000 | Tel.: (84) 3342-4800 | E-mail: alexandre.pimenta@ufrn.br Alexandre Santos Pimenta
Engenharia Florestal, Escola Agrícola de Jundiaí | Universidade Federal do Rio Grande do Norte | Rodovia RN 160, Km 03 s/n, Distrito de
Jundiaí | Macaíba – RN – Brasil | CEP 59280-000 | Tel.: (84) 3342-4800 | E-mail: alexandre.pimenta@ufrn.br Vollbrecht, L. T.; Mascarenhas, A. R. P.; Melo, R. R. de; Lima, M. F. de M.; Soteil, R. P.; Pimenta, A. S. 102
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Del(10q23)/PTEN Gene Locus Deletion Analysis
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Definitions
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Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Del(10q23)/PTEN Gene Locus Deletion
Analysis National Cancer Institute National Cancer Institute Qeios ID: 8FI3C4 · https://doi.org/10.32388/8FI3C4 Source National Cancer Institute. del(10q23)/PTEN Gene Locus Deletion Analysis. NCI Thesaurus. Code C158866. A procedure used to detect and identify deletion of the PTEN gene located in the vicinity
of 10q23. Qeios ID: 8FI3C4 · https://doi.org/10.32388/8FI3C4 1/1
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Ab Initio and Theoretical Study on Electron Transport through Polyene Junctions in between Carbon Nanotube Leads of Various Cuts
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Yiing-Rei Chen1 ✉, Ming-Kuan Lin1, Dun-Hao Chan1, Kuan-Bo Lin2,3 & Chao-Cheng Kaun3,4 Yiing-Rei Chen1 ✉, Ming-Kuan Lin1, Dun-Hao Chan1, Kuan-Bo Lin2,3 & Chao-Cheng Kaun In this study we look into the interference effect in multi-thread molecular junctions in between carbon-
nanotube (CNT) electrodes of assorted edges. From the tube end into the tube bulk of selected CNTs, we
investigate surface Green’s function and layer-by-layer local density of states (LDOS), and find that both
the cross-cut and the angled-cut armchair CNTs exhibit 3-layer-cycled LDOS oscillations. Moreover,
the angled-cut armchair CNTs, which possess a zigzag rim at the cut, exhibit not only the oscillations,
but also edge state component that decays into the tube bulk. In the case of cross-cut zigzag CNTs,
the LDOS shows no sign of oscillations, but prominent singularity feature due to edge states. With
these cut CNTs as leads, we study the single-polyene and two-polyene molecular junctions via both ab
initio and tight-binding model approaches. While the interference effect between transport channels is
manifested through our results, we also differentiate the contributions towards transmission from the
bulk states and the edge states, by understanding the difference in the Green’s functions obtained from
direct integration method and iterative method, separately. Since the discovery of carbon nanotubes (CNTs) in 19911, the properties of these fascinating quasi-one-dimensional,
nano-scaled materials have been extensively pursued2–4. Such a trend has been enhanced further as graphene was
rediscovered in 2004, which brought even more attention toward all different materials based on the honeycomb
carbon structure. Later as the theoretical research turned to look at the boundaries of these materials, such as
edges of graphene, graphite or nano ribbons5–11, or finite-sized CNTs of different tips12–15, experiments kept up as
well. Scanning tunneling microscopy studies16–20 on LDOS of different graphite edges have been performed, and
the edge states of graphite are confirmed. However, similar experimental efforts on CNTs boundaries21,22 are even
more challenging and yet to be solidly realized.ii g
g
y
y
Due to the relatively well-defined lead-molecule covalent bonding and the finite transport channel involved,
prospected nano-devices23 using CNTs is an alternative and interesting choice, compared with the largely studied
single-molecule junctions with metal-molecule linkages24–26. The CNTs, proposed to serve as the junction con-
necting the electrodes, or the electrodes themselves, or forming a heterostructured junction, give rise to peculiar
properties in transport through these devices27–30. g
In recent years, molecular junctions employing CNT electrodes have been fabricated and measured by much more
controlled and developed strategies. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports | (2020) 10:8033 | https://doi.org/10.1038/s41598-020-63363-3 Yiing-Rei Chen1 ✉, Ming-Kuan Lin1, Dun-Hao Chan1, Kuan-Bo Lin2,3 & Chao-Cheng Kaun3,4 In some cases a single protein can be site-specifically attached to the leads31, while
in others the linkage morphology, or the question of whether the linkage is formed by multiple molecules32, becomes
important and therefore requires further confirmation. As the idea of a single-molecule transistor is gradually realized
in these systems, the properties of the CNT leads and the details of the molecule-CNT contact are both crucial for fur-
ther pursuit. Interference effect33–35, among all the above, should accompany the formation of multi-molecule junctions
in between CNT leads, and is an example that illustrates the interplay between the two roles.i In this study, we aim at understanding the assorted cross-cut or angled-cut semi-infinite CNTs and the trans-
port through molecular junctions bridging such leads. On the one hand we use a one-parameter tight-binding
(TB) model that describes the
π
pp
(
) hopping in the CNTs’ honeycomb network, on the other hand we perform ab 1Department of Physics, National Taiwan Normal University, Taipei, 11677, Taiwan. 2Department of Materials
Science and Engineering, National Cheng Kung University, Tainan, 70101, Taiwan. 3Research Center for Applied
Sciences, Academia Sinica, Taipei, 11529, Taiwan. 4Department of Physics, National Tsing-Hua University, Hsinchu,
30013, Taiwan. ✉e-mail: yrchen@ntnu.edu.tw Scientific Reports | (2020) 10:8033 | https://doi.org/10.1038/s41598-020-63363-3 www.nature.com/scientificreports/ Figure 1. Labeling of the sites on the rim of an angled-cut n n
( ,
) CNT electrode, the correspondence to angle θ,
and the illustration for nodes (circles with a cross inside). Here we literally use =
n
8 to illustrate the labeling. The black dots and the white dots represent sites from two different graphene sublattices. Angle θ is also used to
describe the relative positions of two contact sites, when two polyenes are in parallel and bridging the
electrodes. Figure 1. Labeling of the sites on the rim of an angled-cut n n
( ,
) CNT electrode, the correspondence to angle θ,
and the illustration for nodes (circles with a cross inside). Here we literally use =
n
8 to illustrate the labeling. The black dots and the white dots represent sites from two different graphene sublattices. Angle θ is also used to
describe the relative positions of two contact sites, when two polyenes are in parallel and bridging the
electrodes. initio calculations. With these tools we examine the surface Green’s function, whose projection onto contact sites
helps to explain the transmission behavior of molecular junctions. Method
h
b i The ab initio approach. We use the geometry relaxation process implemented in the SIESTA package36 to
optimize the CNT bulk structure, with a force criterion of 0.02 eV/Å, an energy cutoff of 200 Ryd, the Double-ζ
plus polarization (DZP) basis, and the Ceperley-Alder local density approximation (LDA) for the exchange and
correlation functional. A rectangular supercell of 20 Å × 20 Å × a is used, where a is the lengthwise z-dimension
of the unit cell, to be optimized. With this choice of supercell, the distance between nearest tube walls is 9 Å,
which allows enough vacuum to pass the bond length convergence test. g
p
g
g
In a similar way, the structures of the junctions are then relaxed. For each junction we use a rectangular supercell
of 20 Å × 20 Å × D Å, where D is the span of the junction. It contains 3 to 3.5 CNT unit cells from each lead, the
hydrogen atoms that saturate the dangling bonds at the rims, and the bridging polyene molecule(s). Except for the
carbon atoms in the CNT unit cells that sit farthest from the polyene molecules, which are kept fixed such that they
hold the bulk CNT symmetry and bond lengths, all other atom positions are relaxed with no symmetry restriction. The relaxed junction structures are then put into the ab initio transport calculations, which are performed
by the Nanodcal package37 with the density functional LDA_PZ81 and the DZP basis. Non-equilibrium Green’s
function (NEGF) method38 is adopted in this ab initio process, where the leads’ self-consistent field (SCF) calcu-
lation uses the bulk CNT structure, and is followed by the SCF and transmission calculation for the open system
of CNT-junction-CNT. Having done the transmission convergence test, we choose a 3-primitive-unit-cell buffer
layer to sit at both sides of the scattering region, to form the linkages between the junction and the leads. Note that
the Brillouin zone is sampled by a 1 × 1 × 200 Monkhorst-Pack grid, while a supercell of 30 Å × 30 Å × D Å and a
corresponding real-space grid of 450 × 450 × 15 D are used to ensure enough vacuum and accurate transmission
calculations performed by Nanodcal. g
g
g
In a similar way, the structures of the junctions are then relaxed. Yiing-Rei Chen1 ✉, Ming-Kuan Lin1, Dun-Hao Chan1, Kuan-Bo Lin2,3 & Chao-Cheng Kaun3,4 We also look into the LDOS, the by-product of
the surface Green’s function. For different cuts of CNTs we carry out the LDOS comparison that shows the
dependence of edge state contribution on the type of cut. The transmission in double-threaded molecular junc-
tions shows clearly the interference effect, namely, how the pick of contact sites, where the molecules attach to the
CNT leads, affact the transport. This is an effect that has been studied for the cross-cut armchair CNTs, and now
for the angled-cut armchair CNTs and cross-cut zigzag CNTs. Again, we realize that the interference is a conse-
quence of superposition from the properly chosen even and odd channels. Method
h
b i On the rim of a cross-cut n
( , 0) CNT electrode, the correspondence of relative positions to angle θ
and the illustration for nodes. eads, the polyene molecule(s), and the coupling between the molecules and the leads. We also consider the on-site
nergy on all carbon atoms to be a constant that equals the CNTs’ Fermi energy EF, and
≡
E
0
F
. leads, the polyene molecule(s), and the coupling between the molecules and the leads. We also consider the on site
energy on all carbon atoms to be a constant that equals the CNTs’ Fermi energy EF, and
≡
E
0
F
. We calculate the surface Green’s function of the electrodes with the TB Hamiltonian described above, via two
different paths: gy
q
gy
F
F
We calculate the surface Green’s function of the electrodes with the TB Hamiltonian described above, via two
different paths: (i) The iterative method: All CNT leads considered here have the semi-infinite symmetry. Based on such a
symmetry, and this symmetry alone, one gets the self-consistent formula: α
β
β
=
−
−
g E
g E
( )
[
( ) ]
s
s
1
† where α
η
=
−
+
E
h
i and h is the Hamiltonian of the surface layer (i.e., the unit cell at the rim), β
describes the coupling between the surface layer and the bulk (what is left as the surface layer is peeled off),
and gs is the surface Green’s function.hii gs
(ii) The integration method: Out of the electronic states of the infinite 2-D graphene, one finds the allowed k
-point lines in the Brillouin zone, for any specific n m
( ,
) indices that describe the chirality of the CNT of
interest3. By doing so one obtains the n m
( ,
) CNT band structure. Any specific cut of the n m
( ,
) CNT
determines a specific set of boundary conditions, namely, the nodal rim that defines the cut. From the pool
of the n m
( ,
) CNT’s electronic states, we construct all possible linear combinations that give states
vanishing at the nodal rim. Method
h
b i For each junction we use a rectangular supercell
of 20 Å × 20 Å × D Å, where D is the span of the junction. It contains 3 to 3.5 CNT unit cells from each lead, the
hydrogen atoms that saturate the dangling bonds at the rims, and the bridging polyene molecule(s). Except for the
carbon atoms in the CNT unit cells that sit farthest from the polyene molecules, which are kept fixed such that they
hold the bulk CNT symmetry and bond lengths, all other atom positions are relaxed with no symmetry restriction.h The relaxed junction structures are then put into the ab initio transport calculations, which are performed
by the Nanodcal package37 with the density functional LDA_PZ81 and the DZP basis. Non-equilibrium Green’s
function (NEGF) method38 is adopted in this ab initio process, where the leads’ self-consistent field (SCF) calcu-
lation uses the bulk CNT structure, and is followed by the SCF and transmission calculation for the open system
of CNT-junction-CNT. Having done the transmission convergence test, we choose a 3-primitive-unit-cell buffer
layer to sit at both sides of the scattering region, to form the linkages between the junction and the leads. Note that
the Brillouin zone is sampled by a 1 × 1 × 200 Monkhorst-Pack grid, while a supercell of 30 Å × 30 Å × D Å and a
corresponding real-space grid of 450 × 450 × 15 D are used to ensure enough vacuum and accurate transmission
calculations performed by Nanodcal. The tight-binding (TB) approach. Theoretical results shown in this article are derived from a single-parameter
model, where a uniform (
π
pp ) hopping energy t is considered for all the C-C nearest neighbor links in the entire CNT Scientific Reports | (2020) 10:8033 | https://doi.org/10.1038/s41598-020-63363-3 www.nature.com/scientificreports/ Figure 2. On the rim of a cross-cut n n
( ,
) CNT electrode, the correspondence of relative positions to angle θ,
and the illustration for nodes (circles with a cross inside). Figure 2. On the rim of a cross-cut n n
( ,
) CNT electrode, the correspondence of relative positions to angle θ,
and the illustration for nodes (circles with a cross inside). Figure 3. On the rim of a cross-cut n
( , 0) CNT electrode, the correspondence of relative positions to angle θ,
and the illustration for nodes. Figure 3. Scientific Reports | (2020) 10:8033 | https://doi.org/10.1038/s41598-020-63363-3 Method
h
b i These linearly combined states, satisfying the boundary condition of the cut, gs
(ii) The integration method: Out of the electronic states of the infinite 2-D graphene, one finds the allowed k
-point lines in the Brillouin zone, for any specific n m
( ,
) indices that describe the chirality of the CNT of
interest3. By doing so one obtains the n m
( ,
) CNT band structure. Any specific cut of the n m
( ,
) CNT
determines a specific set of boundary conditions, namely, the nodal rim that defines the cut. From the pool
of the n m
( ,
) CNT’s electronic states, we construct all possible linear combinations that give states
vanishing at the nodal rim. These linearly combined states, satisfying the boundary condition of the cut, Scientific Reports | (2020) 10:8033 | https://doi.org/10.1038/s41598-020-63363-3 www.nature.com/scientificreports/ lead to the expression for the surface Green’s function of the cut. For the angled-cut armchair CNTs
th
l
(
′)
j
ti
f th
f
G
’ f
ti
i
Figure 4. The LDOS of the n
4 carbon atoms in the surface unit cell (grey, from iterative method), for (a)
cut (8, 8), (b) cross-cut (9, 9), (c) angled-cut (8, 8), (d) angled-cut (9, 9), (e) angled-cut (10, 10), (f) angle
(12, 12), (g) cross-cut (8, 0), (h) cross-cut (9, 0), (i) cross-cut (10, 0), and (j) cross-cut (12, 0), compared
corresponding bulk unit cell DOS (orange). Edge states appear in all angled-cut n n
( ,
) CNTs, and all cros
n
( , 0) CNTs. Figure 4. The LDOS of the n
4 carbon atoms in the surface unit cell (grey, from iterative method), for (a)
cut (8, 8), (b) cross-cut (9, 9), (c) angled-cut (8, 8), (d) angled-cut (9, 9), (e) angled-cut (10, 10), (f) angle
(12, 12), (g) cross-cut (8, 0), (h) cross-cut (9, 0), (i) cross-cut (10, 0), and (j) cross-cut (12, 0), compared w
corresponding bulk unit cell DOS (orange). Edge states appear in all angled-cut n n
( ,
) CNTs, and all cros
n
( , 0) CNTs. Figure 4. Method
h
b i The LDOS of the n
4 carbon atoms in the surface unit cell (grey, from iterative method), for (a) cross-
cut (8, 8), (b) cross-cut (9, 9), (c) angled-cut (8, 8), (d) angled-cut (9, 9), (e) angled-cut (10, 10), (f) angled-cut
(12, 12), (g) cross-cut (8, 0), (h) cross-cut (9, 0), (i) cross-cut (10, 0), and (j) cross-cut (12, 0), compared with the
corresponding bulk unit cell DOS (orange). Edge states appear in all angled-cut n n
( ,
) CNTs, and all cross-cut
n
( , 0) CNTs. lead to the expression for the surface Green’s function of the cut. For the angled-cut armchair CNTs, e.g.,
the real space (r, r′) projection of the surface Green’s function is g E k l s
l s
l s
P
E
i
P
l s
( ,
;
,
)
s
n
k
k
n
k
k
k
k
k
1 1
2 2
4
1 1
4
2 2
k
k
k
∑
∑
λ
λ
λ
λ
λ
η
λ
λ
=
〈
| ′ 〉〈′ |
×
|
〉〈
|
−
+
| ′′ 〉〈′′ |
〉
λ λ
λ
′
′′ Scientific Reports | (2020) 10:8033 | https://doi.org/10.1038/s41598-020-63363-3 www.nature.com/scientificreports/ Figure 5. The layer-by-layer LDOS from iterative method (grey line and circle dots) at
=
E
EF for (a) cro
(8, 8), (b) cross-cut (9, 9), (c) angled-cut (8, 8), (d) angled-cut (9, 9), (e) angled-cut (10, 10), (f) angled-cu
12), (g) cross-cut (8, 0), (h) cross-cut (9, 0), (i) cross-cut (10, 0), and (j) cross-cut (12, 0). In each case the
sliced according to the shape of the cut, and each layer contains 4n carbon atoms, same as a bulk unit cell. lines and cross symbols present results from the integration method. When in agreement with the bulk re
(orange), only the integration method result is shown. Figure 5. The layer-by-layer LDOS from iterative method (grey line and circle dots) at
=
E
EF for (a) cross-cut
(8, 8), (b) cross-cut (9, 9), (c) angled-cut (8, 8), (d) angled-cut (9, 9), (e) angled-cut (10, 10), (f) angled-cut (12,
12), (g) cross-cut (8, 0), (h) cross-cut (9, 0), (i) cross-cut (10, 0), and (j) cross-cut (12, 0). In each case the layer is
sliced according to the shape of the cut, and each layer contains 4n carbon atoms, same as a bulk unit cell. Method
h
b i Green
lines and cross symbols present results from the integration method. When in agreement with the bulk result
(orange), only the integration method result is shown. Figure 5. The layer-by-layer LDOS from iterative method (grey line and circle dots) at
=
E
EF for (a) cross-cut
(8, 8), (b) cross-cut (9, 9), (c) angled-cut (8, 8), (d) angled-cut (9, 9), (e) angled-cut (10, 10), (f) angled-cut (12,
12), (g) cross-cut (8, 0), (h) cross-cut (9, 0), (i) cross-cut (10, 0), and (j) cross-cut (12, 0). In each case the layer is
sliced according to the shape of the cut, and each layer contains 4n carbon atoms, same as a bulk unit cell. Green
lines and cross symbols present results from the integration method. When in agreement with the bulk result
(orange), only the integration method result is shown. where ∑τ
τ
=
| 〉〈|
τ
P
,
n
k
k
2
k ∑τ
τ
=
| 〉〈|
τ
P
,
n
k
k
2
k and we use notations λ, λ′ and λ″ to label the eigen-states of the bulk tube. The real space position is labeled by
layer l( ) and site s( ). Note that the n
2 τk states, are all the linear combinations of the original n
4 states of wavevec- Scientific Reports | (2020) 10:8033 | https://doi.org/10.1038/s41598-020-63363-3 www.nature.com/scientificreports/ Figure 6. TB (iterative, in grey) and ab initio (red) transmission through angled-cut (8, 8) CNT leads bridged
by one single polyene molecule at site (a) 1, (b) 3, (c) 5, (d) 7, (e) 9, (f) 11, (g) 13, and (h) 15. From the most out-
poking to the most in-tucking, in order. Figure 6. TB (iterative, in grey) and ab initio (red) transmission through angled-cut (8, 8) CNT leads b
by one single polyene molecule at site (a) 1, (b) 3, (c) 5, (d) 7, (e) 9, (f) 11, (g) 13, and (h) 15. From the
poking to the most in-tucking, in order. Figure 6. TB (iterative, in grey) and ab initio (red) transmission through angled-cut (8, 8) CNT leads bridged
by one single polyene molecule at site (a) 1, (b) 3, (c) 5, (d) 7, (e) 9, (f) 11, (g) 13, and (h) 15. From the most out-
poking to the most in-tucking, in order. Figure 7. An illustration of a two-polyene junction, using contact site combination (9, 24), on the angled-cut
(8, 8) electrodes. Method
h
b i Figure 7. An illustration of a two-polyene junction, using contact site combination (9, 24), on the angled-cu
(8, 8) electrodes. tors k and −k, composing the complete orthonormal basis that satisfies the angled-cut boundary conditions. Obviously the boundary conditions of the angled-cut armchair CNTs’ rim are more complicated than those of the
cross-cut tubes, in the sense that there are n
2 non-equivalent sites appearing as nodes on the angled-cut rim,
while all nodal sites of the cross-cut rim correspond to the same boundary condition. See illustrations in Figs. 1,
2 and 3. Scientific Reports | (2020) 10:8033 | https://doi.org/10.1038/s41598-020-63363-3 www.nature.com/scientificreports/ Figure 8. TB (iterative, in grey) and ab initio (red) transmission through angled-cut (8, 8) CNT leads bridged
by two polyene molecules at site combination (a) θ
π
=
n
8 /3 (3–30), (b)
π
n
14 /3 (5–28), (c)
π
n
20 /3 (7–26), (d)
π
n
26 /3 (9–24), (e)
π
n
32 /3 (11–22), and (f)
π
n
38 /3 (13–20). Figure 8. TB (iterative, in grey) and ab initio (red) transmission through angled-cut (8, 8) CNT leads bridged
by two polyene molecules at site combination (a) θ
π
=
n
8 /3 (3–30), (b)
π
n
14 /3 (5–28), (c)
π
n
20 /3 (7–26), (d)
π
n
26 /3 (9–24), (e)
π
n
32 /3 (11–22), and (f)
π
n
38 /3 (13–20). Figure 8. TB (iterative, in grey) and ab initio (red) transmission through angled-cut (8, 8) CNT leads bridged
by two polyene molecules at site combination (a) θ
π
=
n
8 /3 (3–30), (b)
π
n
14 /3 (5–28), (c)
π
n
20 /3 (7–26), (d)
π
n
26 /3 (9–24), (e)
π
n
32 /3 (11–22), and (f)
π
n
38 /3 (13–20). With this integration method, the Green’s function thereby derived comes from the bulk’s Bloch states only. In other words, evanescent waves are excluded in the integration method, while the iteration method includes
everything. Our inspection via the integration method is therefore valuable in the sense that it helps to separate
the contributions from the edge states and the bulk states. Results and Discussionsf Even(crimson) channel and odd(blue) channel contribution for the transmission through angled-cut
(8, 8) CNT leads bridged by two 17-carbon polyene molecules at site combination (a)..(3–30) and (e) θ
π
=
n
32 /3
(11–22), cases selected from Fig. 8. Figure 9. Even(crimson) channel and odd(blue) channel contribution for the transmission through angled-cut
(8, 8) CNT leads bridged by two 17-carbon polyene molecules at site combination (a)..(3–30) and (e) θ
π
=
n
32 /3
(11–22), cases selected from Fig. 8. n
( ,0) CNT decays monotonically to the bulk value, in cases of the angled-cut n n
( ,
) CNTs with ≠
n
m
3 ,
∈
m
,
oscillations of a three-layered cycle emerge from the decay of the edge state. As for the angled-cut n n
( ,
) CNTs
with =
n
m
3 , the edge state LDOS decays and converges to a single value, instead. However, the oscillating LDOS
is the signature feature of all cross-cut n n
( ,
) CNTs, and all angled-cut n n
( ,
) CNTs. Even for angled-cut cases of
=
n
m
3 , the oscillations are still vaguely seen, before the iterative LDOS fully converges to the single bulk value. n
( ,0) CNT decays monotonically to the bulk value, in cases of the angled-cut n n
( ,
) CNTs with ≠
n
m
3 ,
∈
m
,
oscillations of a three-layered cycle emerge from the decay of the edge state. As for the angled-cut n n
( ,
) CNTs
with =
n
m
3 , the edge state LDOS decays and converges to a single value, instead. However, the oscillating LDOS
is the signature feature of all cross-cut n n
( ,
) CNTs, and all angled-cut n n
( ,
) CNTs. Even for angled-cut cases of
=
n
m
3 , the oscillations are still vaguely seen, before the iterative LDOS fully converges to the single bulk value. Transmission. With the =
n
8 angled-cut armchair CNT leads, we consider C17H19, the 17-carbon polyene
molecule(s), and perform the ab initio calculations for transmission through all non-equivalent one-polyene
junctions (shown in Fig. 6) and selected two-polyene junctions (shown in Figs. 7 and 8), excluding two cases
where the two polyenes sit too close in reality. Results and Discussionsf The LDOS at the rims of differently cut CNTs. The surface LDOS of selected CNTs are shown in Fig. 4. The data shown in grey are obtained from the iterative method. In order to compare with the bulk unit cell DOS
(in orange), we sum up the contributions from the n
4 carbon atoms (same number of atoms as in a bulk unit cell)
of the outermost layer along the cut. As the tubes are cut, bulk features are suppressed in various ways, subject to
different boundary conditions: In the cross-cut n n
( ,
) (armchair) cases only the van Hove singularities originated
from =
k
0 band extrema are suppressed, while in the angled-cut n n
( ,
) cases, the boundary condition leads to
entanglement among different bands, and therefore all van Hove singularities are suppressed. In the cases of even-
n cross-cut n
( , 0) (zigzag) CNTs, we also see the only un-suppressed singularities at
= ±
E
t. This comes from the
dispersionless bands in the TB model, where no other =
k
0 band extrema are present. Moreover, for cases of cross-cut n
( , 0) CNTs and angled-cut n n
( ,
) CNTs, considerable amount of states pop up
at EF. The peak of these EF states is wider in all cases of angled-cut n n
( ,
) CNTs, and not as singular as that in any
cross-cut n
( , 0) CNT case. The cross-cut n n
( ,
) CNTs don’t have such a peak. h
Next we investigate the layer-by-layer LDOS, for energy at EF only. Results from path (i) and path (ii) are both
shown in Fig. 5, to compare with the bulk value. It is clearly seen in (c)~(j) that the peak at EF is due to edge states,
and this is true for all angled-cut n n
( ,
) CNTs and all cross-cut n
( , 0) CNTs. While the LDOS of every cross-cut Scientific Reports | (2020) 10:8033 | https://doi.org/10.1038/s41598-020-63363-3 www.nature.com/scientificreports/ Figure 9. Even(crimson) channel and odd(blue) channel contribution for the transmission through angled-cut
(8, 8) CNT leads bridged by two 17-carbon polyene molecules at site combination (a)..(3–30) and (e) θ
π
=
n
32 /3
(11–22), cases selected from Fig. 8. Figure 9. Results and Discussionsf Some surface Green’s function elements, from iterative method (grey) and integration method
(green), are shown for the cross-cut zigzag CNTs: (a) (12, 0) Re g(
)
s,11 , (b) (12, 0) Im g(
)
s,11 , (c) (12, 0) Re g(
)
s,17 ,
(d) (12, 0) Im g(
)
s,17 , (e) (13, 0) Re g(
)
s,11 , (f) (13, 0) Im g(
)
s,11 , (g) (13, 0) Re g(
)
s,17 , and (h) (13, 0) Im g(
)
s,17 . Figure 10. Some surface Green’s function elements, from iterative method (grey) and integration method
(green), are shown for the cross-cut zigzag CNTs: (a) (12, 0) Re g(
)
s,11 , (b) (12, 0) Im g(
)
s,11 , (c) (12, 0) Re g(
)
s,17 ,
(d) (12, 0) Im g(
)
s,17 , (e) (13, 0) Re g(
)
s,11 , (f) (13, 0) Im g(
)
s,11 , (g) (13, 0) Re g(
)
s,17 , and (h) (13, 0) Im g(
)
s,17 . Molecular orbitals and features. Note that the hopping energy t is fitted by the bulk’s band feature in the
vicinity of EF. With this energy scale, we show the ab initio transmission data on top of the iterative method data. For all cases it appears that the TB polyene molecular level spacings are smaller than those in the ab initio
version. To see this, we do the ab initio calculation for polyene C17H19 in vacuum, via the SIESTA package with the
same standard we use for the junctions. The HOMO and LUMO of C17H19 suggest an intra-molecular hopping
that is 1.3 times the CNT hopping. As the polyenes act as the bridge in the junction, the average molecular C-C
bond length increases by only 0.5%, while the standard deviation shrinks to under 22% of the vacuum value. Although the increased average and the shrunken standard deviation of bond length should bring smaller molec-
ular level spacings, all C-C bonds of the polyene in the junction are shorter than the bulk CNT’s two different
bond lengths (1.412 Å and 1.415 Å). This means, the intra-molecular hopping is still larger than the CNT hopping,
even in the junction form, which explains the disagreement between the TB and the ab initio results on energy
spacings of the molecular level resonance. Edge states from the difference between path (i) and path (ii). Results and Discussionsf Depending on contact sites, the gap span is tuned to allowed the
C17H19 polyene to fit in, whereas the choice of C17H19 is meant to prevent the two leads to touch at their most
out-poking sites, even when the polyene is bridging their most in-tucking sites. For each junction the ab initio
result is shown on top of that from the iterative method, and the contact site combinations are labeled with the
index illustrated in Fig. 1. In all cases, the transmission results from both approaches reach agreement. Especially,
via either approach, the two-polyene cases clearly show the transmission’s dependence on contact combination,
and therefore reveals the interference effect. The ab initio central (EF) features exhibit shifts with respect to the iterative method results. This is due to
charge transfer, as discussed before in the literature34, and maps to the TB model as a slightly negative effective
molecular on-site energy. Other than this, the most obvious discrepancy mainly lies in the energy scale, as shown
by the shifts between relative off-center features from both approaches. This is discussed in the next subsection.h tfh
The 2-polyene transmission is composed of contributions from the even and the odd channels, where the
even (odd) channel is the even (odd) combination of the real-space two parallel polyenes. These two channels
diagonalize the effective two-dimensional subspace characterizing the junction of two threads. In Fig. 9 we use
the most destructive case (a) and the most constructive case (e) from Fig. 8, to illustrate how the individual con-
tributions from the even and the odd channels are simply summed up to give the total transmission. Scientific Reports | (2020) 10:8033 | https://doi.org/10.1038/s41598-020-63363-3 www.nature.com/scientificreports/ Figure 10. Some surface Green’s function elements, from iterative method (grey) and integration method
(green), are shown for the cross-cut zigzag CNTs: (a) (12, 0) Re g(
)
s,11 , (b) (12, 0) Im g(
)
s,11 , (c) (12, 0) Re g(
)
s,17 ,
(d) (12, 0) Im g(
)
s,17 , (e) (13, 0) Re g(
)
s,11 , (f) (13, 0) Im g(
)
s,11 , (g) (13, 0) Re g(
)
s,17 , and (h) (13, 0) Im g(
)
s,17 . Figure 10. Results and Discussionsf We use the two cross-cut zigzag
CNTs, (12, 0) and (13, 0), to illustrate the difference between the iterative method and the integration method. In
Fig. 10, results from both methods are shown for selected matrix elements of the surface Green’s function, where
the site indices follow the labeling illustrated in Fig. 3. The disagreement occurs at the vicinity of EF for all cases
shown. With the detailed energy dependence of the surface Green’s function matrix elements readily calculated, we
then consider C8H10, the 8-carbon polyene(s) as the junction, and investigate the one-polyene case and the
two-polyene cases, as shown in Fig. 11. Note that the reason of choosing a polyene species with even number of
carbon atoms is inevitably the geometry of the cut, and such a choice gives no molecular level at EF. For the 9 Scientific Reports | (2020) 10:8033 | https://doi.org/10.1038/s41598-020-63363-3 www.nature.com/scientificreports/ Figure 11. TB transmission through cross-cut zigzag CNT leads bridged by parallel 8-carbon polyene(s), from
iterative method (grey) and integration method (green): (a) (12, 0) CNT leads and one-polyene junction, (b)
(12, 0) CNT leads and two-polyene junction of site combination (1, 7), (c) (13, 0) CNT leads and one-polyene
junction, (d) (13, 0) CNT leads and two-polyene junction of site combination (1, 7). Figure 11. TB transmission through cross-cut zigzag CNT leads bridged by parallel 8-carbon polyene(s), from
iterative method (grey) and integration method (green): (a) (12, 0) CNT leads and one-polyene junction, (b)
(12, 0) CNT leads and two-polyene junction of site combination (1, 7), (c) (13, 0) CNT leads and one-polyene
junction, (d) (13, 0) CNT leads and two-polyene junction of site combination (1, 7). Figure 12. TB transmission through cross-cut (12, 0) zigzag CNT leads bridged by two parallel 8-carbon
polyenes, from iterative method (grey) and integration method (green), with contact site combinations
θ
π
= 4 /12(1, 5), θ
π
= 6 /12(1, 7), θ
π
= 8 /12(1, 9), θ
π
= 10 /12(1, 11), and θ
π
= 12 /12(1, 13). Figure 12. TB transmission through cross-cut (12, 0) zigzag CNT leads bridged by two parallel 8-carbon
polyenes, from iterative method (grey) and integration method (green), with contact site combinations
θ
π
= 4 /12(1, 5), θ
π
= 6 /12(1, 7), θ
π
= 8 /12(1, 9), θ
π
= 10 /12(1, 11), and θ
π
= 12 /12(1, 13). Figure 12. Results and Discussionsf TB transmission through cross-cut (12, 0) zigzag CNT leads bridged by two parallel 8-carbon
polyenes, from iterative method (grey) and integration method (green), with contact site combinations
θ
π
= 4 /12(1, 5), θ
π
= 6 /12(1, 7), θ
π
= 8 /12(1, 9), θ
π
= 10 /12(1, 11), and θ
π
= 12 /12(1, 13). cross-cut (13, 0) leads, the TB transmission results from both methods echo the gap of the bulk CNT, in other
words, the edge states at EF does not make up for the gap, but does modify the transmission, especially near both
band edges. For the cross-cut (12, 0) leads, the original un-cut bulk (12, 0) CNT is a semi-metal. However, the
transmission in the vicinity of EF given by the integration method is taken away by the edge states. cross-cut (13, 0) leads, the TB transmission results from both methods echo the gap of the bulk CNT, in other
words, the edge states at EF does not make up for the gap, but does modify the transmission, especially near both
band edges. For the cross-cut (12, 0) leads, the original un-cut bulk (12, 0) CNT is a semi-metal. However, the
transmission in the vicinity of EF given by the integration method is taken away by the edge states. y
F g
y
g
y y
g
In Fig. 12 we show the transmissions of all 2 polyene cases (except contact combination (1, 3) that is geometri-
cally too close for the two parallel polyene threads) with the cross-cut (12, 0) leads. The results from the iterative
method and the integration method give the transmissions with and without the edge states. Within each individ-
ual method, the results from different contact combinations show the effect of interference. www.nature.com/scientificreports/ www.nature.com/scientificreports/ In the follow-up calculations for transmissions through molecular junctions in between angled-cut (8,8) CNT
electrodes, we compare the ab initio results with the iterative method results. In the two-thread cases, the agree-
ment between both approaches displays the effect of interference between the even and the odd channels. The
discrepancy between the two approaches mainly results from overlooking the difference between the
intra-molecular hopping and intra-CNT hopping, and is explained by our observation on the molecular bond
lengths. We also present a transmission study on one- and two-C8H10 junction in between cross-cut (12, 0) and (13, 0)
CNT electrodes, where we focus on the comparison between the iterative method and the integration method. For the =
n
m
3 cases, where the original bulk is gapless, the presence of edge states even takes away the transmis-
sion in the vicinity of the EF. Looking into the two-polyene cases with all possible combinations of contact sites
on the (12, 0) CNT leads, we show that the interference effect is present via either path, and the comparison
between both paths’ results reveals the edge state influence in the vicinity of EF. l
As the fabrication of CNT-junction-CNT systems is becoming more developed and controlled, theoretical
understanding of electronic structures at CNT edges, contact and molecule properties, and the interplay between
the previous two aspects, are practically needed. Our study reveals the importance of the interference effect,
through the discussion of the LDOS of CNT edges, and the interplay between the molecule and the leads via the
contact selections. Received: 6 December 2019; Accepted: 30 March 2020;
Published: xx xx xxxx Received: 6 December 2019; Accepted: 30 March 2020;
Published: xx xx xxxx References 22 Mi
E D Y i h Y S
V & M E
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i
i
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i
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y
2. Minot, E. D., Yaish, Y., Sazonova, V. & McEuen, P. L. “Determination of electron orbital magnetic moments in carbon nanotubes
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5. Tulevski, G. S., Myers, M. B., Hybertsen, M. S., Steigerwald, M. L. & Nuckolls, C. Formation of Catalytic Metal-Molecule Contacts
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28. Laird, E. A. et al. Quantum transport in carbon nanotubes. Rev. Mod. Phys. 87, 703–764 (2015). 28. Laird, E. A. et al. Quantum transport in carbon nanotubes. Rev. Mod. Phys. 87, 703–764 (2015) 9. Kaun, C.-C., Larade, B., Mehrez, H., Taylor, J. & Guo, H. Current-voltage characteristics of carbon nanotubes with substitutiona
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32. Zhu, J. et al. Conclusion We present in this article a further study for the interference effect in multi-thread molecular junctions in between
CNT leads of various cuts. To this purpose, we first show our calculations concerning the surface Green’s func-
tions of cross-cut and angled-cut n n
( ,
) CNTs, as well as cross-cut n
( , 0) CNTs, using both the iterative method
and the integration method within the TB model. The results from both methods, in comparison with the bulk
values, show the effect brought by the formation of different cuts. The contributions from bulk states and edge
states can be differentiated by the comparison between the 2 methods. While the cross-cut n n
( ,
) CNTs present no
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38. Datta, S. Quantum Transport: Atom to Transistor. (Cambridge University Press, New York, 2005). Acknowledgementsh This work was supported by the Ministry of Science and Technology of the Republic of China under Grant Nos. MOST 104-2112-M-003-002-, MOST 106-2112-M-009-003- and MOST 107-2112-M-001-036-MY3, and the
National Center for Theoretical Sciences of Taiwan. This work was supported by the Ministry of Science and Technology of the Republic of China under Grant Nos. MOST 104-2112-M-003-002-, MOST 106-2112-M-009-003- and MOST 107-2112-M-001-036-MY3, and the h Competing interestsh p
g
The authors declare no competing interests. Author contributions Yiing-Rei Chen wrote the main manuscript text. Yiing-Rei Chen, Ming-Kuan Lin and Dun-Hao Chan prepared
the figures. Kuan-Bo Lin completed the computational work for the response to reviewers. Chao-Cheng Kaun
provided computational help. All authors reviewed the manuscript. Additional information Correspondence and requests for materials should be addressed to Y.-R.C. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-
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material. If material is not included in the article’s Creative Commons license and your intended use is not per-
mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the
copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2020 Scientific Reports | (2020) 10:8033 | https://doi.org/10.1038/s41598-020-63363-3
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https://openalex.org/W2789733605
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https://www.beilstein-journals.org/bjnano/content/pdf/2190-4286-9-88.pdf
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English
| null |
Correction: Computing the T-matrix of a scattering object with multiple plane wave illuminations
|
Beilstein journal of nanotechnology
| 2,018
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cc-by
| 329
|
Correction Address:
1Institute of Theoretical Solid State Physics, Karlsruhe Institute of
Technology, Wolfgang-Gaede-Strasse 1, 76131 Karlsruhe, Germany,
2Institute of Nanotechnology, Karlsruhe Institute of Technology, P.O. Box 3640, 76021 Karlsruhe, Germany and 3Department of Physics,
National Technical University of Athens, Heroon Polytechniou 9,
15780 Zografou, Greece doi:10.3762/bjnano.8.66
hat
23
as
for
License and Terms
This is an Open Access article under the terms of the
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(http://creativecommons.org/licenses/by/4.0), which
permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. The license is subject to the Beilstein Journal of
Nanotechnology terms and conditions:
(https://www.beilstein-journals.org/bjnano)
The definitive version of this article is the electronic one
which can be found at:
doi:10.3762/bjnano.9.88 This correction refers to Beilstein J. Nanotechnol. 2017, 8, 614–626. doi:10.3762/bjnano.8.66 This correction refers to Beilstein J. Nanotechnol. 2017, 8, 614–626. doi:10.3762/bjnano.8.66 This correction refers to Beilstein J. Nanotechnol. 2017, 8, 614–626. doi:10.3762/bjnano.8.66 In the original publication, the unnumbered equation that
appears in the top right corner on page 624 before Equation 23
contains an error. The order of the svd vectors should be as
follows: In the original publication, the unnumbered equation that
appears in the top right corner on page 624 before Equation 23
contains an error. The order of the svd vectors should be as
follows: Martin Fruhnert*1, Ivan Fernandez-Corbaton2, Vassilios Yannopapas3
and Carsten Rockstuhl1,2 Open Access
Beilstein J. Nanotechnol. 2018, 9, 953. doi:10.3762/bjnano.9.88
Received: 27 February 2018
Accepted: 09 March 2018
Published: 22 March 2018
Guest Editor: H. Hahn
© 2018 Fruhnert et al.; licensee Beilstein-Institut. License and terms: see end of document. License and Terms This is an Open Access article under the terms of the
Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0), which
permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. The authors would like to acknowledge Mr. Philipp Gutsche for
pointing out this error. The authors would like to acknowledge Mr. Philipp Gutsche for
pointing out this error. 953
953
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English
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Effect of Weathering and Parent Materials on Clay Mineralisation—Part III
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Zenodo (CERN European Organization for Nuclear Research)
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cc-by
| 3,224
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J. Indian Chem. Soc.
Vol. L, January 1973 pp. 54-58 J. Indian Chem. Soc. Vol. L, January 1973 pp. 54-58 Effect of Weathering and Parent Materials on Clay
Mineralisation-Part III B. DATTA* AND M. ADHIKARI B. DATTA* AND M. ADHIKARI
Department of Agriculture & Department of Applied Chemistry, Calcutta University, Calcutta
(Manuscript received 28 JUly 1972; accepted 10 November 1972) Clay minerals from soils of humid tropical zones of India are analysed by chemical and
physical methods. The formation of clay from parent materials are genetically interpreted in
relation to ionic environment of the weathering zone. Acid igneous rock is the principal parent
material, though, there are some calcium and magnesium bearing matamorphics. The former
is highly rich in muscovite and K-felspars. Ca, Mg and K that are set free from the parent rocks,
under the weathering intensity have been removed from the weathering zone and hydrogen ions
has entered dominantly in the soil exchange complex. The influence of acid igneous parent
material and acidic ionic environment are responsible for the formation of kaolinite in such soils. Mainly K-felspars and possibly muscovite in fine sand, under this condition of acid weathering,
appear to be associated with kaolinite iu clay. T reaction, and nature and content of exchangeable
ion species. T HOUGH the present position regarding the
exact reaction-mechanism leading to the forma·
tion of clay mineral is still obscure, the role of
ionic environment yielded by the weathera.ble rock·
forming minerals in the weathering zone has been
unequivocally emphasised and is considered as one
of the approaches towards clay mineral genesis. According to Grim10, in ionic ~nvironment rich in
alkaline earth cations set free by the basic igneous
rock under condition of insufficient leaching, mont-
morillonite is the product, while in presence of
alkali cations, particularly potassium, illite will
be formed. reaction, and nature and content of exchangeable
ion species. *Agnl. Eng. Dept., I, I. T., Kharagpur. Material and Method Besides kaolinite, there is a trace amount of
illite, the existence and quantity of which is detected
from its very weak (001) reflection. (Fig. 2). Chemical analysis-A complete chemical analysis
of the entire clay fraction was carried out by Na2C03
fusion method (RobinsonlB). 0
<5 0
+I
... a:
:> ,_ .. a:
~ 0
::; ... ,_
..J .. ,_
z
"' 0
a:
UJ
u. ~
0
0
0
A
8
c
0
E
_,_- :-.... r-
t--1\ J
0·1!5 em
~
v ~
0
I
1/"
·60 c " ~
'\ /' f-'v'
~
L
..:::::
~
;·
• 120 em
I
\
v
/
I
I
v
Vo·
0
I
"-
>c_n:
I
I
rQ
120
m
0
\
0
0
200
400
600
800
1000
TEMPERATURE •c
Fig. 2. Differential 'l'hermographs of B-elays. A,B,C,-Manchkund;
D,E,-Padwa. Free oxides were extracted by the reduction-chela-
tion method (Augilera and Jackson, Zoe. cit.) and free
iron from it determined volumetrically. Coarse silt was fused with Na2C03.K and was
determined by flame photometer. Ca and Mg were
estimated by versenate titration. Cation exchange capacity was determined by
Ammonium acetate method as given by Peach et al.U
Exchangeable Ca and Mg were determined by ver-
senate, Na flame photometrically and K by oobalt.i-
nitrite method (Jacksonu). Material and Method The soils included in this study were the red fer-
ruginous soils from Manchkund and Padwa (Koraput,
Orissa) belonging to humid regions. Characteristic
physical and chemical data of the profiles and other
details about the olimate were described earlier
(Datta and Adhikari6). Fig. I. "Fig. 1. Geological Sketch Map around Manchkund and Padwa Dist. Koraput, Orissa. (After Map. G,S.I., 1961). Pleistocene & Recent
Khondalites
Cuddapah
Charnockites
Granites
Unclassified Crystalhnes
Dharwarian "Fig. 1. Drastic leaching condition tending to remove these
bases away from weathering zone, favours the forma-
tion of kaolinite. As is the case of the basic igneous
rocks, the same holds equally good for the acid
igneous rocks containing alkali and alkaline earth
cations under conditions of differential weathering. The purpose of this paper is to relate the mineral
species of clays isolated from two Indian red ferru-
ginous soils on distinct rock types (with the mineralo-
gical make up of their sand and silt separates) and to
study, in the light of discussion presented above, the
nature of ionic environment that led to the forma-
tion of secondary mineral in their clays under the
prevailing humid tropical weathering condition. To t,his end, profile samples of soils are analysed for
the mineralogical composition of their clays by
X-ray,
D.T.A. chemical
and physico-chemical
methods. The mineralogy of coarse silt is studied
by X-ray and is supplemented by chemical analysis. The mineral content,s of fine sand (50-lOOfL), degree
of alteration of weatherable minerals in them and a
more precise assessment as to the parentage of the
soils through provenance study, are worked out
(Datta and Adhikari5). A measure of ionic environ-
ment in the soil forming zone is deduced from soil "Fig. 1. Geological Sketch Map around Manchkund and Padwa Dist. Koraput, Orissa. (After Map. G,S.I., 1961). Pleistocene & Recent
Khondalites
Cuddapah
Charnockites
Granites
Unclassified Crystalhnes
Dharwarian Geology-The
ancient country rock is mainly
ferruginous-schists (Krishnan15) containing much hae-
matite and limonite and bands of garnet-magnetite AR!: WEATHERING AND PARENT MATERIALS ON CLAY MINERAL!SATION. PART Ill DA'tTA & ADHIKAR!: WEATHERING AND PARENT MATERIALS ON CLAY MINERAL rooks. The country rocks are intervened in mono-
tonous repetition by alternating bands of a. host of
well-developed Archaean rocks of Eastern Ghats. illitio cla.y mineral. The low Y-values of 9 (Manch-
kund) and 5·8 (Padwa) are, however indicative of
kaolinite occurring also as an important component
together with illite (Tables 3 & 4). Material and Method illitio cla.y mineral. The low Y-values of 9 (Manch-
kund) and 5·8 (Padwa) are, however indicative of
kaolinite occurring also as an important component
together with illite (Tables 3 & 4). The principal groups are garnetsillimanite-sohists
and gneisses (Khondalite series), Charnockite series,
calc-gneisses and gneissosegranites (Fig. 1). Chemical analysis of soils clays are given in Table 3,
where silica and silica. alumina ratio clearly indicates
the presence of 1 : 1 type clay mineral in both the
soils. A good amount of K 20 in Manchkund clay
points to the presence of illite as well, in it. Sample preparation for X-ray and D.T.A.-Clay
fractions ( < 2f.') obtained by the interna.tional
method were converted into R-elay by treatment
with N/20 HCl and subsequent
washing
and
centrifuging. Minerals in soil clays, X-rays: Lattice spacings
and their relative intensities are given in Table 5. The diffra.ction pattern for Manchkund clay shows
strong reflection at 7·1l.A (001), 3·55A (002) and
2·57 A and (060) reflection at 1·49A. All these
indicate presence of kaolinite. The line intensity
and sharpness further disclose that kaolinite present
is of well crystallised form. Kaolinite is confirmed
on heating to 600°C which marked by the elimina-
tion of the pattern. A diffuse spacing at lOA with
other reflections at 4·38, 3·55 (003) and 2·67 A (004)
indicate illite. In view of the diffuse nature of (001)
reflection and its intensity, it ma.y be inferred that
the illite is degraded. Coarse silt (20-501') separated from sand by wet-
sieving- through 300 mesh sieve and repeated decanta-
tion (Jackson11) were cleansed of free oxides by the
method of Aguilera and Jackson1• The entire clay ( < 21' size) and coarse silt were
subjected to X-ray using nickel filtered CuK« radia-
tion and Philip's large Debye-Scherrer powder camera
(dia. 114·83 rom). Differential thermal diagrams of organic matter
free clay in the H-form were obtained with the help
of a manually operated D.T.A. apparatus. Surface area-Total and external surfaces of "oxides
free clays" were determined following the procedure
of Dyal and Hendricks8. Y-value of soil clays was
calculated according to the formula given by Martin
and Russells. In Padwa clay, the mineral appears to be essen-
tially kaolinitic which is inferred from the strong
intensity first order basal reflection at 7·12A and
[002] and [003] spacings at 3·57 and 2·40A, respec-
tively. Results Base-exchange behaviour of wlwle soil : The results
are presented in Table 1. In Manchkund and Padwa
soils (soils being of humid zone) favourable leaching
condition prevails so that H constitutes a major
fraction of the total exchangeable cations. Per-
centage adsorption of exchangeable calcium and
magnesium are fairly low in comparison to those in
arid tropical soils (Datta and Adhikari, loc. cit.). Potassium saturation is also low. The distribution
of exchangeable Ca, Mg and K along the profile is
essentially the same in both the soils (Tables 1 & 2. Fig. 2. Differential 'l'hermographs of B-elays. A,B,C,-Manchkund;
D,E,-Padwa. O.E.O., internal surface and Y-value: The results
a.re given in Table 2. The values of c.e.c. of Manch-
kund soil clays compare closely ·with those of illite
(Grim10). These values in conjunction with internal
surface a.reas further point to the same. Padwa soil
clay as judged from its characteristic exchange
oa.pacity a.nd internal surface is most likely to contain D.T.A. Study: The d.t.a. curves (Fig. 2) of .Manch-
kund (15-60 em and 60-120 em) and Padwa (30-120
em) are a.lmost flat upto about 500°C, showing little
loss of water at low temperature but give sharp
endothermic peak between 567 a.nd 602°C corres-
ponding to dehydroxylation rea.ction. The latter 65 j, INDIAN CHE~I. SOC. VOL. L, JANUARY 1973 TABLE l. BASE EXCHANGE PROPERTIES OF WHOLE SOILS (OVEN DRY BASIS) TABLE 2. ETHYLENE GLYCOL RETENTION, C.E.C. AND Y·VAI.UE OF SOIL CLAY
Depth
Total surface
External surface
Internal surface
C.E.C. Y-valuef
m
em
g.fg. sq.m.fg. g.fg. . Manchkund
0-15
0.0423
136.B
0.0163
15-60
0.0451
145.7
0.0266
60-120
0.0478
154.2
0.0321
Padwa
0-15
0.0322
103.7
0.0273
30-120
0.0392
126.4
0.0173
TABLE 3
ELEMEN1'AL ANALYSIS OF
meq.fiOO
surface
sq.m.fg. g.fg. sq.m.fg. g . layer
52.6
0.0260
84.0
20.5
86.0
0.0185
59.7
18.2
9.0
103.5
0.0157
50.7
21.7
88.1
0.0048
15.6
29.3
5.8
55.9
0.0219
70.5
26.3
SOU~ CLAY (OVEN DRY BASIS) TABLE 2. ETHYLENE GLYCOL RETENTION, C.E.C. AND Y·VAI.UE OF SOIL CLAY TABLE 3. ELEMEN1'AL ANALYSIS OF
SOU~ CLAY (OVEN DRY BASIS) endothermic feature followed by an exothermic
reaction at 980°C indicates that the major mineral
is well crystallised kaolinite in these clays. endothermic feature followed by an exothermic
reaction at 980°C indicates that the major mineral
is well crystallised kaolinite in these clays. Potash content and simultaneous absence of Ca
indicate that felspars present are only of K-bearing
types and no Ca-plagioclase is present. Results Mica in these
samples does not appear to be one of typical pri-
mary micas which normally give well defined basal The Q-15 om samples from both the soils show,
unlike the other layers, a very weak low tem:r;erature
endotherm. This character together with the broad
and hump-like dehydroxylation endotherm not typi-
cally sharp and intense like that usually encountered
with kaolinite, and absence of exothermic reaction
may indicate the presence of illite as impurity in a
predominantly kaolinitic clay. A similar type of
curve behaviour with the D.T.A. curves of mixtures
of illite and kaolinite was also noted by Grim9 • '!'ABLE 4. PABTIAL CHEMICAL ANALYSIS OF COARSE SILT
(OVEN DRY BASIS)
Depth
C.E.C. Constituents(%)
Sample
in em
moqjlOO
gm
K
Mg
Ca
Manchkund
0-15
2.2
1.6
0.56
15-60
2.4
1.8
0.39
60-120
2.2
2.3
0.33
Padwa
0-15
3.9
2.4
30-120
3.5
2.2
reflections. Presence of mica here, is detected from
a reflection at l0·987A and ll·354A in Manchkund
and Padwa, respectively. No subsequent lower '!'ABLE 4.
PABTIAL CHEMICAL ANALYSIS OF COARSE SILT
(OVEN DRY BASIS) '!'ABLE 4. PABTIAL CHEMICAL ANALYSIS OF COARSE SILT
(OVEN DRY BASIS) .Jl inerals in coarse silt ; X-ray data are given in
Table 5. The study shows that quartz is the pre-
dominant mineral in 20-50,u fractions of both the
soils. Accompanying minerals are felspars (Brown3)
and mica, with a greater abundance of the former in
}lanchkund silt. Potash content (cf, Table 4) of this
size fraction in different layers also substantiates
that a
fair
amount of K-bearing
minerals is
present. reflections. Presence of mica here, is detected from
a reflection at l0·987A and ll·354A in Manchkund
and Padwa, respectively. No subsequent lower 56 DUTTA & ADHIKARI: WEATHERING AND PARENT MATERIALS ON CLAY MINERALISATION. PART III RI: WEATHERING AND PARENT MATERIALS ON CLAY MINERALISATION. PART III DUTTA & ADHIKARI: WEATHERING AND PARENT MATERIALS ON CLAY MINERALI TABLE 5. LATTICE SPACING IN A AND THEIR RELATIVE INTENSITIES IN POWDER DIAGRAMS
H·clay
Coarse silt
Soil
Lattice spacing
Lattice spacing
Lattice spacing
Lattice spacing
comparable with
comparable with
comparable with
comparable with
kaolinite
illite
felspar group
quartz
dnkl
I
dkkl
I
dnk! I
dhk! I
Manchkund
7. II
7
10.0
(diffuse)
6.495!:! ! 3.3285
10
(0-15 em)
3.5503
3
4.3879
5
5.8087
-~-
2. 7809
1
2.5743
l
3.5503
4-
4.2467
6
2.5916
2
1.4955
!·
2.6742
t
3.754
4
2.4609
1
1.4955
t
3.474
1
2.2939
l
3.2268
3
2.251
·:!-
2.9975
I! 2.1281
2
2.9042
~~-
l. 9910
l
1.9363
) ,. 1.8253
4,1
l. 8064
4!-
1.6822
:;
1.5480
5
1.5025
) ·::·
1.4588
1
1.3789
7
l. 2901
1:}
Padwa
7.1204
10
(very diffuse)
3.3285
10
(0-15 em)
4.48
5
10.0
3
4.2029
7
2.4518
5
4.1295
3
4.5997
1
2.2805
5
3.5785
8
3.3507
6
2.2317
3
2.40
5
1.4955
2.1252
5
2.4926
4
1.9811
5
2.2939
2
1.8151
6
2. 0112
2
l. 6749
5
l. 4955
6
1.5438
6
1.4572
2! 1.3774
6! 1.2925
3
1.259
4
1.2325
3
1.2031
5
1.185
5! 1.157
3! I = Intensity of lines. I = Intensity of lines. chiefly basic (towards anorthite.in Padwa) and fairly
altered. order basal spacings could be obtained. The (001)
spacings as obtained, being lar~er. than t.he
us1~al
values (9·9A, lOA) for mica, mchcate that mrca
present is possibly hydrated or interstratified. Exchange capacity (cf. table) also accounts for the
presence of a small amount of such secondary mineral
in silt. '!'ABLE 4.
PABTIAL CHEMICAL ANALYSIS OF COARSE SILT
(OVEN DRY BASIS) Adherence of a fair amount of K-felspars and total
extinction of plagioclase in silt size fraction indicates
more weatheribilit.y of soda-lime felspars compared
to their potassium analogues. This may also suggest
that silt fractions of these soils are chemically more
reactive than their sand fractions. References l. N. H. AGVILERA and M. L. JACKSON, Soil Sci. Amer. P,·oc., 1953, 17, 359-364. ,
z. T. F. BATTES, .11-fineral Industries, Pennsylvania State
Univ., 1960, 29(8), l. 3. G. BROWN, in "The X-ray identification and crystal
structures of clay minerals", pp. 467-488, Mineralog. Soc. (clay minerals group), London, 1961. 4. C. W. CoRRENS, lOth Conf. of clays and clay minerals,
pp. 443-459, Pergamon Press, New York, 1963. l\1
A
l
i
T l ji 5. B. DATTA and l\1. ADHIKARI, Agrolcemia es Talajitan
(supplementum), 1968, 17, 125-141. Whatever may be the mechanism, it may be inferred
that acid granitic parent material and pronounced
leaching .veathering condition leading to an acid
environment, have favoured the formation of kaolin
in these soils. 6. B. DATTA and M. ADHIKARI, Indian J. Appl. Chem.,
1968, 31, 143-150. ,
7. C. DE KrMPE, M. C. GASTUCHE and S. W. BmNDLEY,
Am. Min., 1961, 46, 1370-1382. 8. R. S. DYAL and S. B. HENDRICKS, Soil. Sci., 1950, 69,
421-432. 9. R. E. GRnr, Am. Min., 1947, 32, 493-501. Results go to show that though the mineral com-
position in both these soils is essentially kaolinitic,
the proportion is relatively more in Padwa clay,
while in Manchkund it is diluted by a little or more
illite. 10. R. E. GRIM, "Clay Mineralogy", McGraw-Hill, New
York, 1953. 11. M. L. JACKSON, "Soil Chemical Analysis: Advance
Course", Wisconsin Univ., Madison, 1956. 12. M. L. JACKSON, "Soil Chemical Analysis", Prentice Hall,
New Jersey, 1958. V y
13. vV. D. KELLER, "Soil Clay Mineralogy." (Eds. c. I. Rich
and G. W. Kunze), Univ. North Carolina Press, 1964. Formed on the same nature of parent material and
identical climate being within a latitudinal and longi-
tmlinal variation range of 9' and 7' respectively
(situated at a short distance), this minor but tangible
variation is interesting to note. This anomaly can
be explained if we take into account the greater
intensity of leaching weathering condition in Padwa
soil as evidenced from comparatively lower pH,
and higher clay content and free oxides of iron asso-
ciated with clay. Variation in these properties
under similar set of climatic factors is presumed to 14. W. D. KELLER, W. D. BALGORD and A. L. REESMAN,
J. Sed. petml., 1963, 33, 191-204. p
15. :M.S. KRISHNAN, "Geology of India and Burma", Higgin-
botham, Madras, 1960. 16. R. T. MARTIN and M. B. J". IN"DIAN CHEM. SOC.
VOL. L, JANUARY 1973 Under the prevailing acidic soil environment the
hydrolytic decomposition of the felspars might result
in a pronounced leaching of bases, particularly K and
Ca, leading finally to the production of kaolinite
(Kellerl3). Similar conclusion was arrived at by
De Kime et al.7, Keller et al.13 and Correns4 • have been brought about by some microclimatic in-
fluence inside Padwa soil and possibly, the latter
bringing forth an enhanced leaching effectiveness
that has been responsible for a relatively more con-
tent of kaolin as compared to that in Manchkund
soil clay. When mica weathers, H+ gradually replaces K+
and the mineral is hydrated forming hydro-mica
(Bates2) ultimately passing on to kaolin. Large
abundance of muscovite in fine sands and appear-
ance of hydro-mica phase (Datta and Adhikari,
loc. cit.) in them, indicate mica weathering and its
possible alteration to kaolin in these soils. This
may be another reasonable mechanism of kaolin
formation in these soils. It has also been observed
by Sand19 that micas weather to kaolinite under
fairly intensive weathering environment. Discussions Distinctly acid reaction and good content of free
oxides of iron (Table 3) in associatibn with clay
particles indicate an intense weathering condition of
leaching. Whatever Ca++, Mg++ and K f- are set
by the basic and K-bearing components of the
parent rock, might have been, under these circum-
stances, removed away from the weat,hering zone of
the soils. As a result, influence of parent material
containing basic component (of. calc. schist) whatever
little is there is masked and H-ions constitute a
large proportion of the exchangeable bases. These soils are developed in sit1t from the Archaean
rock'! of the Eastern Ghats15 under humid climatic
condition. The underlying parent material is com-
posed mainly of acid igneous and some mixed meta-
morphic rocks (Datta and Adhikari"). The latter
are mainly mica-schist w·ith a minor amount of tre-
molite-schist in Manchkund and calc-gneiss in case
of Padwa. The dominating influence of the parent
rocks is reflected in tho accessory mineral suite of
the fine sands where a conspicuous abundance of
zircon
in
particular, point.s to the
presence
of acidic parent material. The latter are also highly
rich in K-felspars and muscovite, K-felspars being
in states of alteration. Compared to K-felspars
plagioclase occurs in very small amount. It is The dominance of kaolinite in these clays appears
to be associated with the weathering of felspars,
mainly K-felspars, present in fair amount of both
sand and silt fractions. 57 J". IN"DIAN CHEM. SOC. VOL. L, JANUARY 1973 References RussEL, Soil Sci., 1952, 74,
267-269. 17. M. PEECH, USDA Circ. No. 757, 1947. 18. W. C. RoBINSON, "Method and Procedure of Soil Analysis
med in the Division of Soil Chemistry and Physics",
USDA Circ. 1939. 19. L. B. SAND, Am. Min., 1956, 41, 28-40.
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https://openalex.org/W4283804228
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https://diagnosticpathology.biomedcentral.com/counter/pdf/10.1186/s13000-022-01228-1
|
English
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Atypical primary biliary cholangitis results in vanishing bile duct syndrome with cutaneous xanthomas: a case report
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Diagnostic pathology
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Abstract Background: Vanishing bile duct syndrome (VBDS) is a rare but potentially severe acquired chronic cholestatic liver
disease. Bile duct deficiency is a reduction of bile ducts in the liver, which can eventually lead to cholestatic liver
disease and progress to biliary cirrhosis. Primary biliary cholangitis (PBC) is one of the causes of bile duct deficiency. In addition, 75% of PBC patients may have dyslipidemia, and in case of secondary dyslipidemia, cutaneous
xanthomas may occur. Case summary: A 49-year-old woman was admitted with jaundice and multiple subcutaneous nodules. She
received diagnosis of autoimmune liver disease 2 years before. Although she was treated with liver-protecting
drugs, such as Essentiale and ursodeoxycholic acid, jaundice occurred repeatedly, and the color of her skin was
becoming darker and more yellow. Conclusion: This case highlights that the positivity of ANA that in PBC have a well diagnostic and prognostic
significance and antinuclear antibodies giving the ‘multiple nuclear dots’ or the ‘rim-like/membranous’ pattern scan
ca diagnose primary biliary cirrhosis accurately. Since the liver biopsy of PBC alone may not be sufficient to
establish the diagnosis, serum antibodies should also be examined. PBC can also lead to intrahepatic cholestasis,
which can cause dyslipidemia and cutaneous xanthomas. Keywords: Vanishing bile duct syndrome, Primary biliary cholangitis, Cutaneous xanthomas, Cas Atypical primary biliary cholangitis results
in vanishing bile duct syndrome with
cutaneous xanthomas: a case report Yuebo Jia1†, Lin Liu2†, Baocheng Deng3, Yu Huang2, Jiaqi Zhao1 and Guang Bai2* Chief complaints 2Department of Gastroenterology, Affiliated Hospital of Liaoning University of
Traditional Chinese Medicine, Shenyang, China
Full list of author information is available at the end of the article A 49-year-old woman presented to the TCM Gastro-
enterology Clinic of our hospital complaining of jaundice Department of Gastroenterology, Affiliated Hospital of Liaoning U
Traditional Chinese Medicine, Shenyang, China
Full list of author information is available at the end of the article Introduction specimens, both of which identify bile duct components
[2]. VBDS is not an independent disease, but rather a
pathologic feature of continuous progressive destruction
of intrahepatic bile ducts due to various factors. Some
patients with the absence of intrahepatic bile ducts may
have symptoms of cholestasis, such as jaundice, itching,
and fatigue, while others may be asymptomatic [3]. Here,
we described a case of VBDS diagnosed by liver biopsy
and serological analysis, finally diagnosed with PBC, who
also had cutaneous xanthomas [4]. Vanishing bile duct syndrome (VBDS) is defined as a
finding in specimens containing portal areas; absence of
interlobular bile duct is found in more than 50% of the
portal area. The etiology of VBDS is variable, including
drugs, immunity, congenital malformation, tumor, infec-
tion, and ischemia and hypoxia. Among them, immune
factors causing biliary system injury are an important
mechanism of VBDS [1]. Diagnosis is improved by im-
munostaining
for
CK7
and
CK19
in
liver
biopsy †Yuebo Jia and Lin Liu contributed equally to this work. * Correspondence: lnzyxhkbg@sohu.com Jia et al. Diagnostic Pathology (2022) 17:57
https://doi.org/10.1186/s13000-022-01228-1 Jia et al. Diagnostic Pathology (2022) 17:57
https://doi.org/10.1186/s13000-022-01228-1 and multiple subcutaneous nodules that had been lasting
for 2 years. Imaging examinations Computed tomography (CT) scan of the whole abdomen
revealed hepatosplenomegaly, widened portal veins, hep-
atic cysts, cholecystolithiasis, and ascites. 3-D CT scan
showed multiple old fractures of right ribs. Her lumbar
spine magnetic resonance (MR) images showed L1–L5
vertebral compression fractures. © 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://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. Page 2 of 4 Jia et al. Diagnostic Pathology (2022) 17:57 Jia et al. Diagnostic Pathology to 40), γ-glutamyl-transpeptidase (γ-GGT) level of 824
U/L(normal range,7 to 32), alkaline phosphatase (ALP)
level of 1879 U/L(normal range,70 to 150), prothrombin
time (PT) of 21.2 s(normal range,10 to 14), international
normalized ratio (INR) of 1.85(normal range,0.8 to 1.24),
activated partial thromboplastin time (APTT) of 52.8
s(normal range,23 to 35), total bilirubin (TBIL) level of
515.76 μmol/L(normal range,3.42 to 20.52), direct biliru-
bin (DBIL) level of 249.88 μmol/L(normal range,<=6.91),
indirect bilirubin (IBIL) level of 265.88 μmol/L(normal
range,2
to
15.22),
total
bile
acid
(TBA)
level
of
255.5 μmol/L0.(normal range,14 to 9.66), and free tri-
iodothyronine
(FT3)
level
of
1.83 pmol/L(normal
range,3.1 to 6.8). Blood tests showed no obvious abnor-
malities. Triglyceride (TG) level of 5.28 mmol/L(normal
range,0.7 to 1.7), total cholesterol (CHOL) level of 18.17
mmol/L(normal range,<=5.2),HDL cholesterol(HDL-C)
level
of
1.56 mmol/L(normal
range > =1.03),
LDL-
Cholesterol
(LDL-C)
level
of
0.55 mmol/L(normal
range < −3.62), C-reactive protein (CRP) level of 103.59
mg/L(normal range,<=8), IgA level of 4.16 g/L(normal
range,2.01 to 2.69), and IgG level of 15.31 g/L(normal
range,11.52 to 14.22) were found. Antibody screening re-
vealed positive antinuclear antibody (ANA), antimito-
chondrial (AMA), anti-mitochondrial M2 (M2-3E), and
hepatitis E IgG (HEV-IgG) antibodies. Tumor markers
showed positive CA12–5 (37.28 U/mL)(normal range,<=
35) and CA72–4 (24.45 U/mL)(normal range,<=6.9). and multiple subcutaneous nodules that had been lasting
for 2 years. History of present illness The patient’s symptoms started 2 years ago with mani-
festations of jaundice and multiple subcutaneous nod-
ules, and worsened over the last 3 days. Two years ago,
she received diagnosis of autoimmune hepatitis (AIH),
but the specific type was unknown. Although she re-
ceived Polyene phosphatidylcholine 228 mg oral tid and
ursodeoxycholic acid 250 mg oral qid, jaundice occurred
repeatedly. The patient received a detailed medical
examination and treatment at the current hospital. Personal and family history The patient did not abuse alcohol or substances. There
was no family history of liver disease. Laboratory examinations Blood samples revealed alanine aminotransferase (ALT)
level of 103 U/L(normal range,5 to 35), serum aspartate
aminotransferase (AST) level of 199 U/L(normal range,8 Fig. 1 Numerous xanthomas in the patient. Histopathological
assessment of a subcutaneous nodule from the elbow joint revealed
that it was xanthoma Physical examination The patient had yellow skin and icteric sclera, and her
elbow, wrist, finger joints, and metacarpophalangeal
joints presented multiple, scattered, soft non-tender sub-
cutaneous nodules (Fig. 1). The inferior border of the
spleen exceeded the lower costal margin by 3 cm; there
was sternal tenderness and L2–L3 spinal percussive
pain. History of past illness The patient had type 2 diabetes for 4 years, and had been
using metformin sustained-release tablets intermittently
to control blood glucose. Pathological examination Percutaneous liver needle biopsy revealed 25 fibrosis ex-
panded small and medium portal areas and an incom-
plete large one (Figs. 2, 3). Among them, the epithelium
of small bile ducts was not complete in three large and
incomplete large portal areas, and lymphocytes infiltra-
tion was observed. Hepatocyte swelling and bile salt ac-
cumulation were noted, but no arterioles accompanying
small bile ducts in other portal areas were found. Mul-
tiple confluent fields of necrosis in lobules, collapse of
reticular scaffolds, and perisinusoidal fibrosis were also
found. Some hepatocytes had cholestasis, and some hep-
atic sinusoids nonobstructively expanded with aberrant
erythrocyte morphology. Fig. 1 Numerous xanthomas in the patient. Histopathological
assessment of a subcutaneous nodule from the elbow joint revealed
that it was xanthoma Histopathological assessment of a subcutaneous nod-
ule from the elbow joint revealed that it was xanthoma. Jia et al. Diagnostic Pathology (2022) 17:57 Page 3 of 4 Jia et al. Diagnostic Pathology Fig. 2 Hepatic tissue of Hematoxylin and eosin staining. Hematoxylin and eosin staining showed 25 fibrosis expanded small and medium portal
areas and an incomplete large one Fig. 2 Hepatic tissue of Hematoxylin and eosin staining. Hematoxylin and eosin staining showed 25 fibrosis expanded small and medium portal
areas and an incomplete large one Fig. 2 Hepatic tissue of Hematoxylin and eosin staining. Hematoxylin and eosin staining showed 25 fibrosis expanded small and medium portal
areas and an incomplete large one Clinical diagnosis combined with liver pathology and
serum antibodies: primary biliary cholangitis (PBC), pha-
seII (Inflammation and/or fibrosis limited to and around
the portal area); Treatment Treatment included ursodeoxycholic acid 250 mg oral
qid and fenofibrate 200 mg oral qd, in combination with calcium 600 mg and vitamin D
500 units oral tid. The patient continued to take ursodeoxycholic acid 250
mg oral qid and fenofibrate 200 mg oral qd after discharge. Outcome and follow-up Based on the results of 25-day-long treatment, the pa-
tient responded poorly to ursodeoxycholic acid. Consid-
ered low drug response rate, jaundice did not abate. Liver transplantation is considered the only effective Diagnostic procedure Based on the patient’s pathological examination, the diag-
nosis of VBDS was made. Histological lesions were graded
G2S2 as per the modified Scheuer score. Combined with
the laboratory examinations, she received the diagnosis of
PBC. After symptomatic treatment, we discharged the pa-
tient on the 25th day of hospitalization with the following
serum results: AST 128 U/L, ALT 51 U/L, γ-GGT 670 U/
L,
TBIL
515.76 μmol/L,
DBIL
249.88 μmol/L,
IBIL
268.88 μmol/L, and ALP 720 U/L. Availability of data and materials All data, models, and code generated or used during the study appear in
the submitted article. Discussion The etiology of VBDS is diverse, including congenital
and genetic diseases, ischemia and hypoxia, tumors, in-
fections, immune disorders, idiopathic adult intrahepatic
bile duct deficiency, and other factors. Biliary system in-
jury caused by immunological factors is an important
mechanism of VBDS, and PBC is an autoimmune liver
disease. With the progression of PBC, small bile ducts
affected by immunological process may gradually dis-
appear, thereby leading to VBDS. Meanwhile, given that
cholestasis affects lipid metabolism, individuals with
PBC may show elevated cholesterol levels and develop
cutaneous xanthomas. Received: 11 November 2021 Accepted: 30 April 2022 In this case, liver biopsy indicated disappearance of
intrahepatic bile ducts but did not find well-defined
pathological features of PBC. Yet, PBC is one of the
causes of disappearance of intrahepatic bile ducts, and it
was confirmed via serological tests showing positive
AMA and AMA2-3E antibodies. Therefore, for more ac-
curate
diagnosis,
combining
multiple
assessment
methods is needed in clinical practice. Conclusion This case highlights that the positivity of ANA that in
PBC have a well diagnostic and prognostic significance
[5] and antinuclear antibodies giving the ‘multiple nu-
clear dots’ or the ‘rim-like/membranous’ pattern scan ca
diagnose primary biliary cirrhosis accurately [6]. Since
the liver biopsy of PBC alone may not be sufficient to es-
tablish the diagnosis, serum antibodies should also be
examined. PBC can also lead to intrahepatic cholestasis,
which can cause dyslipidemia and cutaneous xanthomas. 8. Pusl T, Beuers U. Ursodeoxycholic acid treatment of vanishing bile duct
syndromes. World J Gastroenterol. 2006;12:3487. 8. Pusl T, Beuers U. Ursodeoxycholic acid treatment of vanishing bile duct
syndromes. World J Gastroenterol. 2006;12:3487. References
h 1. Zhao Z, Bao L, Yu X, Zhu C, Xu J, Wang Y, et al. Acute vanishing bile duct
syndrome after therapy with cephalosporin, metronidazole, and
clotrimazole: a case report. Medicine (Baltimore). 2017;96:e8009. 2. Nakanuma Y, Tsuneyama K, Harada K. Pathology and pathogenesis of
intrahepatic bile duct loss. J Hepatobiliary Pancreat Surg. 2001;8:303–15. 3. Ludwig J, Wiesner RH, LaRusso NF. Idiopathic adulthood ductopenia. A
f h
i
h l
t ti li
di
d bili
i h
i
J H
t l 1. Zhao Z, Bao L, Yu X, Zhu C, Xu J, Wang Y, et al. Acute vanishing bile duct
syndrome after therapy with cephalosporin, metronidazole, and
clotrimazole: a case report. Medicine (Baltimore). 2017;96:e8009. 2. Nakanuma Y, Tsuneyama K, Harada K. Pathology and pathogenesis of
intrahepatic bile duct loss. J Hepatobiliary Pancreat Surg. 2001;8:303–15. 3. Ludwig J, Wiesner RH, LaRusso NF. Idiopathic adulthood ductopenia. A
cause of chronic cholestatic liver disease and biliary cirrhosis. J Hepatol. 1988;7:193–9. 4. Harris J, Cao S, Hile G, Eshaq M. Diffuse xanthomas in a patient with primary
biliary cholangitis and lipoprotein X. JAAD Case Rep. 2020;7:30–2. 5. Granito A, Muratori P, etc. Antinuclear antibodies as ancillary markers in
primary biliary cirrhosis. Expert Rev Mol Diagn. 2012;12(1):65–74. 6. Granito A, Muratori P, etc. Antinuclear antibodies giving the 'multiple
nuclear dots' or the 'rim-like/membranous’ patterns: diagnostic accuracy for
primary biliary cirrhosis. Aliment Pharmacol Ther. 2006;24(11–12):1575–83. 7. Gluud C, Christensen E. Ursodeoxycholic acid for primary biliary cirrhosis. Cochrane Database Syst Rev. 2002;1:CD000551. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Ursodeoxycholic acid should be used first for patients
with VBDS caused by PBC [7, 8]; for the patients with
high lipids or even cutaneous xanthomas, fenofibrate
should be added. Many patients with VBDS eventually
require
liver
transplantation
due
to
poor
capillary
regeneration. Data availability All data included in this study are available upon request by contact with
the corresponding author. Competing interests The authors declare that there are no conflict of interests. Consent for publication
f
d 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. Acknowledgements We thanks Dr. Baocheng Deng for the helpful discussions and assistance of
the diagnosis. Final diagnosis Liver biopsy pathology only: Vanishing bile duct syndro-
me.G2S2.This suggested liver fibrosis but not cirrhosis. Fig. 3 Hepatic tissue of IHC, immunohistochemistry for CK7. It can improve the diagnosis of VBDS and PBC more reliable Fig. 3 Hepatic tissue of IHC, immunohistochemistry for CK7. It can improve the diagnosis of VBDS and PBC more reliable Page 4 of 4 Page 4 of 4 Page 4 of 4 Jia et al. Diagnostic Pathology (2022) 17:57 Jia et al. Diagnostic Pathology (2022) 17:57 Jia et al. Diagnostic Pathology treatment for end-stage PBC.However in low income pa-
tients, liver transplantations will not be performed. Availability of data and materials Author details
1
d
d 1Graduate Studies, Liaoning University of Traditional Chinese Medicine,
Shenyang, China. 2Department of Gastroenterology, Affiliated Hospital of
Liaoning University of Traditional Chinese Medicine, Shenyang, China. 3The
First Affiliated Hospital, China Medical University, Shenyang, China. Received: 11 November 2021 Accepted: 30 April 2022 Authors’ contributions Lin Liu contributed significantly to analysis and manuscript preparation,
Yuebo Jia analyzed the data and wrote the manuscript. Baocheng Deng
contributed to the pathological diagnosis of the patient. Yu Huang and Jiaqi
Zhao analyzed and interpreted the patient data regarding the disease. Guang Bai helped perform the analysis with constructive discussions. All
authors read and approved the final manuscript. Yuebo Jia and Lin Liu are
co-first authors of the article.
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https://openalex.org/W2128447046
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Ca<sup>2+</sup>influx-linked protein kinase C activity regulates the β-catenin localization, micromere induction signalling and the oral–aboral axis formation in early sea urchin embryos
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Zygote
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Zygote 23 (June), pp. 426–446. c⃝Cambridge University Press 2014. The online version of this article is published within an
Open Access environment subject to the conditions of the Creative Commons Attribution licence <http://creativecommons.
org/licenses/by/3.0/>. Zygote 23 (June), pp. 426–446. c⃝Cambridge University Press 2014. The online version of this article is published within an
Open Access environment subject to the conditions of the Creative Commons Attribution licence <http://creativecommons. org/licenses/by/3.0/>. Zygote 23 (June), pp. 426–446. c⃝Cambridge University Press 2014. The online version of this article is published within an
Open Access environment subject to the conditions of the Creative Commons Attribution licence <http://creativecommons. org/licenses/by/3 0/> g
y
doi:10.1017/S0967199414000033
First Published Online 9 April 2014 Summary Sea urchin embryos initiate cell specifications at the 16-cell stage by forming the mesomeres, macromeres
and micromeres according to the relative position of the cells in the animal–vegetal axis. The most
vegetal cells, micromeres, autonomously differentiate into skeletons and induce the neighbouring
macromere cells to become mesoendoderm in the -catenin-dependent Wnt8 signalling pathway. Although the underlying molecular mechanism for this progression is largely unknown, we have
previously reported that the initial events might be triggered by the Ca2+ influxes through the egg-
originated L-type Ca2+ channels distributed asymmetrically along the animal–vegetal axis and through
the stretch-dependent Ca2+channels expressed specifically in the micromere at the 4th cleavage. In
this communication, we have examined whether one of the earliest Ca2+ targets, protein kinase C
(PKC), plays a role in cell specification upstream of -catenin. To this end, we surveyed the expression
pattern of -catenin in early embryos in the presence or absence of the specific peptide inhibitor of
Hemicentrotus pulcherrimus PKC (HpPKC-I). Unlike previous knowledge, we have found that the initial
nuclear entrance of -catenin does not take place in the micromeres, but in the macromeres at the 16-cell
stage. Using the HpPKC-I, we have demonstrated further that PKC not only determines cell-specific
nucleation of -catenin, but also regulates a variety of cell specification events in the early sea urchin
embryos by modulating the cell adhesion structures, actin dynamics, intracellular Ca2+ signalling, and
the expression of key transcription factors. Keywords: -catenin, Ca2+ influx, Oral–aboral axis formation, Protein kinase C, Sea urchin embryo Ca2+ influx-linked protein kinase C activity regulates the -catenin
localization, micromere induction signalling and the oral–aboral
axis formation in early sea urchin embryos Ikuko Yazaki1,7, Toko Tsurugaya3, Luigia Santella4, Jong Tai Chun4, Gabriele Amore4,
Shinichiro Kusunoki5, Akiko Asada2, Tatsuru Togo6 and Koji Akasaka3
Department of Biological Sciences, Tokyo Metropolitan University, Hachiohji-shi, Tokyo, Japan; Misaki Marine Biological
Station, University of Tokyo, Miura, Japan; Stazione Zoologica Anton Dohrn, Napoli, Italy; LSL Co. Ltd, Nerima-ku Tokyo,
Japan; and Department of Anatomy, St Marianna University School of Medicine, Sugao, Kawasaki, Kanagawa, Japan Ikuko Yazaki1,7, Toko Tsurugaya3, Luigia Santella4, Jong Tai Chun4, Gabriele Amore4,
Shinichiro Kusunoki5, Akiko Asada2, Tatsuru Togo6 and Koji Akasaka3
Department of Biological Sciences, Tokyo Metropolitan University, Hachiohji-shi, Tokyo, Japan; Misaki Marine Biological
Station, University of Tokyo, Miura, Japan; Stazione Zoologica Anton Dohrn, Napoli, Italy; LSL Co. Ltd, Nerima-ku Tokyo,
Japan; and Department of Anatomy, St Marianna University School of Medicine, Sugao, Kawasaki, Kanagawa, Japan Date submitted: 16.08.2013. Date revised: 11.11.2013. Date accepted: 23.12.2013 Date submitted: 16.08.2013. Date revised: 11.11.2013. Date accepted: 23.12.2013 1All
correspondence
to:
Ikuko Yazaki.
Department
of
Biological
Sciences,
Tokyo
Metropolitan
University,
Minamiohsawa 1–1, Hachiohji-shi, Tokyo 192–0397, Japan.
Tel:
+81–426–76–6194.
Fax:
+81–426–77–2559.
e-mail:
yazaki-ikuko@tmu.ac.jp
2Department of Biological Sciences, Tokyo Metropolitan
University, Minamiohsawa 1–1, Hachiohji-shi, Tokyo 192–
0397, Japan.
3Misaki Marine Biological Station, University of Tokyo,
Miura, Japan.
4Stazione Zoologica Anton Dohrn, Villa Comunale 1–80121
Napoli, Italy.
5LSL Co. Ltd, Nerima-ku Tokyo 178–0061, Japan.
6Department of Anatomy, St. Marianna University School of
Medicine, Sugao, Kawasaki, Kanagawa 216–8511, Japan.
7Present address: Bessyo 2–51–1-1105, Hachiohji, Tokyo 192–
0363, Japan. y
jp
2Department of Biological Sciences, Tokyo Metropolitan
University, Minamiohsawa 1–1, Hachiohji-shi, Tokyo 192–
0397, Japan. Introduction We have
found that: (i) PKC activity regulates the nuclear
localization of -catenin by means of the cortical
actin filaments; (ii) PKC controls the early endoderm
induction signal of micromeres by modulating the
intercellular adhesion system and intracellular Ca2+
signalling; and (iii) the nuclear -catenin activates
not only the endodermal genes but also ectodermal
genes, depending on their timing and location, without
interfering with the formation of the animal–vegetal
and oral–aboral axes. accepted that these abilities of the micromeres coincide
with nuclear localization of -catenin at the 16-cell
stage (Logan et al., 1999), although it is still unclear
exactly how the -catenin enters the nuclei (Davidson
et al., 2002). -Catenin is a multifunctional protein that
is involved not only in cell–cell adhesion but also in
gene expression (MacDonald et al., 2009). In epithelial
cells, the cytoplasmic domains of cadherin link to the
cortical actin cytoskeleton via -, - and -catenins
(Takeichi, 1991; Gumbiner, 1996). In several cell types,
the ‘myristoylated alanine-rich C kinase substrate’
(MARCKS) cross-links actin filaments and thereby
anchors the actin network to the plasma membrane
(Eliyahu et al., 2005). In sea urchin eggs, cadherin
and -catenin are co-localized in the cytoplasm and
near the plasma membrane, and have been shown to
accumulate at the sites of cell–cell contact following
fertilization and cleavage (Miller & McClay, 1997a,
b). Thus, the subcellular localization of -catenin is a
significant marker for the control of cell division and
gene expression. In many animals, inositol phospholipid turnover is
involved in the regulation of many cellular processes. Phospholipase C (PLC) hydrolyses phosphatidylinos-
itol 4,5-bisphosphate (PIP2) in the plasma membrane
to produce 1,2-diacylglycerol (DAG) and inositol
1,4,5-trisphosphate (InsP3). It has been shown that
DAG activates protein kinase C (PKC), and that
InsP3 promotes an increase in intracellular Ca2+
concentration (Nishizuka, 1984; Berridge, 1993). PKC
from sea urchin embryos was cloned from Lytechinus
pictus (Rakow & Shen, 1994), and its roles have been
well demonstrated during egg activation (Shen &
Buck, 1990), skeletogenesis (Mitsunaga et al., 1990)
and apoptosis in gastrulae (Dickey-Sims et al., 2005). Livingston & Wilt (1992, 1995) suggested that the
InsP3-PKC signalling pathway played an important
role in vegetalizing the ectodermal cells by showing
that 10–15 min exposure of the embryos at the 16-cell
stage to a PKC activator 12-O-tetradecanoyl phorbol-
13-acetate (TPA) is sufficient to vegetalize the embryos. Animals and gametes Individual animals of Hemicentrotus pulcherrimus were
supplied from the Misaki Marine Biological Station,
the University of Tokyo, and Paracentrotus lividus from
the Stazione Zoologica Anton Dohrn in Naples, Italy. Gametes were obtained by intracoelomic injection
of 0.5 M KCl for H. pulcherrimus. P. lividus eggs
were released from the dissected gonad into in
natural seawater filtered with a Millipore-filter set. Eggs were washed and inseminated in seawater that
contained 5 mM p-aminobenzoic acid (PABA-SW). Fertilization envelopes were removed by pipetting,
and the embryos were cultured in filtered seawater at
15°C or 18°C (H. pulcherrimus), or at room temperature
(20–23°C) for P. lividus. Introduction In addition, the presumptive ectoderm blastomeres
could be vegetalized by a lithium ion (Li+) that
modulates the metabolism of inositol phospholipids,
and the vegetalizing effect of lithium was reversed
by preinjection of the blastomere with myo-inositol
(Livingston & Wilt, 1995). However, Li+ is also known
to inhibit glycogen synthase kinase 3 (GSK-3), which
regulates the activity of the transcription factors and
-catenin (Li et al., 2007), and to increase the levels
of DAG and PKC activity (Drummond and Raeburn,
1984). Introduction 1All
correspondence
to:
Ikuko Yazaki. Department
of
Biological
Sciences,
Tokyo
Metropolitan
University,
Minamiohsawa 1–1, Hachiohji-shi, Tokyo 192–0397, Japan. Tel:
+81–426–76–6194. Fax:
+81–426–77–2559. e-mail:
yazaki-ikuko@tmu.ac.jp
2 In sea urchin embryos, cell specifications take place
along the two embryonic axes: the animal–vegetal (AV)
axis and the oral–aboral (OA) axis. The AV axis can
be traced back to the unfertilized egg (Boveri, 1901;
Hörstadius, 1939), and the OA axis to the zygote
(Coffman et al., 2004). By the end of the 4th division,
three different sizes of blastomeres comprising eight
mesomeres, four macromeres and four micromeres
are formed and eventually become the ectoderm,
the mesoendoderm and the mesoderm, respectively. The vegetal cells, micromeres, emit signals for the
macromeres
to
form
the
endoderm
(Ransick
&
Davidson, 1993), while they themselves develop into
skeletogenic cells (Okazaki, 1975). It is generally 2Department of Biological Sciences, Tokyo Metropolitan
University, Minamiohsawa 1–1, Hachiohji-shi, Tokyo 192–
0397, Japan. 3 3Misaki Marine Biological Station, University of Tokyo,
Miura, Japan. p
4Stazione Zoologica Anton Dohrn, Villa Comunale 1–80121
Napoli, Italy. p
y
5LSL Co. Ltd, Nerima-ku Tokyo 178–0061, Japan. 6Department of Anatomy, St. Marianna University School of
Medicine, Sugao, Kawasaki, Kanagawa 216–8511, Japan. https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press Ca2+-PKC signalling in sea urchin cell specification 427 Ca2+oscillation for at least 10 min (Yazaki, 2001; Yazaki
et al., 2004). It is plausible that these Ca2+ influxes or
their amplified signals could lead to PKC activation. In
the present paper, to elucidate the role of PKC activity
in the functions of micromere and in the development
of embryo, we have used a PKC pseudosubstrate
inhibitor (HpPKC-I) constructed from the amino acid
sequence of the Hemicentrotus pulcherrimus PKC and a
specific antibody against -catenin of H. pulcherrimus
(Hp-catenin). First of all, contrary to a previous report
(Logan et al., 1999), we found that the initial nuclear
entrance of -catenin takes place in the macromeres,
not in the micromeres, as judged by the co-staining of
-catenin and the DNA-binding dye propidium iodide
(PI). Secondly, we have found that the inhibition of
PKC leads to appreciable deregulation of the cortical
actin cytoskeleton in the blastomeres. In addition,
we have examined the morphology and the gene
expression pattern of embryos after the treatment of
with HpPKC-I in the early cleavage stage. https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press Chemical treatments of embryos LiCl was added to the 4-cell stage H. pulcherrimus
embryos at the final concentration of 40 mM as
described by Ciapa & Maggio (1993), and a batch
of
the
embryos
were
grown
up
to
the
16-cell
stage (2.5 h) or until vegetalization (5 h) in the
LiCl-seawater. PKC activator, phorbol-12-myristate-
13-acetate ester (PMA; CALBIOCHEM) was added 5
min before the 4th cleavage at the final concentration
of 8 nM (Livingston & Wilt, 1992) and incubated
for 20 min. HpPKC-I was added to the embryos
20 min before the 4th division until 20 min after
the 6th division with the final concentration of
5–7
M. A
Ca2+-dependent
PKC-specific
inhib-
itor, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-
5-oxo-5-idolo (2,3-a)pyrrolo(3,4-c)-carbazole (Gö6976;
CALBIOCHEM) was applied at a concentration of
400 nM during the same period as the treatment
with HpPKC-I. Gadolinium chloride (GdCl3·6H2O;
Sigma), a stretch-activated calcium channel blocker
(Yang & Sacks, 1989), was used for suppression of
Ca2+ elevation in the micromeres (Yazaki et al., 2004)
by application at the concentration of 10–30 M from Immunoblotting Whole lysate of H. pulcherrimus embryos was prepared
by sonication in Laemmli’s sample buffer (62.5 mM
Tris–HCl,
pH
6.8,
2%
sodium
dodecyl
sulphate
(SDS), 10% glycerol, 0.005% bromophenol blue, 5%
-mercaptoethanol) and boiled for 5 min. SDS-PAGE
was
carried
out
with
10%
polyacrylamide
gels. Proteins were transferred to polyvinylidene difluoride
(PVDF) membranes (Millipore, Bedford, MA, USA)
using a semi-dry blotting apparatus. Membranes
were
probed
with
the
rabbit
antiserum
against
Hp-catenin (1:10,000 dilution) and the horseradish
peroxidase (HRP)-conjugated swine anti-rabbit IgG
(Dako, Glostrup, Denmark). Immunoreactivity signals
were detected with the Millipore Immobilon western
chemiluminescent HRP substrate (Millipore). Preparation of HpPKC inhibitor (HpPKC-I) and
anti-Hp-catenin antibody Sea urchin embryos exhibit polarized Ca2+ influxes
in every cell division after fertilization (Yazaki et al.,
1995; Dale et al., 1997). The micromere-specific Ca2+
influxes occur at the 4th cleavage and are followed by The HpPKC pseudosubstrate domain (FARRGALRQ)
was synthesized according to GenBank information on https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press Ikuko Yazaki et al. 428 H. pulcherrimus PKC (AB699356, amino acids 11–19)
and myristoylated at the N-terminus of the peptide
(Peptide Institute Inc., Osaka, Japan) to enable it
to permeate cell membranes (Eichholtz et al., 1993). Recombinant protein of Hp-catenin was expressed
bacterially by using the sequence data from the cDNA
clone (AB699355) isolated from the H. pulcherrimus
gastrula cDNA library (Fuchikami et al., 2002). The
cDNA fragment encoding the N-terminal region of
Hp-catenin (1–250 amino acids) was inserted into
the pET42b/GST vector and expressed in E. coli BL21
(Stratagene). The overexpressed protein was extracted
and purified by affinity chromatography using a ‘GST-
Bind Resin’ column (Novagen). The GST-tag was
removed using the Thrombin Cleavage Capture Kit
(Novagen), and the remaining polypeptide was used
to immunize rabbits to generate polyclonal antibodies
against Hp-catenin. H. pulcherrimus PKC (AB699356, amino acids 11–19)
and myristoylated at the N-terminus of the peptide
(Peptide Institute Inc., Osaka, Japan) to enable it
to permeate cell membranes (Eichholtz et al., 1993). Recombinant protein of Hp-catenin was expressed
bacterially by using the sequence data from the cDNA
clone (AB699355) isolated from the H. pulcherrimus
gastrula cDNA library (Fuchikami et al., 2002). The
cDNA fragment encoding the N-terminal region of
Hp-catenin (1–250 amino acids) was inserted into
the pET42b/GST vector and expressed in E. coli BL21
(Stratagene). The overexpressed protein was extracted
and purified by affinity chromatography using a ‘GST-
Bind Resin’ column (Novagen). The GST-tag was
removed using the Thrombin Cleavage Capture Kit
(Novagen), and the remaining polypeptide was used
to immunize rabbits to generate polyclonal antibodies
against Hp-catenin. dilution of the stock solution) in 0.1 M Na-phosphate,
0.1 M NaCl, 5 mM NaN3, pH 7.5. In the last
10 min of the immuno-reaction, a DNA-binding dye
PI (KPL) was added at the 1:600 dilution ratio of the
stock solution (1 mg/ml PBS). After four washes in
PBS, the specimens were placed in a well on a glass
slide surrounded by lens paper soaked in mineral oil
and were monitored with an Olympus laser-scanning
confocal microscope (LSM-GB200). Visualization of F-actin P. lividus embryos were used for the observation
of F-actin in accordance with the method described
previously (Kyozuka et al., 2008). Control embryos
and the embryos treated with PKC modulators were
fixed for 30 min in seawater that contained 4%
formaldehyde and 0.1% glutaraldehyde, starting from
20 min after the 4th cleavage. After three cycles of rinse
in the washing buffer (50 mM HEPES, pH 7.0, 50 mM
PIPES, 600 mM mannitol, 3 mM MgCl2) that contained
0.1% Triton X-100, the embryos were incubated for
30 min with 3–10 U/ml of Alexa Fluor 488-conjugated
phalloidin in PBT (137 mM NaCl, 2.7 mM KCl, 15 mM
KH2PO4, 8 mM NaHPO4, 0.1% Triton X-100, pH 7.2)
and then rinsed in PBT for three times. All steps were
performed at room temperature. The embryos stained
with fluorescent phalloidin were observed under an
Olympus Fluoview 200 laser-scanning microscope
using the BP510540 emission filter. Transmitted light
and fluorescent images were acquired from the same
confocal plane. Results Total RNA was extracted from Strongylocentrotus
purpuratus embryos at pertinent developmental stages
with the RNeasy mini kit columns (Qiagen), and
subjected to cDNA synthesis by use of the SuperScript
First-Strand
Synthesis
System
(Invitrogen). Polymerase chain reaction (PCR) was carried out with
specific primers synthesised from mRNA sequences
of target genes in Strongylocentrotus purpuratus. The
sequences (5′→3′) of each primer set (forward and
reverse) are as follows: Gsc, F_ggattgatggcgatacgact
and R_gcagaggaagacaccgagac; Delta, F_tgtgcaataac-
cagggtgaa and R_tgcaagagtttggtgagtcg; Nodal, F_ga-
cctgctctggacctcttg and R_agtggtcgaagtttggttgg; Lim,
F_gtatctttcgggcaaggaca
and
R_ctcttccatcggatgtggat;
Gcm, F_atcgttcaccatcgacaaca and R_aggcatggtaggca-
gctaag; FoxG, F_gtgaaagtccctcgccatta and R_tcagctt-
acccgttgtaccc; Brain 1/2/4, F_gacgcattaagctcggctac and Statistical analysis Paired
t-test
and
one-way
analysis
of
variance
(ANOVA) with Tukey’s multiple comparison test were
performed using Prism 6.0 software (Graph Pad
Software); P-values less than 0.05 were considered to
be statistically significant. Ca2+ measurements Paracentrotus lividus eggs were dejellied with acid
seawater and washed in 10 ml seawater that contained
3 mg p-aminobenzoic acid (PABA; Sigma) and then
lined on a protamine sulphate-coated dish filled with
PABA seawater. A mixture of 10 mg/ml of calcium
green R⃝BAPTA conjugated with 10 kDa of dextran
(Molecular Probe) and 3 mg/ml of rhodamine red was
prepared in the injection buffer (0.45 M KCl, 10 mM
HEPES, pH 7.0), and microinjected into eggs within 5
min after fertilization. The eggs were then collected by
a mouth pipette and transferred to agar-coated dishes
filled with Millipore-filtered natural seawater. About
10
min
before
the
2nd
cleavage,
zygotes
were
transferred into the Ca2+-free seawater (CFSW; 530
mM NaCl, 10 mM KCl, 27 mM MgCl2, 29 mM MgSO4,
2 mM NaHCO3, pH 8.0). Blastomeres were separated at
the 4-cell stage by gentle pipetting. These cells give rise
to quarter embryos, which are able to configure their
cells as 1-cell layer: two mesomeres, one macromere
and one micromere to enable photometry on each cell. Ca2+ imaging was performed in the period from 5 to
20 min after the 4th cleavage, the time of the
micromere-specific
Ca2+elevation
had
established
in normal embryos, using a charged coupled device
(CCD) camera with a ×63 objective and MetaMorph
analyzing software. Calcium levels were expressed as
the ratios of fluorescence intensities between calcium
green R⃝BAPTA (Ex 485 nm/Em 535 nm for 200 ms)
and the internal control rhodamine red (Ex 555
nm/Em 565 nm for 100 ms exposure). When a time
course measurement was needed, the time resolution
was 5 s. Whole-mount immunostaining Following the methodology by Logan et al. (1999),
embryos of H. pulcherrimus were fixed in artificial
seawater (ASW; 425 mM NaCl, 9.3 mM KCl, 10 mM
CaCl2, 24.5 mM MgCl2, 25.5 mM MgSO4, 2.5 mM
NaHCO3, pH 8.0) containing 2% paraformaldehyde
for 10 min at room temperature, and then passed
briefly through 100% MeOH on ice to permeabilize
the cell membrane. After three cycles of washing
in phosphate-buffered saline (PBS) by sedimentation,
embryos were treated with 3% bovine serum albumin
in PBS containing 0.1% Tween 20 (TPBS) for 30 min,
and then incubated with rabbit polyclonal antiserum
against Hp-catenin (1:10,000 dilution) for 1 h with
gentle agitation. Following one wash in TPBS and
three subsequent washes in PBS, embryos were
incubated for 1 h in a solution that contained the
secondary antibody: the Alexa Fluor 488-conjugated
anti-rabbit IgG (Molecular Probe) at 1 g/ml (1:2000 https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press 429 Ca2+-PKC signalling in sea urchin cell specification R_aagctcaactgcaaggcttc; FoxA, F_caattcgccacagtctctca
and R_ctcggacatattcccagcat; NK22, F_actttggttcggt-
tggtcag and R_ttcacggaaaagctcgtctt; Coquilette, F_ct-
gaccaggcatttcatgtg and R_gtgcaccagggcactatttt; Blimp,
F_cagggaatctccgattcaaa
and
R_tggcacctcctcttctctgt;
Deadringer, F_cagagctgcaatcagccata and R_gggaacca-
gaaggggtatgt;
Sm
30,
F_caaccagtctggatcggtct
and
R_ataggggccatcattccttc; Sm 50, F_gccttttcgcaggataa-
tca and R_ccgtttggataagctggtgt; G-cadhedrin, F_cg-
gtggaacttgtgttgatg and R_cgtaaccgttgttgaagctg; Bra,
F_aggacacggaattgtggaag
and
R_gcgatgttagccgagag-
aac. The automated thermal cycler used was the
Chromo4TM
Real-Time Detector (Biorad) utilizing
Sybr Green in the reaction mixture that is intercalated
into
double-stranded
PCR
amplicons
and
emits
fluorescence. For each query transcript, the cycle
threshold value (Ct) was normalized to the Ct value
obtained
on
the
same
cDNA
preparations
with
ubiquitin primers: i.e., Ct = Ct (sample)/Ct (ubiquitin). To
evaluate the effect of the PKC modulator on the
transcript level of each query gene, the difference
(D)
between
the
Ct
values
of
the
control
and
experimental samples were obtained, i.e., D = Ct (control)
– Ct (experimental). Thus, positive D values indicate an
increase in the transcript after treatment. To convert D
into the ratio (R) of the transcript concentrations, we
applied the formula R = 1.94 × D, where 1.94 is the
mass amplification coefficient per PCR cycle for this
particular case. 10 min before the 1st, 3rd, 4th and 7th cleavages,
respectively. Specificity of the sea urchin-specific PKC inhibitor
and anti -catenin antibody To study the roles of the PKC pathway in the early
development of Hemicentrotus pulcherrimus embryos,
we prepared a specific peptide inhibitor of PKC and
antibody against -catenin, which is a potential down-
stream target of PKC in the micromeres (Livingston
& Wilt, 1992, 1995; Wikramanayake et al., 2004). As
described in Materials and methods, the peptide in-
hibitor elected from H. pulcherrimus PKC (HpPKC) was
completely identical to Lytechinus pictus PKC (LpPKC;
Rakow & Shen, 1994), differing from the human PKC
pseudosubstrate by only one amino acid (Fig. 1A). When HpPKC-I (7 M) was applied to the embryo
20 min before cleavage, the next cell division stopped, https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press 430 Ikuko Yazaki et al. Figure 1 Inhibition of PKC interferes with the normal distribution of actin cytoskeleton in blastomeres. (A) Preparation of the
HpPKC inhibitor (HpPKC-I). The amino acid sequence of Hemicentrotus pulcherrimus PKC was aligned with Lytechinus pictus
(LpPKC) and human PKC (HuPKC) (1–68) to define the pseudosubstrate region (underlined nine residues). Asterisks denote
identical amino acid residues. The synthetic peptides of the pseudosubstrate were myristoylated to use as HpPKC-I. (B) Effects
of PKC on actin distribution. F-actin was visualized with Alexa Fluor 488-conjugated phalloidin; 8 nM PMA was added to the
embryos (P. lividus) for 20 min starting from 7 min before the 4th division; 5 M of HpPKC-I was added 20 min before the
4th division. Optical (upper) and fluorescent images (lower) were taken from the same confocal plane. In either case, cell
specifications were modified, but cell division progressed normally. Figure 1 Inhibition of PKC interferes with the normal distribution of actin cytoskeleton in blastomeres. (A) Preparation of the
HpPKC inhibitor (HpPKC-I). The amino acid sequence of Hemicentrotus pulcherrimus PKC was aligned with Lytechinus pictus
(LpPKC) and human PKC (HuPKC) (1–68) to define the pseudosubstrate region (underlined nine residues). Asterisks denote
identical amino acid residues. The synthetic peptides of the pseudosubstrate were myristoylated to use as HpPKC-I. (B) Effects
of PKC on actin distribution. F-actin was visualized with Alexa Fluor 488-conjugated phalloidin; 8 nM PMA was added to the
embryos (P. lividus) for 20 min starting from 7 min before the 4th division; 5 M of HpPKC-I was added 20 min before the
4th division. Optical (upper) and fluorescent images (lower) were taken from the same confocal plane. In either case, cell
specifications were modified, but cell division progressed normally. Specificity of the sea urchin-specific PKC inhibitor
and anti -catenin antibody although the nuclear division progressed at the normal
schedule (Fig. 2C). At a lower dose (5 M), we found
that treatment with HpPKC-I strikingly reorganized
the actin cytoskeleton in the blastomeres. Whereas the
Alexa 488-phalloidin-stained F-actin filaments in the
normal blastomeres were localized predominantly at
the plasma membrane, the distribution of F-actin
in the blastomeres of the HpPKC-I-treated embryos
was shifted more towards the cytoplasm (Fig. 1B). In
contrast, when the myristoylated pseudosubstrate of
human PKC differing by only one amino acid was
used (Calbiochem), cell division of sea urchin embryos
was not affected, even at 28 M (data not shown). This finding suggested that the inhibitory effect on the
target enzyme requires highly stringent specificity. Antibody to Hp-catenin was prepared against amino
acids 1–250, in consideration of the antigenic region of
Lv-catenin (1–173 amino acids, underlined). In this
region, four residues of serine/threonine (asterisked)
matched the consensus substrate sequences of the
glycogen synthase kinase-3 (GSK-3) (Fig. 2A). As
expected, when a western blot of the proteins extracted
from H. pulcherrimus eggs was probed with the Hp-
catenin antibody, a single band of 103 kDa was
recognized as Hp-catenin (Fig. 2B). To the best of
our knowledge, this finding is the first information for
the molecular weight of sea urchin -catenin. Taken
together, these results assured that our PKC inhibitor
and anti -catenin antibody were highly specific. As
to
the
specificity
of
the
anti-Hp-catenin
antibody, we used a part of the Hp-catenin as
the epitope. To this end, the homologous protein to
Lytechinus variegatus -catenin (Lv-catenin) (Miller &
McClay, 1997a) was cloned and verified by sequence
comparison. As shown in Fig. 2A, the amino acid
sequence of Hp-catenin (825 amino acids) was 95%
homologous to that of Lv-catenin (821 amino acids). https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press Hp-catenin initially nucleated in the macromeres
at the 16-cell stage Indeed, Hp-
catenin immunoreactivity was present in the spindle
regions but absent in the astral regions (Fig. 3B, b). (Miller & McClay, 1997a). From the 16-cell stage, the
cell cycle of the embryo became asynchronous; the
micromeres took longer time to arrive at the next
(5th) cleavage than other blastomeres. To monitor the
mitotic phase of blastomeres, every embryo stained
with Hp-catenin antibody was counterstained with
a DNA-binding dye PI. At prophase, the PI stain
displayed a thread or spireme to construct chro-
mosomes (Fig. 3A1, a1). Interestingly, Hp-catenin
appeared to be concentrated at the place where
amphiaster is located (Wilson, 1937). At metaphase,
the chromosomal plate was centred while spindles
were formed. At anaphase, chromatids were separated
within the spindles (Fig. 3B1, b1). We found Hp-
catenin either surrounding the equatorial plate of
metaphase chromosomes (the right cell marked with
asterisk in Fig. 3B1, B2) or localized in the space
between the separating chromosomes (the left cell
marked with asterisk in Fig. 3B1, B2). In the latter case,
-catenin was scarce near the mitotic asters. In Fig. 3C,
four micromeres in the centre were evident with
metaphase chromosomes, but the eight surrounding
cells were already in telophase. When the chromo-
somes construct the nuclei in the newly divided cells
at telophase, the Hp-catenin was evidently outside
the constructing nuclei (Fig. 3c1–c3). In Fig. 3D, the
outer eight macromere derivatives are in interphase,
and Hp-catenin accumulated in the interphase nuclei
(Fig. 3d1, d2). Hp-catenin changed its subcellular dis-
tribution during mitosis presumably by its interaction
with the actin cytoskeletons through cadherin (Bienz,
2005) rather than with microtubules. Indeed, Hp-
catenin immunoreactivity was present in the spindle
regions but absent in the astral regions (Fig. 3B, b). were in metaphase with more condensed stain with
PI, while Mac had already divided and attained the
telophase. It is noticeable that -catenin accumulated
around the newly formed metaphase chromosomal
plates of mic (Fig. 4C1, C2). When the Mac and
meso had completed the 5th division and formed
interphase nuclei, the micromeres belatedly entered
anaphase of the 5th division, displaying nuclear--
catenin-like distribution (Fig. 4D). Nuclear -catenin
was still detected only in the daughter cells of the
macromeres. At the 56-cell stage (Fig. 4E), four small
(s-mic) and four large micromeres (l-mic) were formed,
and the macromere descendants were located in the
veg1 and veg2 cell layers. Hp-catenin initially nucleated in the macromeres
at the 16-cell stage Nuclear -catenin was now
detected in the interphase nuclei of both s-mic and l-
mic, and as well as in the veg2 cell layer. A small weak
signal seemed to be present in the nuclei of veg1 cells. However, nuclei of animal cells (an) always exhibited
no -catenin signal. (shaded amino acid residues). The consensus serine/threonine targets for GSK-3 were marked with asterisks. Hp-catenin
antibody was prepared by using the underlined region (1–250 amino acids), which included the epitope for Lv-catenin
antibody (1–173 amino acids). (B) Western blot analysis with anti-Hp-catenin antibody: lane 1, molecular weight marker;
lane 2, cell lysate prepared from the fertilized eggs of H. pulcherrimus; lane 3, the blot stained with Ponceau-S. Hp-catenin
protein sized 103 kDa was marked with an arrow. (C) 7 M HpPKC-I inhibits cell division. HpPKC-I was applied to H.
pulcherrimus embryos from 20 min before the 4th cleavage. Embryos were fixed when untreated embryos arrived at the 32-cell
stage, and stained with the DNA dye PI (red) and Hp-catenin antibody (green). Cytokinesis was blocked, but nuclear division
progressed normally. Hp-catenin initially nucleated in the macromeres
at the 16-cell stage In the normal sea urchin embryos, we found Hp-
catenin protein localized in the cytoplasm and in the
apical and intercellular regions of plasma membrane
by the 8-cell stage (Fig. 2C). This result is consistent
with immunostaining with anti-Lv-catenin antibody https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press Ca2+-PKC signalling in sea urchin cell specification 431 Figure 2 Specificity of the antibody against Hp-catenin. (A) Deduced amino acid sequences of -catenin from H. pulcherrimus
(Hp) and Lytechinus variegatus (Lv; Miller & McClay, 1997b) were aligned. Hp-catenin and Lv-catenin were 95% identical Figure 2 Specificity of the antibody against Hp-catenin. (A) Deduced amino acid sequences of -catenin from H. pulcherrimus
(Hp) and Lytechinus variegatus (Lv; Miller & McClay, 1997b) were aligned. Hp-catenin and Lv-catenin were 95% identical https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press Ikuko Yazaki et al. 432 (Miller & McClay, 1997a). From the 16-cell stage, the
cell cycle of the embryo became asynchronous; the
micromeres took longer time to arrive at the next
(5th) cleavage than other blastomeres. To monitor the
mitotic phase of blastomeres, every embryo stained
with Hp-catenin antibody was counterstained with
a DNA-binding dye PI. At prophase, the PI stain
displayed a thread or spireme to construct chro-
mosomes (Fig. 3A1, a1). Interestingly, Hp-catenin
appeared to be concentrated at the place where
amphiaster is located (Wilson, 1937). At metaphase,
the chromosomal plate was centred while spindles
were formed. At anaphase, chromatids were separated
within the spindles (Fig. 3B1, b1). We found Hp-
catenin either surrounding the equatorial plate of
metaphase chromosomes (the right cell marked with
asterisk in Fig. 3B1, B2) or localized in the space
between the separating chromosomes (the left cell
marked with asterisk in Fig. 3B1, B2). In the latter case,
-catenin was scarce near the mitotic asters. In Fig. 3C,
four micromeres in the centre were evident with
metaphase chromosomes, but the eight surrounding
cells were already in telophase. When the chromo-
somes construct the nuclei in the newly divided cells
at telophase, the Hp-catenin was evidently outside
the constructing nuclei (Fig. 3c1–c3). In Fig. 3D, the
outer eight macromere derivatives are in interphase,
and Hp-catenin accumulated in the interphase nuclei
(Fig. 3d1, d2). Hp-catenin changed its subcellular dis-
tribution during mitosis presumably by its interaction
with the actin cytoskeletons through cadherin (Bienz,
2005) rather than with microtubules. PKC regulates nuclear entrance of Hp-catenin (C) Cells at telophase in the 28-cell stage
embryo from the ventral view. Macromeres have just divided to be in telophase, showing an irregular, chambered structure
in the process of reconstruction of the daughter nuclei. (D) A ventral view of 28-cell stage embryo. Macromeres were at
interphase. Fi
3 Di t ib ti
f H
t
i
d
i
it
i
E
b
i
ll
t
i
it ti
t
(H
l h
i
) 3 Di t ib ti
f H
t
i
d
i
it
i
E
b
i
ll
t
i
it ti
t
(H
l h
i
) Figure 3 Distribution of Hp-catenin during mitosis. Embryonic cells at various mitotic stages (H. pulcherrimus) were stained
with Hp-catenin antibody (green) and PI (red). Cells marked with asterisks in each embryo of (A), (B), (C) and (D) respectively
were enlarged in the right-side panel (a, b, c, d). (A) At the 56-cell stage, most macromere and all mesomere derivatives shown
were in prophase. (B) Cells at metaphase and anaphase in the 28-cell stage embryo. (C) Cells at telophase in the 28-cell stage
embryo from the ventral view. Macromeres have just divided to be in telophase, showing an irregular, chambered structure
in the process of reconstruction of the daughter nuclei. (D) A ventral view of 28-cell stage embryo. Macromeres were at
interphase. AV axis polarity (Yazaki et al., 1995). Ca2+ currents
were highest in mesomeres, but nearly absent in
micromeres. Another type of Ca2+ influx starts to take
place at the 4th cleavage through a stretch-dependent
Ca2+
channels
in
the
micromere
(Yazaki,
2001). This micromere-specific Ca2+influx was followed by
Ca2+oscillations through the InsP3/PKC signalling
pathway, and thereby attained the intracellular Ca2+
level in the micromeres to about 10 % higher than
in other blastomeres (Yazaki et al., 2004). While
the Ca2+ increase is expected to activate the PKC
pathway, it is not well known whether PKC in turn
may contribute to modulate the intracellular Ca2+
levels in the micromeres. Here we have tested if
PKC plays a role in the micromere-specific Ca2+
elevation. Using a fluorescent Ca2+ indicator (see of all blastomeres (Fig. 5D, E). Embryos treated with
another PKC inhibitor Gö6976 produced virtually the
same results as in the embryos treated with HpPKC-I
(Fig. 5F). PKC regulates nuclear entrance of Hp-catenin Hence, whereas a pharmacological activator
of PKC mimics some of the effects displayed by Li+ in
terms of -catenin distribution, the peptide inhibitor
of the PKC interfered with the normal subcellular
distribution of -catenin, negating its cell-specific
nuclear localization. of all blastomeres (Fig. 5D, E). Embryos treated with
another PKC inhibitor Gö6976 produced virtually the
same results as in the embryos treated with HpPKC-I
(Fig. 5F). Hence, whereas a pharmacological activator
of PKC mimics some of the effects displayed by Li+ in
terms of -catenin distribution, the peptide inhibitor
of the PKC interfered with the normal subcellular
distribution of -catenin, negating its cell-specific
nuclear localization. PKC regulates nuclear entrance of Hp-catenin In the 28-cell embryo, nuclear localization of the
-catenin was evident preferentially in the cells at the
vegetal side, although the micromeres had not yet
arrived at interphase and therefore exhibited no clear
sign of nuclear -catenin (Fig. 4). When the embryos
were treated with LiCl, which is a well known vegetal-
izing agent, the overall immunoreactivity of -catenin
was increased throughout the cytoplasm (Fig. 5B), and
the nuclei were more densely stained in the cells at the
vegetal side in comparison with the control, although
the nuclei of the cells in the animal side were largely
devoid of -catenin (Fig. 5B2; Logan et al., 1999). Similarly, addition of the activator of PKC (phorbol
12-myristate 13-acetate, PMA; also known as TPA)
markedly increased -catenin immunoreactivity in
the cytoplasm and nucleus, and the cells in the animal
side were largely missing -catenin immunoreactivity
in the nucleus (Fig. 5C). Thus, the addition of LiCl
and PMA appeared to intensify the AV gradient of
nuclear -catenin, which is found in the control. By
contrast, 5 M HpPKC-I markedly increased -catenin
predominantly near the plasma membrane with the
concomitant decrease in the cytoplasm. In addition,
it was noted that -catenin was present in the nuclei Figure 4 shows the Hp-catenin distribution in
embryos from the 16- to 56-cell stages. At the 16-
cell stage, nuclear -catenin was usually detected
only in the macromeres (Mac) (Fig. 4A), although in
some batches of embryos it was detectable also in
the micromeres, but its fluorescence intensity was
always much weaker than in the macromeres (data
not shown). In the late 16-cell stage (Fig. 4B), the
macromeres and mesomeres entered the next cleavage
cycle (anaphase chromosomes were visible in Mac)
and the four micromeres (mic) were still in the
interphase. At the early 28-cell stage, the micromeres https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press 433 Ca2+-PKC signalling in sea urchin cell specification Figure 3 Distribution of Hp-catenin during mitosis. Embryonic cells at various mitotic stages (H. pulcherrimus) were stained
with Hp-catenin antibody (green) and PI (red). Cells marked with asterisks in each embryo of (A), (B), (C) and (D) respectively
were enlarged in the right-side panel (a, b, c, d). (A) At the 56-cell stage, most macromere and all mesomere derivatives shown
were in prophase. (B) Cells at metaphase and anaphase in the 28-cell stage embryo. Effects of HpPKC inhibitor on micromere-specific
Ca2+ rise and gastrulation Sea urchin embryos display two types of Ca2+influxes. L-type Ca2+ channels derived from the egg plasma
membrane function from the first cleavage and show https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press 434 Ikuko Yazaki et al. e 4 Localization of the nuclear -catenin in the 16-cell to 56-cell stage embryos. (A1–E1) double staining of
e (PI) (red) and -catenin antibody (green). (A2–E2) images of -catenin staining only. (A3–E3) Drawing
s to illustrate the nuclei and the contour lines of blastomeres. Abbreviations: mic; micromeres, Mac; macrom
meres. (A) H. pulcherrimus embryo at the 16-cell stage: all cells were at interphase. Nuclear -catenin was pre
nt in Mac. (B) Late 16-cell stage: mic remained at the interphase, but Mac progressed to anaphase. No -c
in mic nuclei. (C) Early 28-cell stage: Mac divided to the telophase, and mic formed chromosomal plate of m
t the 28-cell stage: mic at metaphase or anaphase. Mac and meso derivatives were at interphase. Nuclear -c
ed only in Mac derivatives. (E) At the 56-cell stage: s-mic, small micromeres; l-mic, large micromeres. Veg1 an
acromere derivatives locating at the animal side (an) or next to l-mic, respectively. All cells shown were at
uclear -catenin was detected in every cell except for ‘an (animal side)’ cells. Abbreviations: An, animal side; V Figure 4 Localization of the nuclear -catenin in the 16-cell to 56-cell stage embryos. (A1–E1) double staining of propidium
iodide (PI) (red) and -catenin antibody (green). (A2–E2) images of -catenin staining only. (A3–E3) Drawing of (A1–E1)
images to illustrate the nuclei and the contour lines of blastomeres. Abbreviations: mic; micromeres, Mac; macromeres, meso;
mesomeres. (A) H. pulcherrimus embryo at the 16-cell stage: all cells were at interphase. Nuclear -catenin was preferentially
present in Mac. (B) Late 16-cell stage: mic remained at the interphase, but Mac progressed to anaphase. No -catenin was
found in mic nuclei. (C) Early 28-cell stage: Mac divided to the telophase, and mic formed chromosomal plate of metaphase. (D) At the 28-cell stage: mic at metaphase or anaphase. Mac and meso derivatives were at interphase. Nuclear -catenin was
detected only in Mac derivatives. (E) At the 56-cell stage: s-mic, small micromeres; l-mic, large micromeres. Veg1 and Veg2 are
the macromere derivatives locating at the animal side (an) or next to l-mic, respectively. All cells shown were at interphase,
and nuclear -catenin was detected in every cell except for ‘an (animal side)’ cells. Effects of HpPKC inhibitor on micromere-specific
Ca2+ rise and gastrulation Abbreviations: An, animal side; Vg, vegetal
side. https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press Ca2+-PKC signalling in sea urchin cell specification 435 Figure 5 LiCl, PKC activator and PKC inhibitors affect the distribution of -catenin. Localization of -catenin (green) and
nuclear staining (red) in the H. pulcherrimus embryos at the 16-cell, 28-cell and 56-cell stages. (A) Control embryos. (B) Embryos
treated with 40 mM LiCl starting from the 4-cell stage. (C) Embryos exposed to PKC activator (8 nM PMA for 20 min starting
from 5 min before the 4th cleavage). (D, E) Embryos exposed to 5 M HpPKC-I starting from 20 min before the 4th division
(16-cell stage). (F) Embryos exposed to 400 nM Gö6976, a calcium-dependent PKC inhibitor, for the same period as HpPKC-I. Abbreviations: An, animal side; Vg, vegetal side; mic; micromeres, Mac; macromeres, meso; mesomeres. Figure 5 LiCl, PKC activator and PKC inhibitors affect the distribution of -catenin. Localization of -catenin (green) and
nuclear staining (red) in the H. pulcherrimus embryos at the 16-cell, 28-cell and 56-cell stages. (A) Control embryos. (B) Embryos
treated with 40 mM LiCl starting from the 4-cell stage. (C) Embryos exposed to PKC activator (8 nM PMA for 20 min starting
from 5 min before the 4th cleavage). (D, E) Embryos exposed to 5 M HpPKC-I starting from 20 min before the 4th division
(16-cell stage). (F) Embryos exposed to 400 nM Gö6976, a calcium-dependent PKC inhibitor, for the same period as HpPKC-I. Abbreviations: An, animal side; Vg, vegetal side; mic; micromeres, Mac; macromeres, meso; mesomeres. comparison with the control (P < 0.05; Table 1). In
line with this result, the addition of 400 nM Gö6976
(a pharmacological inhibitor of PKC) significantly
increased the Ca2+ levels in all blastomeres, and
suggested that PKC is involved in intracellular Ca2+
homeostasis. Interestingly, when the embryos were
treated with 5 M HpPKC-I, the disparity of the
Ca2+ levels in the three types of blastomeres was
abolished, as the level in the micromere was reduced
significantly (P < 0.05) to the levels in the macromeres
and mesomeres, which were virtually unchanged in
comparison with the controls (Table 1). Materials and methods), we have monitored changes
in the intracellular Ca2+ levels 5–10 min after the 4th
cleavage. The Ca2+ levels of each blastomere were
monitored by single photometry at the 16-cell stage
(Table 1). HpPKC inhibitor enlarged the oral ectoderm in both
H. pulcherrimus and P. lividus embryos without
affecting mesoendoderm specification We have studied how HpPKC-I would affect the
development of the embryo at later stages. To this end,
embryos of H. pulcherrimus and P. lividus were treated
with 5 M HpPKC-I (Fig. 7). In comparison with the
control, the embryos treated with HpPKC-I displayed:
(i) disruptions of the cell-cell contact in the early
blastula; (ii) delay of gastrulation; and (iii) enlargement
of the oral ectoderm in the pluteus. However, the gut
developed normal in these embryos, and the pigment
cells were well discernible. When quartet micromeres
isolated from a 16-cell stage embryo were recombined
to an animal cap, the plutei that derived from the
recombinant embryo displayed a normal distribution
of alkaline phosphatase-positive cells (gut marker) and
formation of functional guts regardless of pretreatment
with HpPKC-I (data not shown). Thus, HpPKC-
I does not appear to interfere with the ability of
micromeres to induce mesoderm. However, HpPKC-I
caused morphological shifts at the pluteus stage. When
the skeletal size of plutei was measured with regard to
the post-oral rod (por), body rod (br) and antero-lateral
rod (alr) (nomenclature identified in Fig. 7C), the total
length of the rod structures in the embryos treated with
HpPKC-I was about 10% reduced compared with the
control embryos, while the skeleton supporting oral
ectoderm, alr, remained the same (Table 2). Thus, the Similar to finding with GdCl3, HpPKC-I affected the
timing of gastrulation as shown with H. pulcherrimus
embryos exposed to 6 M HpPKC-I (Fig. 6B, C). To
quantify the progression of gastrulation, we rated the
embryos into four grades based on the length of the
invaginated archenteron: 0G, uninvaginated embryo;
1/4G, embryo with an indent at the vegetal pole;
2/4G, embryo with the archenteron elongated to the
midpoint; 4/4G, embryo with the archenteron attained
to the animal pole. The control embryos underwent
gastrulation by 23 h (Fig. 6B). About 30% of embryos
nearly completed gastrulation (3/4G + 4/4G), and
the rest elongated their archenteron and arrived at
the 2/4G stage. In contrast, the embryos exposed to
HpPKC-I barely started gastrulation by 23 h. Two
hours later (Fig. 6B, at 25 h), all the control embryos
had finished gastrulation, but the embryos treated
with HpPKC-I were still gastrulating (most embryos
were estimated to be at 2/4G). The extent of the delay
was not changed by the duration of the treatment,
from 70 min (8–16-cell stage) to 150 min (8–60-cell
stage). Effects of HpPKC inhibitor on micromere-specific
Ca2+ rise and gastrulation As expected from a previous study using
intracellular recording (Yazaki, 2001), the Ca2+ levels in
the micromeres (0.99 relative fluorescence unit; RFU)
were slightly, but significantly, higher than those of the
macromeres (0.91 RFU, p < 0.05) and the mesomeres
(0.92 RFU, p < 0.05). In embryos treated with the
PKC activator PMA (8 nM), the intracellular Ca2+
levels in all blastomeres were significantly lowered in Materials and methods), we have monitored changes
in the intracellular Ca2+ levels 5–10 min after the 4th
cleavage. The Ca2+ levels of each blastomere were
monitored by single photometry at the 16-cell stage
(Table 1). As expected from a previous study using
intracellular recording (Yazaki, 2001), the Ca2+ levels in
the micromeres (0.99 relative fluorescence unit; RFU)
were slightly, but significantly, higher than those of the
macromeres (0.91 RFU, p < 0.05) and the mesomeres
(0.92 RFU, p < 0.05). In embryos treated with the
PKC activator PMA (8 nM), the intracellular Ca2+
levels in all blastomeres were significantly lowered in https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press 436 Ikuko Yazaki et al. Table 1 Effects of HpPKC inhibitors or activator on micromere-specific Ca2+ elevation (P. lividus)
Treatments
Micromeres
Macromeres
Mesomeres
Control
0.99 ± 0.02 (24)
0.91 ± 0.01 (23)
0.92 ± 0.02 (33)
5 M HpPKC-I
0.91 ± 0.02 (17)
0.89 ± 0.02 (16)
0.90 ± 0.02 (26)
400 nM Gö6976
1.06 ± 0.02 (5)
0.98 ± 0.03 (5)
0.96 ± 0.02 (7)
8 nM PMA
0.76 ± 0.00 (2)
0.72 ± 0.04 (2)
0.69 ± 0.03 (2)
Ca2+ levels of each blastomere were reported as mean ± standard deviation (SD) of the ratios of fluorescence
intensity of Ca2+-sensitive dye (calcium green BAPTA) to the intensity of the Ca2+-insensitive internal control
dye (rhodamine red) in the same cell. The numbers of monitored cells were given in parentheses. appeared to have a critical period of about 70 min
that was between the 8-cell and 16-cell stages. Indeed,
inhibition of PKC from the 7th cleavage (124-cell stage)
caused no delay in gastrulation (Fig. 6B), whereas
the largest delay in gastrulation was provoked by
the treatment during the 8–60-cell stages (Fig. 6C). In contrast, in all these conditions, PMC ingression
took place on a normal schedule (data not shown),
analogous to the results obtained with GdCl3 (Fig. 6A). Effects of HpPKC inhibitor on micromere-specific
Ca2+ rise and gastrulation Hence, inhibition of the stretch-activated Ca2+ channel
(GdCl3) and PKC (HpPKC-I) both led to a delay in
gastrulation but not to alteration of the micromere
specifications. The earliest function of the micromeres appears to
be endoderm induction, as the signal is emitted from
the micromeres during the 16–60-cell stages (Ransick &
Davidson, 1995). We have examined whether the Ca2+
influx and PKC activity are essential for this micromere
signal. As the Ca2+ influx at the micromere formation
is suppressed with GdCl3, an inhibitor of stretch-
activated ion channels (Yazaki et al., 2004), we exposed
P. lividus embryos to 25 M GdCl3 and examined
the frequency of the embryos that clearly exhibited
the expected phenotypes: ingression of the primary
mesenchyme cells (PMCs) and gastrulation (Fig. 6A). We found that gastrulation was delayed by GdCl3
when it was applied at the 4th and 5th cleavages, but
not before or after the period. When GdCl3 was added
from the 5th cleavage, which included micromere
cleavage to form small and large micromeres during
treatment, gastrulation was delayed to a lesser extent
than when treatment was carried out at the 4th
cleavage (Fig. 6A). By contrast, the ingression of PMCs
into the blastocoel was not altered in all treatment
schemes. HpPKC inhibitor enlarged the oral ectoderm in both
H. pulcherrimus and P. lividus embryos without
affecting mesoendoderm specification Thus, the delay of gastrulation by HpPKC-I https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press Ca2+-PKC signalling in sea urchin cell specification 437 Ca +-PKC signalling in sea urchin cell specification Figure 6 Inhibition of Ca2+infux and of PKC activity delayed gastrulation, but not PMC ingression. (A) P. lividus embryos wer
treated with 25 M GdCl3 for 50 min starting from 15 min before the 2nd, 4th, 5th and 7th cleavages at room temperature. Ce
oi.org/10.1017/S0967199414000033 Published online by Cambridge University Press Figure 6 Inhibition of Ca2+infux and of PKC activity delayed gastrulation, but not PMC ingression. (A) P. lividus embryos were
treated with 25 M GdCl3 for 50 min starting from 15 min before the 2nd, 4th, 5th and 7th cleavages at room temperature. Cell https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press 438 Ikuko Yazaki et al. treatment (Table 3). We have also found that HpPKC-
I increased Nodal but suppressed Deadringer (Dri)
transcripts. Together with Lim, Nodal and Deadringer
(Dri)
are
involved
in
the
development
of
oral
ectoderm, but the expression times of these genes
are known to be slightly different. In the beginning
of gastrulation, Nodal declines by half (Duboc et al.,
2004), but Dri increases (Amore et al., 2003). Likewise,
genes involved in aboral ectoderm formation (NK2.2
and
Coquilette)
are
expressed
from
the
blastula
stage (Takacs et al., 2004; Croce et al., 2003), and
accordingly we have found that HpPKC-I treatment
suppressed their expression. Thus, our data with
HpPKC-I are in line with these previous observations,
and suggest that PKC signalling may contribute
to the fine regulation of gene expression before
gastrulation. effects of HpPKC-I on the skeleton size were restricted
to br and por in the aboral ectoderm region. In contrast,
LiCl showed a tendency to suppress skeletogenesis
as a whole, whereas another PKC inhibitor, Gö6976,
enhanced the growth of skeletons at the concentration
used. Instead, the addition of PKC activator, PMA
made no difference. As mentioned for Fig. 6C, when HpPKC-I was
added to the embryos by the 8-cell stage, the timing
of gastrulation was not changed, but it was noted
that the shape of embryos seemed to be changed
considerably (Fig. 8B). To discriminate the shape
clearly, the embryos marked with asterisks in the left
panels were delineated on the right to indicate the
oral (arrowheads) and the aboral ectoderms (arrow)
in the normal embryos (Fig. 8). cycles went on every 30–35 min. Control and GdCl3-treated embryos were both fixed at the same time, and the percentage
of PMC-ingressed embryos or gastrulation-initiated embryos were calculated from circa 100 embryos from two or three
independent experiments, respectively. (B, C) H. pulcherrimus embryos were treated with HpPKC-I at 6 M (B) or 5 M (C)
during the period indicated by the horizontal bars. Gastrulation levels were estimated only from the side-viewed embryos.
Embryos were cultured at 15°C (B), at 18°C (C). The cell cycles of these experiments were 45–48 min (B) and 39 min (C). Discussion In the present study, we have demonstrated that the
PKC pathway plays an important role during the early
embryonic development of sea urchin. Suggestive of
a conserved mechanism of control, the PKCs from
the two species of sea urchins (H. pulcherrimus and
P. lividus) shared the same myristoylated pseudosub-
strate region that we have employed as a specific PKC
inhibitor (HpPKC-I). HpPKC-I was highly effective
and completely blocked cell division at 7 M concen-
tration. At 5 M, phalloidin-stained F-actin filaments
were apparently released from the plasma membrane
region, disrupting the intercellular adhesion system
(Figs. 1 and 7). Oral ectoderm specifications were also
affected by HpPKC-I in both embryos (Fig. 7). Our
studies using HpPKC-I have thus revealed that the
PKC pathway is involved in the regulation of many
aspects of early development such as cytokinesis,
dynamic rearrangement of the actin cytoskeleton,
nucleation of -catenin, gastrulation, intracellular Ca2+
signalling, and the expression of the genes accountable
for the formation of oral ectoderm. Taken together
with previous findings that blastomeres of 16-cell stage
sea urchin embryos have Ca2+ influx (Yazaki, 2001),
the results of our studies using HpPKC-I suggested
the Ca2+/DAG-dependent protein kinase PKC as the
central piece of the signalling pathway that links HpPKC inhibitor enlarged the oral ectoderm in both
H. pulcherrimus and P. lividus embryos without
affecting mesoendoderm specification When HpPKC-I was
added before the 3rd cleavage (4-cell stage), the
embryo displayed a much decreased aboral ectoderm
in comparison with the control, forming embryos
in which the oral ectoderm was disproportionally
larger (Fig. 8B). Similar morphological changes were
observed in the embryos treated with HpPKC-I after
the 4th cleavage but to a lesser extent (Fig. 8C). Again, added after the 7th cleavage, HpPKC-I had no
effect on the morphology of the embryos (data not
shown). qPCR analysis of the embryos treated with the
HpPKC inhibitor To
study
the
effect
of
PKC
on
the
expression
of the pivotal genes involved in early embryonic
development, we examined the changes in levels of the
17 transcripts with or without treatment with HpPKC-
I. As summarized in Table 3, the genes normally
expressed in the oral ectoderm, Nodal, Deadringer
and Lim, markedly increased their expression after
the embryos were exposed to HpPKC-I, while the
transcripts of the aboral ectoderm genes, NK2.2
and Coquilette, decreased. In accordance with the
morphology data (Table 2), the skeletogenic genes also
decreased in expression. However, we need to pay
special attention to those genes whose expression is
linked to the timing of gastrulation. As the addition
of HpPKC-I delayed gastrulation, the genes linked to
gastrulation are likely to display an altered expression
profile in response to HpPKC-I. As expected, the level
of Brain1/2/4, a transcription factor that is expressed
at the initiation of gastrulation and controls endo16
(Yuh et al., 2005), was much lower after HpPKC-I https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press Ca2+-PKC signalling in sea urchin cell specification 439 2B) that was in general agreement
with the molecular weights of the -catenins in
Xenopus (92 kDa) (McCrea & Gumbiner, 1991) and in
starfish embryos (100 kDa) (Miyawaki et al., 2003). Our
antibody also visualized the characteristic cytoplasmic
and membrane distributions of -catenin reported
in L. variegatus (Miller & McClay, 1997a) up to the
4th cleavage. Thus, this difference does not seem
to represent a technical issue on our part. We have
checked the subcellular localization of -catenin in the
context of mitotic cell cycle (Fig. 3), and confirmed
that nuclear entrance of -catenin first occurred in the
macromeres at the 16-cell stage and then expanded g
y
The results of our experiment using HpPKC-I
raised intriguing questions as to how PKC regulates
subcellular distribution of -catenin, and as to the roles
of nuclear -catenin. In sea urchin embryos, it has
been proposed that the specific nuclear entrance of -
catenin in the cells on the vegetal side is mediated
by glycogen synthase kinase 3 (GSK-3), which
is a serine/threonine kinase comprising the Wnt
pathway. In line with this idea, Lhomond et al., (2012)
reported that Wnt6 and its receptor Frizzled 1/2/7
selectively expressed in macromeres were responsible
for nuclear localization of -catenin in macromere
daughter cells after the 5th cleavage. In view of the
fact that the Wnt/Ca2+ signalling pathway activates
Ca2+-dependent kinases such as PKC and CAMK II
in vertebrates (Kühl et al., 2000), it is conceivable that
PKC may serve as a downstream effector of Wnt
and preclude the nuclear entrance of -catenin in
blastomeres on the animal side in sea urchin embryos. Indeed, it has been reported that PKC inactivates GSK-
3 by phosphorylation in test tubes and in fibroblasts
(Goode et al., 1992; Cook et al., 1996). In addition, -
catenin is phosphorylated by the maternally expressed
GSK-3 and rapidly degenerated in blastomeres on
the animal side of the 16-cell stage sea urchin embryo,
whereas -catenin is stable in the vegetal cells because
GSK-3 is inactivated by Dishevelled (Dsh) protein,
a transducer of the canonical Wnt pathway (Weitzel
et al., 2004; Croce et al., 2011). lividus) Table 2 Effects of PKC modulators on the formation of the skeletal rods in the pluteus (P. lividus) In total, 5 M HpPKC-I and 400 nM Gö6976 were added to embryos 20 min before the 4th cleavage; 8 nM PMA was added
to the 16-cell stage embryos for 20 min, and 40 mM LiCl to the embryos at the 4-cell to 60-cell stages (2.5 h). The numbers of
monitored cells are given in parentheses. the intracellular Ca2+ increase to various downstream
events in early embryonic development. to micromeres at the 32-cell stage, whereas nuclear
localization of -catenin in the micromeres clearly
occurred only after the 5th cleavage (Fig. 4). Hence,
whether this subtle difference in H. pulcherrimus and
L. variegatus arises from a real biological difference
in the two sea urchin species or simply reflects
methodological limitations is yet to be resolved. y
y
p
Sea urchin embryos specify cell fates along two
embryonic axes; animal–vegetal (AV) axis and oral–
aboral (OA) axis. Both axes are initially inherent in
the cytoarchitecture of the unfertilized egg (Boveri,
1901; Hörstadius, 1939; Coffman & Davidson, 2001;
Croce et al., 2011). -Catenin is a maternally expressed
protein, and its nuclear entrance in the blastomeres
sets the tone for the AV axis for cell specification
(Wikramanayake et al., 1998; Logan et al., 1999; Croce
et al., 2011). Nuclear entrance of -catenin takes place
in the micromeres and the macromeres (Fig. 4) on
the vegetal side, but not on the animal side (Miller
& McClay, 1997a; Logan et al, 1999). One of our
major findings in this study is that this tight cell-
specific control of nuclear localization of -catenin is
deregulated by the inhibition of PKC, as 5 M HpPKC-
I induced nuclear entrance of -catenin in all cells
at the 16-cell stage (Fig. 5D, E). In addition, we have
found that the initial nuclear localization of -catenin
takes place in the macromeres (Figs. 4A and 5A). While
the significance of this finding is not entirely clear,
our result is at variance with a previous study using
another sea urchin species L. variegatus in which the
initial nuclear entry of -catenin was reported to take
place in the micromeres at the 16-cell stage (Logan
et al., 1999). The specificity of our antibody against
-catenin of H. pulcherrimus was demonstrated in
western blot analysis, which showed a single band
of 103 kDa (Fig. Figure 7 HpPKC inhibitor caused similar morphological changes on embryos of H. pulcherrimus and P. lividus. Embryos were
treated with 5 M HpPKC-I from 20 min before the 4th cleavage to 20 min after the 6th cleavage. This period was about 120
min for H. pulcherrimus culturing at 18°C and 100 min for P. lividus at 23°C. Control and HpPKC-I-treated embryos. (A) H. pulcherrimus embryos; early blastulae at 6 h post fertilization (p.f.), gastrulae at 22 h, and plutei at 52 h (B) P. lividus; morulae
at 5 h, gastrulae at 21 h, and plutei at 47 h. (C) Names of the skeletal parts of the pluteus. Figure 7 HpPKC inhibitor caused similar morphological changes on embryos of H. pulcherrimus and P. lividus. Embryos were
treated with 5 M HpPKC-I from 20 min before the 4th cleavage to 20 min after the 6th cleavage. This period was about 120
min for H. pulcherrimus culturing at 18°C and 100 min for P. lividus at 23°C. Control and HpPKC-I-treated embryos. (A) H. pulcherrimus embryos; early blastulae at 6 h post fertilization (p.f.), gastrulae at 22 h, and plutei at 52 h (B) P. lividus; morulae
at 5 h, gastrulae at 21 h, and plutei at 47 h. (C) Names of the skeletal parts of the pluteus. https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press Ikuko Yazaki et al. 440 Table 2 Effects of PKC modulators on the formation of the skeletal rods in the pluteus (P. lividus)
Body rods (Br)
Post-oral rods (Por)
Antero-lateral rods (Alr)
Total length (m)
Control
103.5 ± 0.95 (102)
51.1 ± 0.90 (115)
64.0 ± 0.67 (118)
219
HpPKC-I
91.5 ± 0.67 (132)
42.2 ± 0.90 (118)
63.8 ± 0.67 (128)
198
Gö6976
103.2 ± 0.53 (163)
61.2 ± 0.59 (145)
75.9 ± 0.73 (146)
240
PMA
103.5 ± 0.39 (129)
55.9 ± 0.76 (125)
68.8 ± 0.56 (130)
228
LiCl
101.2 ± 0.71 (85)
45.4 ± 0.72 (77)
56.5 ± 0.99 (72)
203
In total, 5 M HpPKC-I and 400 nM Gö6976 were added to embryos 20 min before the 4th cleavage; 8 nM PMA was added
to the 16-cell stage embryos for 20 min, and 40 mM LiCl to the embryos at the 4-cell to 60-cell stages (2.5 h). The numbers of
monitored cells are given in parentheses. modulators on the formation of the skeletal rods in the pluteus (P. The idea that the
subcellular localization of -catenin may be mediated
by GSK-3 was further supported by the results of
our experiment using LiCl, which has been reported
to inhibit GSK-3 activity in vitro (Klein & Melton,
1996) and is thus expected to exert the same effect
that PKC has on GSK-3. We found that the PKC
activator PMA and LiCl both stabilized -catenin in https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press Ca2+-PKC signalling in sea urchin cell specification Ca2+-PKC signalling in sea urchin cell specification
441
re 8 Morphological changes of H. pulcherrimus embryos treated with HpPKC inhibitor before the 8-cell stage. (A) Control
ryos. (B) Embryos treated with 5 M HpPKC-I starting from the 4-cell stage (20 min before the 3rd cleavage) to the end of
l stage. (C) Embryos treated with 5 M HpPKC starting from 20 min before the 4th cleavage to the end of 16-cell stage. ryos were cultured at 18°C. The hand-drawn delineations on the right panels represent the embryos marked with asterisks
A–C). Oral ectoderm area and aboral area were indicated by white arrowheads and black arrows, respectively. 441 Figure 8 Morphological changes of H. pulcherrimus embryos treated with HpPKC inhibitor before the 8-cell stage. (A) Control
embryos. (B) Embryos treated with 5 M HpPKC-I starting from the 4-cell stage (20 min before the 3rd cleavage) to the end of
8-cell stage. (C) Embryos treated with 5 M HpPKC starting from 20 min before the 4th cleavage to the end of 16-cell stage. Embryos were cultured at 18°C. The hand-drawn delineations on the right panels represent the embryos marked with asterisks
in (A–C). Oral ectoderm area and aboral area were indicated by white arrowheads and black arrows, respectively. Figure 8 Morphological changes of H. pulcherrimus embryos treated with HpPKC inhibitor before the 8-cell stage. (A) Control
embryos. (B) Embryos treated with 5 M HpPKC-I starting from the 4-cell stage (20 min before the 3rd cleavage) to the end of
8-cell stage. (C) Embryos treated with 5 M HpPKC starting from 20 min before the 4th cleavage to the end of 16-cell stage. Embryos were cultured at 18°C. The hand-drawn delineations on the right panels represent the embryos marked with asterisks
in (A–C). Oral ectoderm area and aboral area were indicated by white arrowheads and black arrows, respectively. the cytoplasm, whereas 5 M HpPKC-I appreciably
reduced -catenin in the cytoplasm (Fig. 5D, E). Table 3 qPCR analysis of embryos treated with HpPKC inhibitor Embryos were treated with 5 M HpPKC-I starting from 20 min before the 4th cleavage until 20 min after the 6th cleavage. Embryos were harvested at time points 20 h (early gastrula), 23 h (late gastrula or prism) and 43 h (two-armed pluteus) post
fertilization. The D values defined in Methods and materials were calculated from the results of qPCR and reported in
positive and negative numbers that respectively represent increases and decreases of the transcripts after the treatment with
the inhibitor. NS, not significant (P > 0.05). Embryos were treated with 5 M HpPKC-I starting from 20 min before the 4th cleavage until 20 min after the 6th cleavage. Embryos were harvested at time points 20 h (early gastrula), 23 h (late gastrula or prism) and 43 h (two-armed pluteus) post
fertilization. The D values defined in Methods and materials were calculated from the results of qPCR and reported in
positive and negative numbers that respectively represent increases and decreases of the transcripts after the treatment with
the inhibitor. NS, not significant (P > 0.05). Embryos were treated with 5 M HpPKC-I starting from 20 min before the 4th cleavage until 20 min after the 6th cleavage. Embryos were harvested at time points 20 h (early gastrula), 23 h (late gastrula or prism) and 43 h (two-armed pluteus) post
fertilization. The D values defined in Methods and materials were calculated from the results of qPCR and reported in
positive and negative numbers that respectively represent increases and decreases of the transcripts after the treatment with
the inhibitor. NS, not significant (P > 0.05). nuclear localization of -catenin in the blastomeres
of the vegetal side, establishing the AV axis. Indeed,
PKC is thought to be present in the eggs throughout
the developmental stages of sea urchin (Rakow &
Shen, 1994); the early embryos exposed to 12-O-
tetradecanoyl phorbol-13-acetate (TPA, also called
PMA), an activator of PKC, promotes the development
of endoderm and mesoderm (Livingston & Wilt, 1992). In our study, we have demonstrated that the same
PKC activator increased -catenin in the cytoplasm
and enlarged the nuclear entrance of -catenin more to
the animal side, as was observed with LiCl (Fig. 5B1,
C1). In view of the fact that -catenin entering the
nucleus functions as a transcriptional activator of
target genes (Miller et al., 1999), it is tempting to
speculate that -catenin in the nucleus of blastomeres
on the vegetal side might lead to production of the cytoplasm, whereas 5 M HpPKC-I appreciably
reduced -catenin in the cytoplasm (Fig. 5D, E). inductive factors that determine the vegetal cell
fates (McClay et al., 2000). A paired homeodomain
transcription factor pmar-1 is expressed exclusively in
micromeres and is known to be a direct target of the
-catenin/TCF complex (Oliveri et al., 2002). Pmar1
in turn accelerates micromere-selective degradation
of SoxB1, which is an ‘animalizing’ transcription y
p
g
In view of the fact that -catenin entering the
nucleus functions as a transcriptional activator of
target genes (Miller et al., 1999), it is tempting to
speculate that -catenin in the nucleus of blastomeres
on the vegetal side might lead to production of https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press 442 Ikuko Yazaki et al. Table 3 qPCR analysis of embryos treated with HpPKC inhibitor Table 3 qPCR analysis of embryos treated with HpPKC inhibitor
Gastrula
Pluteus
Genes
20 h
23 h
43 h
Endomesodermal transcription factors and signal components
Delta
NS
NS
NS
Gcm
−2.73
NS
NS
Fox A
NS
NS
NS
Bra
NS
NS
NS
Blimp/Krox
NS
NS
+1.84
Brain1/2/4
−4.78
−3.91
NS
PMC ingression and skeletogenic genes
G-cadherin
NS
−1.81
NS
Sm50
−2.71
−5.26
NS
Sm37
−2.40
NS
NS
Sm30
−2.34
−1.92
NS
Regulator genes of O–A axis formation
Nodal
+3.81
NS
NS
Deadringer (Dri)
−5.44
−4.00
+3.32
Lim
NS
NS
+3.22
Gsc
−2.02
−2.46
NS
FoxG
−1.95
−1.89
NS
NK2.2
−4.40
−1.70
NS
Coquilette
−2.53
−1.79
NS
Embryos were treated with 5 M HpPKC-I starting from 20 min before the 4th cleavage until 20 min after the 6th cleavage. Embryos were harvested at time points 20 h (early gastrula), 23 h (late gastrula or prism) and 43 h (two-armed pluteus) post
fertilization. The D values defined in Methods and materials were calculated from the results of qPCR and reported in
positive and negative numbers that respectively represent increases and decreases of the transcripts after the treatment with
the inhibitor. NS, not significant (P > 0.05). Instead,
the PKC pathway leading to the pattern determination
appeared to involve the Ca2+ influx that takes place
slightly later at the 4th cleavage through a stretch-
dependent Ca2+ channels in the bulging-out region of
the micromere and thereby elevates the intracellular
Ca2+ levels in the micromeres (Yazaki, 2001; Yazaki
et al., 2004). We found that the abolishment of
micromere-specific
Ca2+
elevation
with
HpPKC-I
(Table 1) and the suppression of the micromere-
specific Ca2+ influx with a blocker of stretch-activated
ion channel, GdCl3, both led to delayed gastrulation,
but not to a delay in ingression of micromere to
the PMCs (Fig. 6). Thus, PKC inhibitor blocked the
micromere signals without interfering with the self-
differentiation of the micromeres. One speculation
on the mechanism enabling this outcome is that PKC
might contribute to the formation of the gap junction-
like adherence junctions, and that the micromeres
effect mesoendoderm specifications through these
junctions with the macromeres during the 16–60-cell
stages. Indeed, the inhibitor for gap junctions 1-octanol
markedly delayed gastrulation when added at the
16-cell stage (Temnopleurus hardwicki) or even at the 16-
to 60-cell stages (H. pulcherrimus) (Yazaki et al., 1999). Interestingly, after the 7th cleavage, by which the fully
passable
gap
junctions
were
already
established
(Yazaki
et
al.,
1999),
PKC
inhibition
no
longer In this study, we have also found that inhibition
of PKC interfered with gene expression in later
embryonic stages (Table 3). HpPKC-I decreased the
expression of aboral ectoderm specification genes,
NK2.2 and Coquilette. The expression of Brain1/2/4,
a regulator for midgut-specific transcriptions, was
markedly delayed in agreement with the delay in
gastrulation, but it was later expressed to form
the gut with the same size and shape as in the
control (Figs. 7 and 8). As for the skeletogenic genes,
HpPKC-I repressed the skeletal matrix genes, Sm-
30, Sm-37 and Sm-50. This finding exhibits a good
correlation with the formation of a smaller skeleton
(Table 2). Likewise, HpPKC-I suppressed Deadringer
(Dri) gene (Table 3), which in sea urchin (SpDri) is
first expressed in the precursor cells of PMCs but
disappears altogether with PMCs ingression. Just
before gastrulation, SpDri restarts its expression in
the presumptive oral ectoderm (Amore et al., 2003). Thus, SpDri suppression in the gastrulae pretreated
with HpPKC-I is in line with our observation of
delayed gastrulation. Amore et al. This might be because LiCl inhibits
inositol monophosphatase activity (Berridge et al.,
1989) and thereby reduces the InsP3 increase that
normally takes place in the embryos during mesoderm
induction (Maslansky et al., 1992; Ciapa & Maggio,
1993). Whether this InsP3-dependent pathway of the
Li+ effect would converge with the aforementioned
GSK-3 pathway is yet to be clarified. In this context, it is noteworthy that HpPKC-I
drastically changed the subplasmalemmal actin cyto-
skeleton (Fig. 1C). The observation that PKC activity
may modulate actin dynamics in a subcellular-specific
manner offers two intriguing implications. Firstly,
the actin network subjacent the plasma membrane
may modulate the functions of adherence junctions. Yazaki (1984, 1991) showed that the sea urchin
blastomeres had two membrane domains: the egg-
originated apical membrane being lined with cortical
actin meshwork and the newly formed membrane
by each cleavage that is virtually devoid of actin
filaments. The desmosomes in the electron micrograph
start in the cell junctions at the 4-cell stage (Spiegel
& Howard, 1983), but they do not display a network
of microfilaments emanating from the desmosomal
plaques unlike in the adult epithelium. This unique
feature of embryonic desmosomes might facilitate
direct cell-to-cell communication. Secondly, the control
of the subplasmalemmal actin meshwork by PKC
might serve as a way to modulate the activity
of the L-type channel and stretch-activated Ca2+
channels (Yazaki et al., 1995; Yazaki, 2001) in view of
recent findings that the actin cytoskeleton may affect
activities of the intracellular Ca2+ channels (reviewed
in Chun et al., 2010). Another
important
layer
of
control
in
early
embryogenesis is the changes in intracellular Ca2+
levels in the blastomeres. We have reported previously
a polarized distribution of L-type Ca2+ channels along
the AV axis of the P. lividus blastula, and that those
activities are linked to the cortical actin network
(Yazaki et al., 1995). This channel was activated during
M-phase of cell cycles, and displayed Ca2+ current
in the mesomeres (7.4 ± 3.8 A/cm2) and in the
macromeres (2.3 ± 2.7 A/cm2) at the 16-cell stage,
but not in the micromeres (Dale et al., 1997). However,
this ion channel might be irrelevant to the PKC
pathway that we described. Whereas a L-type channel
antagonist Nifedipine delayed cleavage and inhibited
spicule formation, 5 M HpPKC-I did not affect cell
division, and the formation of spicule and gut was
normal in these embryos (Fig. 6 and Table 2). In support of the idea that GSK-3-dependent
modulation of -catenin level is largely responsible
for regional specification, overexpression of GSK-
3 produced animalized embryos, while the kinase-
dead GSK-3 markedly vegetalized embryos (Emily-
Fenouil et al., 1998). Consistent with these findings,
the major components of the Wnt pathway have also
recently been discovered in sea urchin embryos, as
SpWnt8 was shown to be selectively expressed in
the micromeres at the 16-cell stage, and SpWnt5 in
mesomeres and macromeres (Stamateris et al., 2010). However, what remains largely unresolved is how LiCl
mediates vegetalization. We found that LiCl expanded
the nuclear entrance of -catenin to a more animal factor counteracting -catenin and thereby carving the
patterning of cell fates along the AV axis (Oliveri
et al., 2003, Angerer et al., 2005). Thus, deregulation
of -catenin expression or its translocation to the
nucleus was expected to result in alteration of the
pattern formation along the AV axis. Indeed, when -
catenin was sequestered in the plasma membrane by
overexpression of the intracellular domain of cadherin
(LvG-cadherin), (Logan et al., 1999), the failure of
-catenin to enter the nucleus led to development of
animalized embryos without formation of endoderm
and mesoderm. Overexpression of LvG-cadherin
also inhibited SpWnt8 expression in the micromeres,
which
is
essential
for
endomesoderm
formation
(Wikramanayake et al., 2004). In the present study
using HpPKC-I, we have found a converse situation in
which -catenin entered the nuclei of all blastomeres
at the 16-cell stage (Fig. 5). Although HpPKC-I did
not vegetalize the embryo, the treatment led to
the development of embryos with reduced aboral
ectoderm (Figs. 7 and 8), implying that the nuclear -
catenin in the cells on the animal side may have a
functionally different meaning from that on the vegetal
side. Considering the results so far, our conceivable
model would be that PKC activity contributes to the https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press Ca2+-PKC signalling in sea urchin cell specification 443 affected
gastrulation
nor
morphogenesis
(Figs. 6
and 7). side (Fig 5B), leading to vegetalization of the sea
urchin embryo (Logan et al., 1999). LiCl vegetalizes
the prospective ectodermal cells, but the effect could
be reversed by injection of myo-inositol (Livingston
& Wilt, 1995). (2003) found that
expression of SpDri in the oral ectoderm is required
to impart the pattering information to the skeletogenic
PMCs that is needed for oral rod (alr) formation. A chimera that consisted of normal micromeres and
micromere-deleted embryo using SpDri morpholino
developed into an alr-deficient pluteus. In the present https://doi.org/10.1017/S0967199414000033 Published online by Cambridge University Press Ikuko Yazaki et al. 444 intercellular communications by constructing cell
adhesive structures by the 60-cell stage; and (iii)
establishment of the embryonic axes, animal–vegetal
and oral–aboral axes. study, we have observed that, as a result of PKC
inhibition during the 16–60-cell stages, alr was larger
than the other skeletons, br and por (Table 2). Increases
in Dri expression should guarantee this preferential
growth of alr probably in the cells on the animal
side (Table 2). We have also found that HpPKC-I
induced a late increase in Lim transcripts by 43 h,
in the early pluteus (Table 3). Overexpression of
HpLim1 directed all embryonic cells to differentiate
into oral ectoderm (Kawasaki et al., 1999). SpLim1
was expressed in the ciliated band along the oral
and aboral ectoderm border with the endoderm of
the pluteus (Su et al., 2009). We have also found that
HpPKC-I increased expression of the oral ectoderm
genes, Nodal, Deadringer (Dri) and Lim. Our result
suggesting that Nodal gene is modulated by PKC
activity is of keen interest, as Nodal is probably the
earliest zygotic marker that determines OA polarity
following the mitochondrial distribution gradient and
the local redox state (Duboc et al., 2004; Coffman
et al., 2004, 2009). To establish the OA axis, animal
cells also need to receive the vegetal signals after
the 4th division to express the aboral ectoderm
(Kominami et al., 2006). Without the vegetal signals,
the explant of the animal half develops into an
epithelial ball consisting of neurogenic ectoderm,
in which the OA axis fails to form due to the
deficiency of aboral ectoderm expression, and the
oral ectoderm marker molecules express all over the
embryo (Hörstadius, 1975; Wikramanayake et al., 1995;
Yaguchi et al., 2006). Interestingly, to express the
aboral ectoderm in these embryos, proper expression
of -catenin and Otx are essential (Wikramanayake
& Klein, 1997; Wikramanayake et al., 1998; Li et al.,
1999), the latter of which is a transcription factor
for aboral ectoderm genes (Mao et al., 1996). Acknowledgements We thank the technical staff at the Stazione Zoologica
Anton
Dohrn:
Mr
G. Gragnaniello
who
helped
us with the photometrical techniques for calcium
measurements, and Mr D. Caramiello who provided
the sea urchin, Paracentrotus lividus. We are also
grateful to Mr M. Sekifuji and Mrs N. Ito (MMBS,
The University of Tokyo) for providing the sea urchin,
Hemicentrotus pulcherrimus. We are indebted to Dr T. Minokawa for his recombination experiments of the
animal cap and the HpPKC-I-treated micromeres, and
to Dr F. Wilt and Dr K. Fukuda for their critical reading
of the manuscript. Thus,
PKC seems to be implicated in the fine regulation
of a variety of developmental genes at different
times, and in part may well modulate cell fate
and pattern formation in the other axis through
Nodal and -catenin. This situation might explain
why the oral ectoderm specification was exaggerated
if the embryo was treated with HpPKC-I before
the 8-cell stage (Fig. 8), although the gastrulation
timing and the gut morphology were not changed
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Psychological and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian orthodontists´ perspective
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Dental Press J Orthod. 2022;27(6):e2221219
Ludmila Mangialardo LIMA1
https://orcid.org/0000-0002-7758-0870
Aron ALIAGA-DEL CASTILLO1,2
https://orcid.org/0000-0003-3963-1742
Camila MASSARO1
https://orcid.org/0000-0001-6011-7946
Caroline Martins GAMBARDELA1
https://orcid.org/0000-0003-0430-5955
Deborah Brindeiro de Araújo BRITO1
https://orcid.org/0000-0002-6327-8021
Felicia MIRANDA1
https://orcid.org/0000-0002-4015-0623
Lorena VILANOVA1
https://orcid.org/0000-0002-7575-1613
Paula COTRIN1
https://orcid.org/0000-0002-6230-0522
Raquel Silva POLETTO1
https://orcid.org/0000-0001-9400-5459
Wilana MOURA1
https://orcid.org/0000-0003-0567-3717
Arnaldo PINZAN1
https://orcid.org/0000-0002-7195-5299
Guilherme JANSON1
https://orcid.org/0000-0001-5969-5175
Fabiola Alvarez AVILA1
https://orcid.org/0000-0003-4136-0638
Psychological and financial impact of the
COVID-19 pandemic during the first stages of the
pandemic: Brazilian orthodontists´ perspective
(1) Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Ortodontia (Bauru/SP, Brazil). (2) University of Michigan, School of Dentistry, Department of Orthodontics and Pediatric Dentistry (Ann Arbor, USA). Submitted: June 30, 2021 • Revised and accepted: December 15, 2021
ludmila_mlima@yahoo.com.br
How to cite: Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. Psychological and financial impact of the COVID-19 pandemic during the first stages of the pandemic:
Brazilian orthodontists´ perspective. Dental Press J Orthod. 2022;27(6):e2221219. Psychological and financial impact of the
COVID-19 pandemic during the first stages of the
pandemic: Brazilian orthodontists´ perspective Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective ABSTRACT Introduction: Brazil faced a catastrophic situation in the corona-
virus pandemic. Due to the high risk of contamination and spread
of COVID-19, dentist have been attending only urgency and emer-
gency services in Brazil at the beginning of the pandemic. Objective: This research aimed to evaluate the psychological
and financial impacts caused by the coronavirus pandemic in
Brazilian orthodontists. Methods: This population-based cross-sectional study collect-
ed demographic data and mental health measurements from 404
orthodontists. Depression, anxiety, insomnia, and distress were
evaluated through Brazilian versions of the 9-item Patient Health
Questionnaire (9-PHQ), the 7-item Generalized Anxiety Disorder
scale and Mini-Tracking (GAD), the 7-item Insomnia Severity In-
dex (ISI), and the 22-item Impact of Event Scale–Revised (IES-R),
respectively. The demographic data of the sample was described
using descriptive statistics. The data was analyzed according
to sex, professional status, and economic income. Comparisons
were performed using Chi-square tests, Mann-Whitney U tests,
and Kruskal-Wallis followed by post-hoc tests. Results: Females, graduate students, and lower incomes sub-
groups showed higher levels of depression, anxiety, insomnia,
and distress. Most orthodontists showed moderate to extreme
financial and professional concerns during the pandemic. Conclusion: The coronavirus pandemic negatively affected the
psychological health and increased the financial concerns of
the Brazilian orthodontists, mainly female, graduate students,
and with income below 10k participants. Keywords: COVID-19. Psychology. Orthodontics. Dental Press J Orthod. 2022;27(6):e2221219 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective RESUMO Introdução: O Brasil enfrentou uma situação catastrófica du-
rante a pandemia do coronavírus. Devido ao alto risco de con-
taminação e disseminação do vírus da COVID-19, os cirurgiões-
-dentistas passaram a realizar apenas atendimentos de urgência
ou emergência no início da pandemia. Objetivo: O presente es-
tudo teve como objetivo avaliar o impacto financeiro e psicoló-
gico causado pela pandemia do coronavírus nos ortodontistas
brasileiros. Métodos: Este estudo transversal de base popula-
cional coletou os dados demográficos e o impacto da pandemia
em 404 ortodontistas. Depressão, ansiedade, insônia e angústia
foram avaliadas, respectivamente, por meio da versão em portu-
guês do Questionário de Saúde do Paciente (PHQ-9), do módulo
de transtorno de ansiedade generalizada (GAD) do Mini-Trac-
king (GAD/Mini-Tracking), do Índice de Severidade de Insônia
(ISI) e o do Impact of Events Scale-Revised (IES-R). As caracte-
rísticas demográficas da amostra foram apresentadas por meio
de estatística descritiva. Os dados foram analisados de acordo
com o sexo, situação profissional e renda econômica. As compa-
rações foram realizadas utilizando os testes de Qui-quadrado,
Mann-Whitney U e Kruskal-Wallis, seguidos de testes post-hoc
(p<0,05). Resultados: Mulheres, estudantes de pós-graduação
e profissionais com menores rendas apresentaram níveis mais
altos de depressão, ansiedade, insônia e angústia. A maioria dos
ortodontistas mostrou preocupação financeira e profissional
moderada a extrema durante a pandemia. Conclusão: A pan-
demia do coronavírus afetou negativamente a saúde psicológi-
ca dos ortodontistas brasileiros e aumentou as preocupações fi-
nanceiras desses profissionais. As mulheres, os estudantes de
pós-graduação e os participantes com renda mensal menor que
R$10 mil foram os grupos mais afetados. Introdução: O Brasil enfrentou uma situação catastrófica du-
rante a pandemia do coronavírus. Devido ao alto risco de con-
taminação e disseminação do vírus da COVID-19, os cirurgiões-
-dentistas passaram a realizar apenas atendimentos de urgência
ou emergência no início da pandemia. Objetivo: O presente es-
tudo teve como objetivo avaliar o impacto financeiro e psicoló-
gico causado pela pandemia do coronavírus nos ortodontistas
brasileiros. Métodos: Este estudo transversal de base popula-
cional coletou os dados demográficos e o impacto da pandemia
em 404 ortodontistas. Depressão, ansiedade, insônia e angústia
foram avaliadas, respectivamente, por meio da versão em portu-
guês do Questionário de Saúde do Paciente (PHQ-9), do módulo
de transtorno de ansiedade generalizada (GAD) do Mini-Trac-
king (GAD/Mini-Tracking), do Índice de Severidade de Insônia
(ISI) e o do Impact of Events Scale-Revised (IES-R). RESUMO As caracte-
rísticas demográficas da amostra foram apresentadas por meio
de estatística descritiva. Os dados foram analisados de acordo
com o sexo, situação profissional e renda econômica. As compa-
rações foram realizadas utilizando os testes de Qui-quadrado,
Mann-Whitney U e Kruskal-Wallis, seguidos de testes post-hoc
(p<0,05). Resultados: Mulheres, estudantes de pós-graduação
e profissionais com menores rendas apresentaram níveis mais
altos de depressão, ansiedade, insônia e angústia. A maioria dos
ortodontistas mostrou preocupação financeira e profissional
moderada a extrema durante a pandemia. Conclusão: A pan-
demia do coronavírus afetou negativamente a saúde psicológi-
ca dos ortodontistas brasileiros e aumentou as preocupações fi-
nanceiras desses profissionais. As mulheres, os estudantes de
pós-graduação e os participantes com renda mensal menor que
R$10 mil foram os grupos mais afetados. Palavras chave: COVID 19 Psicologia Ortodontia Palavras-chave: COVID-19. Psicologia. Ortodontia. Dental Press J Orthod. 2022;27(6):e2221219 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective INTRODUCTION In March 2020, the World Health Organization (WHO) declared
the coronavirus pandemic, due to the significant increase in
the number of reported cases and the global virus spread.1
In the middle of 2020, Brazil was the epicenter of the coronavi-
rus outbreak in Latin America, presenting the second-highest
number of cases and deaths in the world. Until the end of July
2020, more than 2 million cases were confirmed, and 90 thou-
sand deaths were reported in Brazil.2 In dental practice, both patients and professionals are exposed to
a high risk of COVID-19 infections. The frequent exposure to saliva
and blood, the proximity between patient and professional, and
the aerosol spread increase the contamination risks.3,4 For this reason, dentists attended only urgency and emergency
services during the coronavirus pandemic in Brazil, at the begin-
ning of the pandemic. Elective treatments such as Orthodontics
have been postponed until the situation becomes controlled.5,6 Concerns regarding contamination of dentists due to the transmis-
sion of the coronavirus through saliva were previously reported,
and safety measures were recommended.4,7-9 In this context, the
coronavirus pandemic may cause physical and mental effects on
health workers. The pandemic situation negatively affects psycho-
logical health and financial status.10,11 Symptoms as post-traumatic Dental Press J Orthod. 2022;27(6):e2221219 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective
5 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective 5 stress, depression, anxiety, insomnia, and emotional exhaustion
have been reported.10-13 Therefore, this study aimed to evaluate the psychological, financial,
and professional impacts during the first stages of the coronavirus
pandemic in Brazilian orthodontists. MATERIAL AND METHODS 2022;27(6):e2221219 7
Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective 7
Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective All participants also answered 10 additional questions regarding the
impact of coronavirus pandemic in financial and professional concerns. The economic incomes were evaluated in the local currency (Fig 1). MATERIAL AND METHODS This population-based cross-sectional study was approved by the
Ethics Committee on Human Research of University of São Paulo
(protocol number N. 4.023.156 CAAE 30984620.6.0000.5417), and
all participants agreed to participate in the survey. Their identities
were kept confidential. The sample size was calculated considering 80% of test power, a
significance level of 5%, a design effect of 1, and 50% frequency
of psychological symptoms, based on a previous study of the
COVID-19 outbreak.7 According to the Brazilian Federal Council
of Dentistry, the population of orthodontists comprised 27940
subjects.14 Therefore at least 379 completed questionnaires
were necessary. The data was obtained in early May 2020 through the Google Forms
platform (Google Inc, Mountain View, CA, USA). The questionnaire
was sent by e-mail or WhatsApp Messenger (Facebook Inc, Mountain
View, CA, USA) to Brazilian orthodontists and postgraduate students. Dental Press J Orthod. 2022;27(6):e2221219 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective
6 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective The link was available for 10 days. Participants could refuse or with-
draw to participate at any time, leaving the website, without any
penalty or loss. The survey was composed by questionnaires with multiple-choice
answers to evaluate the demographic data, mental health, and
impact in finances and professional activities. The symptoms of depression, anxiety, insomnia and distress for
all participants were assessed through previously validated ques-
tionnaires: the Patient Health Questionnaire (PHQ-9),15-17 the gen-
eralized anxiety disorder (GAD) module of the Mini-Tracking (GAD/
Mini-Tracking),18-20 Insomnia Severity Index (ISI),21 Impact of Events
Scale-Revised (IES-R),22,23 respectively. PHQ values ranged from 0 to 27, and were classified as (0-4)
normal, (5-9) mild, (10-14) moderate, (15-27) severe; GAD/Mini-
tracking values ranged from 0 to 32 and were classified as (0-8)
normal, (9-16) mild, (17-24) moderate and (25-32) severe; ISI scores
ranged from 0 to 28, and were classified as (0-7) absence, (8-14)
subthreshold, (15-27) moderate and (22-28) severe; IES-R scores
ranged from 0 to 88, and were classified as (0-8) normal, (9-25)
mild, (26-43) moderate and (44-88) severe. These classifiers were
adapted from previous studies.7,15-23 Dental Press J Orthod. STATISTICAL ANALYSIS The demographic data of the sample was described using descriptive sta-
tistics. The data was analyzed according to sex, professional status, and
economic income. Comparisons were performed using Chi-square tests,
Mann-Whitney U tests, and Kruskal-Wallis followed by post-hoc tests (p<0.05). Figure 1: Level of concern in Orthodontics about financial and professional activities
during the coronavirus pandemic. Figure 1: Level of concern in Orthodontics about financial and professional activities
during the coronavirus pandemic. Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective
8 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective Statistical analyses were performed with SPSS statistical soft-
ware package (version 21.0; SPSS, Chicago, IL). RESULTS The sample was composed by 404 orthodontists (259 females;
145 males). The demographic characteristics of the sample are
described in Table 1. The severity of depression, anxiety, insomnia, and distress
is reported in Table 2. Most of the sample presented mild
to severe symptoms of depression (62.4%), anxiety (62.6%),
insomnia (50.8%), and distress (82.4%) (Table 2). Table 1: Demographic and professional characteristics of responders. n (%)
TOTAL
PROFESSIONAL STATUS
ECONOMIC INCOME
Professor
Graduate
student
Clinician
<5K
5-10k
> 10k
Overall
404 (100.0)
152 (37.6)
115 (28.5)
137 (33.9)
97 (24.0)
134 (33.2)
173 (42.8)
Sex
Female
259 (64.1)
77 (50.7)
84 (73.0)
98 (71.5)
79 (81.4)
100 (74.6)
80 (46.2)
Male
145 (35.9)
75 (49.3)
31 (27.0)
39 (28.5)
18 (18.6)
34 (25.4)
93 (53.8) Table 1: Demographic and professional characteristics of responders. n (%)
TOTAL
PROFESSIONAL STATUS
ECONOMIC INCOME
Professor
Graduate
student
Clinician
<5K
5-10k
> 10k
Overall
404 (100.0)
152 (37.6)
115 (28.5)
137 (33.9)
97 (24.0)
134 (33.2)
173 (42.8)
Sex
Female
259 (64.1)
77 (50.7)
84 (73.0)
98 (71.5)
79 (81.4)
100 (74.6)
80 (46.2)
Male
145 (35.9)
75 (49.3)
31 (27.0)
39 (28.5)
18 (18.6)
34 (25.4)
93 (53.8) able 1: Demographic and professional characteristics of responders. Dental Press J Orthod. 2022;27(6):e2221219 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective Table 2: Descriptive statistics of the severity categories of depression, anxiety, insomnia,
and distress in Brazilian orthodontists during COVID-19 pandemic. RESULTS PHQ-9 = 9-item Patient Health Questionnaire; GAD = Generalized Anxiety Disorder; ISI = Insomnia Severity
Index; IES-R = Impact of Event Scale–Revised
Question-
naire
Total
n (%)
Sex - n (%)
Professional status - n (%)
Economic income - n (%)
Female
Male
Profes-
sor
Graduate
Clini-
cian
<5k
5-10k
>10k
PHQ-9 - depression symptoms
Normal
(0-4)
128 (31.7)
51 (19.7)
77 (53.1)
69 (45.4)
19 (16.5)
40 (29.2)
11 (11.3)
39 (29.1)
78 (45.1)
Mild (5-9)
121 (30.0)
83 (32.0)
38 (26.2)
41 (27.0)
36 (31.3)
44 (32.1)
30 (30.9)
35 (26.1)
56 (32.4)
Moderate
(10-14)
80 (19.8)
65 (25.1)
15 (10.3)
23 (15.1)
26 (22.6)
31 (22.6)
24 (24.7)
31 (23.1)
25 (14.5)
Severe
(15-27)
75 (18.6)
60 (23.2)
15 (10.3)
19 (12.5)
34 (29.6)
22 (16.1)
32 (33.0)
29 (21.6)
14 (8.1)
GAD module (Mini-Tracking) - anxiety symptoms
Normal
(0-8)
151 (37.4)
70 (27.0)
81 (55.9)
75 (49.3)
25 (21.7)
51 (37.2)
20 (20.6)
45 (33.6)
86 (49.7)
Mild (9-16)
131 (32.4)
94 (36.3)
37 (25.5)
38 (25.0)
45 (39.1)
48 (35.0)
36 (37.1)
43 (32.1)
52 (30.1)
Moderate
(17-24)
95 (23.5)
74 (28.6)
21 (14.5)
32 (21.1)
33 (28.7)
30 (21.9)
31 (32.0)
33 (24.6)
31 (17.9)
Severe
(25-32)
27 (6.7)
21 (8.1)
6 (4.1)
7 (4.6)
12 (10.4)
8 (5.8)
10 (10.3)
13 (9.7)
4 (2.3)
ISI - insomnia symptoms
Absence
(0-7)
199 (49.3)
108 (41.7)
91 (62.8)
84 (55.3)
42 (36.5)
73 (53.3)
37 (38.2)
60 (44.8)
102 (59.0)
Subthresh-
old (8-14)
134 (33.2)
96 (37.1)
38 (26.2)
44 (28.9)
47 (40.9)
43 (31.4)
36 (37.1)
50 (37.3)
48 (27.7)
Moderate
(15-21)
65 (16.1)
51 (19.7)
14 (9.7)
20 (13.2)
26 (22.6)
19 (13.9)
22 (22.7)
23 (17.2)
20 (11.6)
Severe
(22-28)
6 (1.5)
4 (1.5)
2 (1.4)
4 (2.6)
0 (0.0)
2 (1.5)
2 (2.1)
1 (0.7)
3 (1.7)
IES-R - distress symptoms
Normal
(0-8)
71 (17.6)
35 (13.5)
36 (24.8)
41 (27.0)
11 (9.6)
19 (13.9)
12 (12.4)
22 (16.4)
37 (21.4)
Mild (9-25)
162 (40.1)
94 (36.3)
68 (46.9)
62 (40.8)
36 (31.3)
64 (46.7)
34 (35.1)
51 (38.1)
77 (44.5)
Moderate
(26-43)
112 (27.7)
86 (33.2)
26 (17.9)
31 (20.4)
47 (40.9)
34 (24.8)
34 (35.1)
37 (27.6)
41 (23.7)
Severe
(44-88)
59 (14.6)
44 (17.0)
15 (10.3)
18 (11.8)
21 (18.3)
20 (14.6)
17 (17.5)
24 (17.9)
18 (10.4) Table 2: Descriptive statistics of the severity categories of depression, anxiety, insomnia,
and distress in Brazilian orthodontists during COVID-19 pandemic. RESULTS PHQ-9 = 9-item Patient Health Questionnaire; GAD = Generalized Anxiety Disorder; ISI = Insomnia Severity
Index; IES-R = Impact of Event Scale–Revised. Dental Press J Orthod. 2022;27(6):e2221219 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective
10 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective 10 Statistically significant differences were observed in all com-
pared categories (Table 3). Greater median scores were
observed for females, graduate students, and professionals
with lowest incomes (<10k). Table 3: Total scores of depression, anxiety, insomnia, and distress in orthodontists, and com-
parison between sex, professional status, and economic income range. * Statistically significant for p < 0.05. Different capital letters indicate statistically significant differences. PHQ-9 = 9-item
Patient Health Questionnaire; GAD = Generalized Anxiety Disorder; ISI = Insomnia Severity Index; IES-R = Impact of
Event Scale–Revised. IQR = interquartile range. Question-
naire
TOTAL
SCORE
Median
(IQR)
SEX
Median (IQR)
PROFESSIONAL STATUS
Median (IQR)
ECONOMIC INCOME
Median (IQR)
Female
Male
p
value
Professor
Graduate
Clinician
p
value
<5k
5-10k
>10k
p
value
PHQ-9
(0-27)
8
(4.0-13.0)
9
(5.0-14.0)
4
(2.0-8.0)
< 0.001*
5.5A
(2.0-10.0)
10.0B
(6.0-15.0)
7.0A
(4.0-12.5)
< 0.001*
10.0A
(7.0-16.0)
8.0A
(4.0-14.0)
5.0B
(2.0-9.0)
< 0.001*
GAD module
(Mini-Tracking)
(0-32),
anxiety
symptoms
11
(6.0-18.0)
14
(8.0-20.0)
7
(4.0-13.0)
< 0.001*
9.0A
(4.7-17.0)
14.0B
(9.0-20.0)
11.0A
(6.0-17.0)
< 0.001*
15.0A
(9.0-20.0)
12.5A
(6.0-19.0)
9.0B
(4.0-15.0)
< 0.001*
ISI (0-28),
insomnia
symptoms
8
(3.0-12.3)
9
(3.5-13.5)
5
(2.0-11.0)
< 0.001*
6.0A
(2.0-12.0)
10.0B
(5.0-14.0)
7.0A
(3.0-12.0)
< 0.001*
10.0A
(4.0-14.0)
9.0A
(3.2-12.8)
6.0B
(2.0-11.0)
< 0.001*
IES-R
(0-88),
distress
symptoms
22.5
(12.0-36.0)
26
(14.0-39.0)
17
(9.0-27.0)
< 0.001*
18.5A
(8.0-29.0)
30.0B
(17.0-39.5)
21.0A
(12.0-34.0)
< 0.001*
26.0A
(17.0-41.0)
23.0A
(12.0-38.0)
18.0B
(10.0-29.0)
< 0.001* Table 3: Total scores of depression, anxiety, insomnia, and distress in orthodontists, and com-
parison between sex, professional status, and economic income range. Table 3: Total scores of depression, anxiety, insomnia, and distress in orthodontists, and com-
parison between sex, professional status, and economic income range. * Statistically significant for p < 0.05. Different capital letters indicate statistically significant differences. RESULTS PHQ-9 = 9-item
Patient Health Questionnaire; GAD = Generalized Anxiety Disorder; ISI = Insomnia Severity Index; IES-R = Impact of
Event Scale–Revised. IQR = interquartile range. Distinct levels of concern were observed among orthodontists regarding the
impact of the coronavirus pandemic in the financial and professional activities
(Fig 1). Females demonstrated greater concern about payment of office expenses,
delay of treatment end, contamination risks, and emergency appointments
(Table 4). Professors demonstrated lower concern about patient’s dropouts and
delays of treatment end, compared to graduate students and clinicians (Table 4). Dental Press J Orthod. 2022;27(6):e2221219 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective
11 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective Professionals with highest incomes (>10k) were less concerned regarding
the delay of treatment end, contamination risks, and emergency appoint-
ments (Table 4). Different capital letters indicate statistically significant differences. * Statistically significant for p < 0.05. Different capital letters indicate statistically significant differences. RESULTS — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective
12 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective 12 RESULTS Questionnaire
TOTAL
SCORE
Median
(IQR)
SEX
Median (IQR)
PROFESSIONAL STATUS
Median (IQR)
ECONOMIC INCOME
Median (IQR)
Female
Male
p val-
ue
Pro-
fessor
Post-
gradu-
ate
Clini-
cian
p val-
ue
<5k
5-10k
>10k
p value
Payment of of-
fice expenses
(0-4)
3
(1-3)
3
(2-3)
2
(1-3)
0.032*
2
(1-3)
3
(2-4)
3
(2-3)
0.131
3
(2-3)
3
(2-4)
2
(1-3)
0.139
Payment of per-
sonal expenses
(0-4)
2
(1-3)
2
(1-3)
2
(1-3)
0.131
2
(1-3)
2
(1-4)
2
(1-3)
0.077
2
(1-4)
2
(1-3)
2
(1-3)
0.42
Patient non-pay-
ment dues
(0-4)
2
(1-3)
3
(2-4)
3
(2-4)
0.32
2
(1-3)
3
(2-3)
2
(2-3)
0.219
3
(1-3)
2
(1-3)
2
(2-3)
0.917
Decrease in the
number of pa-
tients (0-4)
3
(2-4)
3
(2-4)
3
(2-4)
0.23
2.0A
(1-3)
3.0B
(2-4)
3.0B
(2-4)
0.000*
3
(2-4)
3
(1-4)
2
(2-4)
0.061
Emergency funds
(0-4)
2
(1-3)
2
(1-3)
2
(1-3)
0.164
2
(1-3)
2
(1-3)
2
(1-3)
0.499
2
(1-3)
2
(1-3)
2
(1-3)
0.302
Dropouts of or-
thodontic treat-
ment
(0-4)
3
(1-4)
3
(1-4)
2
(1-3)
0.061
2.0A
(1-3)
3.0B
(2-4)
3.0B
(1-4)
0.000*
3
(1-4)
2
(1-4)
2
(1-3)
0.117
Damages to pa-
tients’ oral health
(0-4)
2
(2-3)
2
(2-3)
2
(1-3)
0.403
2
(2-3)
2
(2-3)
2
(1-3)
0.508
3
(2-3)
3
(1-3)
2
(2-3)
0.497
Delay of treat-
ment end
(0-4)
3
(1-3)
3
(2-3)
2
(1-3)
0.010*
2.0A
(1-3)
3.0B
(2-4)
3.0AB
(1-3)
0.006*
3.0A
(2-4)
3.0AB
(1-3)
2.0B
(1-3)
0.005*
Contamination
risk after social
isolation (0-4)
3
(2-4)
3
(2-4)
2
(1-3)
0.001*
3
(1-4)
3
(2-4)
2
(1-4)
0.092
3.0A
(2-4)
3.0A
(2-4)
2.0B
(1-3)
0.006*
Emergency
appointments
during the isola-
tion period (0-4)
2
(1-3)
3
(1-4)
2
(1-3)
0.000*
2
(1-3)
2
(1-4)
2
(1-3)
0.222
3.0A
(1-4)
3.0A
(1-4)
2.0B
(1-3)
0.008*
Table 4: Perception of orthodontics about income and appointments (Mann-Whitney and
Kruskal-Wallis tests). * Statistically significant for p < 0.05. Table 4: Perception of orthodontics about income and appointments (Mann-Whitney and
Kruskal-Wallis tests). Table 4: Perception of orthodontics about income and appointments (Mann-Whitney and
Kruskal-Wallis tests). * Statistically significant for p < 0.05. Different capital letters indicate statistically significant differences. Different capital letters indicate statistically significant differences. Dental Press J Orthod. 2022;27(6):e2221219 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. DISCUSSION To the best of our knowledge, this is one of the few studies
that evaluated the psychological and financial impacts of the
coronavirus pandemic in orthodontics. Moreover, it is essen-
tial to highlight that the results reported here are relevant to
the time the study was conducted, that is, in the early stages of
the pandemic in Brazil. Several studies conducted at the same
time worldwide showed similar results. It could be stated that
Orthodontics situation was similar all over the world. Most of
the respondents reported perceived economic, psychosocial,
and social impacts due to the pandemic.24-27 An electronic questionnaire was applied in a Brazilian ortho-
dontic population and 404 responses were received. Female
orthodontists comprised the majority of the sample (64.1%). This was expected since most of orthodontists in Brazil are
female.14 Additionally, previous studies showed a greater
female response prevalence in studies involving question-
naires with health professionals.7,28-32 Overall, most of the par-
ticipants were professors or graduate students, suggesting
that academic professionals were most predisposed to answer
the questionnaire. Outbreaks of infectious diseases cause a high psychological impact
on health professionals.28 In the present study, the participants
reported mild to severe symptoms of depression (68.4%), Dental Press J Orthod. 2022;27(6):e2221219 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective
13 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective 13 anxiety (62.2%) insomnia (50.7%), and distress (82.4%), which
suggests a negative impact of COVID-19 pandemic in the men-
tal health of orthodontists (Table 2). DISCUSSION A similar study conducted
in the USA during the same stages of the pandemic showed
that dentists reported symptoms of depression and anxiety as
well.33 Negative psychologic impact in healthcare workers have
been previously reported during coronavirus pandemic.7,11,29,32
Also, anxiety and depression are very associated with sleep
disturbances.34,35 Compared with other occupational groups,
healthcare workers reported the highest rate of poor sleep
quality.36 Therefore, the concern with mental health is directly
related to physical health, and both need attention by the occu-
pational health policies, during the pandemic COVID-19.7,31 Females were statistically significant more affected than males
regarding all psychological symptoms reported in this study
(Tables 2 and 3), which agrees with Gibson et al.37 They stated
that female sex is one of the factors that could predict mental
health inequalities during the COVID-19 pandemic. This was
expected; since females usually report higher anxiety scores
than males.38,39 During the coronavirus pandemic, previous
studies showed that adult females also reported higher anx-
iety scores.31,40 In addition, moderate to severe symptoms of
depression, anxiety, insomnia, and distress was found for female
physicians and nurses.7,41 On the other hand, a similar study con-
ducted in the same period in Turkey showed different results.24 Dental Press J Orthod. 2022;27(6):e2221219 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective
14 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective
14 14 The authors reported that only 16.7% of the orthodontists had
anxiety symptoms, and there was no statistically significant
difference when the prevalence of these symptoms was strati-
fied by sex and age. It could be speculated that although both
studies were conducted simultaneously, the countries were in
different phases of the pandemic. One must also consider the
geographic and cultural differences between the two countries. Graduate participants were significantly more affected than
clinicians and professors regarding all psychological issues
(Tables 2 and 3). DISCUSSION Similarly, graduate students experienced a
negative psychological impact of the coronavirus outbreak in
China.31 The results of this study are in accordance with previous
reports showing that younger people demonstrated a signifi-
cantly higher prevalence of anxiety and depressive symptoms
than older ones.36,42,43 So, it could be speculated that graduated
students were more affected than clinicians and professors
because they are usually younger. The present research showed significantly greater psychological
impact in professionals with income below 10k (Tables 2 and 3). In Brazil, the postponing of elective dental procedures caused
a substantial reduction of orthodontics activities. According to
Cotrin et al.44 the number of jobs reduced for all healthcare
workers in Brazil during the first stages of the pandemic, but
this reduction was significantly greater for dentists. It has been Dental Press J Orthod. 2022;27(6):e2221219 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective
15 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective 15 reported that people with financial stress are more vulnerable
to mental health issues.43 A previous study with dental practi-
tioners showed that the negative economic impact of the office
closure was subsequently associated with concerns about pro-
fessional future, anxiety, and fear.11 Additionally, lower incomes
were previously associated with higher levels of distress.43,45,46 Most orthodontists presented moderate to extreme concerns
regarding financial issues, including dropouts of orthodontic
treatment and patient’s non-payment dues. Interestingly, they
were extremely more concerned with patients’ dropouts than
having a financial emergency personal fund (Fig 1). In addition,
orthodontists also showed concern about contamination risks
after social isolation (Fig 1). Coronavirus outbreak has affected
all sectors in the economy all over the world, and it was not
different in Brazil, which already presented a fragile economic
situation before the pandemic. DISCUSSION The New York Times recently
identified dentists as in the highest risk of contamination at that
time.47 Due to these facts, the postponing of the elective den-
tal procedures was strongly recommended, resulting in severe
monetary implications for dental practitioners worldwide.48
In addition, different from other countries, no governmental
assistance was provided to Brazilian clinicians.48,49 Then, ortho-
dontists can be expected to be dramatically concerned about
the financial impact that the pandemic will cause during the
restart of their dental practices.50 Most orthodontists presented moderate to extreme concerns
regarding financial issues, including dropouts of orthodontic
treatment and patient’s non-payment dues. Interestingly, they
were extremely more concerned with patients’ dropouts than
having a financial emergency personal fund (Fig 1). In addition,
orthodontists also showed concern about contamination risks
after social isolation (Fig 1). Coronavirus outbreak has affected
all sectors in the economy all over the world, and it was not
different in Brazil, which already presented a fragile economic
situation before the pandemic. The New York Times recently
identified dentists as in the highest risk of contamination at that
time.47 Due to these facts, the postponing of the elective den-
tal procedures was strongly recommended, resulting in severe
monetary implications for dental practitioners worldwide.48
In addition, different from other countries, no governmental
assistance was provided to Brazilian clinicians.48,49 Then, ortho-
dontists can be expected to be dramatically concerned about
the financial impact that the pandemic will cause during the
restart of their dental practices.50 Dental Press J Orthod. 2022;27(6):e2221219 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective
16 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective Regarding differences between sexes, the only financial
issue that the female orthodontists were significantly more
concerned than males was regarding the payment of office
expenses (Table 4). This specific concern agrees with Ferneini,51
who evaluated the financial impact of COVID-19 in the dental
practice. The author stated that the pandemic brought over-
head costs because it required the orthodontic team to have
a better and safer working environment for the patients, staff,
and orthodontists. DISCUSSION This will potentially increase orthodontists’
business overhead and reduce the profit margin even further. When evaluating the impact of the coronavirus pandemic on the
orthodontists’ professional lives, the females showed greater
concern regarding the delay of treatment end, contamina-
tions risks and emergency appointments than males (Table 4). These findings reinforce the greater emotional impact of the
pandemic in female orthodontists (Table 3). These sex differ-
ences on risk and resilience to stress are complex and varies
according to characteristics of the stressful factor, such as type,
timing, and duration, as well as changes in brain structure and
function.39 Moreover, health care providers are particularly vul-
nerable to emotional distress in the current pandemic, due to
the exposure risks, and the additional concern about infecting
their family and friends.7,52 Dental Press J Orthod. 2022;27(6):e2221219 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective
17 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective
17 Among the three evaluated groups, professors showed the
lowest level of concern regarding patients’ dropouts, decreas-
ing number of patients and delay of treatment end (Table 4). The delay of treatment end was also a recurring concern among
patients during the pandemic.53,54 It could be thought that grad-
uate students and clinicians have higher levels of patient-re-
lated concerns due to the nature of their clinical routine. In the
other hand, professors usually have a greater workload dedi-
cated to teaching and research. Finally, orthodontists with higher incomes showed a lower
level of concerns regarding the delay of treatment end, con-
tamination risks and emergency appointments during the iso-
lation period (Table 4). It is not surprising that financial security
influences the behavior of orthodontists in other areas of their
lives. DISCUSSION — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective DISCUSSION Higher income has been reported as a beneficial factor
for psychological wellbeing.55,56 A previous study showed that
respondents with higher income were happier, more satisfied
with their lives, health, achievement, future economic situa-
tion, and social conditions.57 It is important to highlight that the present findings directly
inform the effects of the coronavirus pandemic on the men-
tal health and financial impact in orthodontists during the
first stages of the pandemic. At the time when this survey Dental Press J Orthod. 2022;27(6):e2221219 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective
18 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective 18 was conducted, Orthodontics and Dentistry in general were
lost about how to proceed amidst the chaos of the pandemic. There was no precise scientific evidence regarding the safety
of the procedures and care provided. These results may help
to draw attention to the need for prevention and control of
physiological and financial issues during the coronavirus out-
break, which is still ongoing at the time of this research was
conducted. Some changes in the orthodontic practice have
come to stay. Garcia-Camba et al.58 stated that these changes
concern four areas: microbiologic control measures, social dis-
tancing measures by redistributing spaces and decreasing the
number of patients and companions in the clinics, increasing
teleorthodontics and use of appliances and techniques that
requires fewer scheduled and urgent appointments, and bio-
ethical considerations to promote a broader view of the psy-
chological aspects of the patients and the community. A limitation of the present study is the cross-sectional design,
since psychological symptoms may change as a consequence of
the coronavirus crisis variation. Follow-up studies are needed
to complement the present results. Dental Press J Orthod. 2022;27(6):e2221219 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. CONCLUSIONS In this study performed at the beginning of the pandemic: »
Brazilian orthodontists reported high rates of symptoms of
depression, anxiety, insomnia, and distress. Female, gradu-
ate students and income below 10k were the most affected. »
Most orthodontists were from moderate to extremely con-
cerned about financial issues and regarding patient care
during the pandemic. »
Female orthodontists showed a higher level of financial
concern than their male counterparts. »
Professors showed a higher level of financial concern than
postgraduate students and clinicians. »
Orthodontists with higher income showed a low level of
concern with delay in the orthodontic treatment, contami-
nation after social isolation, and caring of urgencies during
the social isolation. Dental Press J Orthod. 2022;27(6):e2221219 Dental Press J Orthod. 2022;27(6):e2221219 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective
0 20 Ludmila Mangialardo Lima (LML)
Aron Aliaga-Del Castillo (AADC)
Camila Massaro (CM)
Caroline Gambardela-Tkacz (CGT)
Deborah B. de A. Brito (DBAB)
Felicia Miranda (FM)
Lorena Vilanova (LV)
Paula Cotrin (PC)
Raquel Silva Poletto (RSP)
Wilana Moura (WM)
Arnaldo Pinzan (AP)
Guilherme Janson (GJ)
Fabiola Alvarez Avila (FAA) Ludmila Mangialardo Lima (LML)
Aron Aliaga-Del Castillo (AADC)
Camila Massaro (CM)
Caroline Gambardela-Tkacz (CGT)
Deborah B. de A. Brito (DBAB)
Felicia Miranda (FM)
Lorena Vilanova (LV)
Paula Cotrin (PC)
Raquel Silva Poletto (RSP)
Wilana Moura (WM)
Arnaldo Pinzan (AP)
Guilherme Janson (GJ)
Fabiola Alvarez Avila (FAA) The authors report no commercial, proprietary or financial interest in the products or
companies described in this article. Dental Press J Orthod. 2022;27(6):e2221219 Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
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and financial impact of the COVID-19 pandemic during the first stages of the pandemic: Brazilian
orthodontists´ perspective Lima LM, Aliaga-Del Castillo A, Massaro C, Gambardela CM, Brito DBA, Miranda F, et al. — Psychological
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Author Correction: Regional and temporal variability of Indian summer monsoon rainfall in relation to El Niño southern oscillation
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www.nature.com/scientificreports www.nature.com/scientificreports Author Correction: Regional
and temporal variability of Indian
summer monsoon rainfall
in relation to El Niño southern
oscillation
OPEN K. S. Athira , M. K. Roxy , Panini Dasgupta , J. S. Saranya , Vineet Kumar Singh & Raju Attada Correction to: Scientific Reports https://doi.org/10.1038/s41598-023-38730-5, published online 04 August 2023 In the original version of this Article, Panini Dasgupta and J. S. Saranya were incorrectly affiliated with ‘Seoul
National University, Seoul, South Korea’. The correct affiliations are listed below: Panini Dasgupta: • Centre for Climate Change Research, Indian Institute of Tropical Meteorology, Ministry of Earth Sciences,
Pune, India. • Department of Meteorology and Oceanography, College of Science and Technology, Andhra University,
Visakhapatnam, India. • ‘Future Innovation Institute, Seoul National University, Siheung 15011, Seoul, Republic of Korea’ J. S. Saranya: • Centre for Climate Change Research, Indian Institute of Tropical Meteorology, Ministry of Earth Sciences,
Pune, India. • College of Climate Change and Environmental Sciences, Kerala Agricultural University, Thrissur, India
• ‘School of Earth and Environmental Sciences/Research Institute of Oceanography, Seoul National University,
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IκB kinases increase Myc protein stability and enhance progression of breast cancer cells
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Molecular cancer
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RESEARCH Open Access © 2011 Yeh et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. * Correspondence: alcheng@ntu.edu.tw
1Department of Oncology, National Taiwan University Hospital, No. 7,
Chung-Shan South Road, Taipei, 100, Taiwan
Full list of author information is available at the end of the article Abstract Background: Both IB kinase (IKK) complex and oncgenic protein Myc play important roles in cancer progression,
including cancer cell invasiveness and metastasis. The levels of Myc is regulated by the phosphorylation of Myc at
Thr58 and Ser62. Results: In this study, we show that the expression of Myc is associated with IKKa and IKKb in breast cancers and
that Myc is an IKKs substrate. Suppression of IKK activity by either chemical inhibitor or transfection of kinase-dead
mutants decreases the phosphorylation of Myc at Ser62 and enhances the degradation of Myc. Consequently,
these treatments decrease the tumorigenic and invasive ability of breast cancer cells. Furthermore, doxorubicin, a
frequently used anticancer drug in breast cancer, activates IKKs and Myc, thereby increasing invasiveness and
tumorigenesis of breast carcinoma MCF7 cells. Inhibition of IKKs prevents these doxorubicin-induced effects. Conclusions: Our study indicates that IKKs tightly regulate Myc expression through prolonging protein stability,
and suggests that IKKs are potentially therapeutic targets and that suppression of IKKs may be used following
chemotherapy to reduce the risk of treatment-induced tumor progression. FOXO3a, and consequently induces FOXO3a nuclear
exclusion and degradation, thereby promoting tumor
survival [11]. Interesting, IKKa and IKKb may have
opposite effect on certain proteins. For example, IKKa
increases but IKKb decreases the transcriptional activity
and protein level of b-catenin [12,13]. The biological
significance of IKKs is getting complicated and requires
further characterization. The identification of new sub-
strates of IKKs is important for the understanding of
IKKs functions in cancer biology. Pei-Yen Yeh1, Yen-Shen Lu1, Da-Liang Ou2,4 and Ann-Lii Cheng1,3,4* Pei-Yen Yeh1, Yen-Shen Lu1, Da-Liang Ou2,4 and Ann-Lii Cheng1,3,4* Yeh et al. Molecular Cancer 2011, 10:53 Yeh et al. Molecular Cancer 2011, 10:53
http://www.molecular-cancer.com/content/10/1/53 http://www.molecular-cancer.com/content/10/1/53 Background The IKK complex is composed of two kinase catalytic
subunits IKKa and IKKb and a non-kinase scaffold pro-
tein IKKg [1-3]. The complex functions as an upstream
kinase involved in the activation of nuclear factor kappa
B(NF-B)by phosphorylation of the NF-B inhibitory
molecule, IBa, resulting in the subsequent degradation
of IBa through the ubiqutin/proteasome pathway. The
released NF-B translocates into the nucleus and then
regulates the expression of multiple genes [1,4,5]. Numerous reports have indicated that the functions of
IKKs are necessary for cancer cell survival and progres-
sion [3,6-8]. The oncogenic Myc protein is a transcription factor
that regulates a wide spectrum of downstream genes
involved in cancer cell metabolism, growth, and progres-
sion [14-17], and it is well documented that Myc plays
an important role in breast cancer metastasis [17-19]. Abnormal expression of Myc is frequently associated
with cancer progression [20-23]. Several transcription
factors, including NF-B, E2F, STAT, and b-catenin, are
involved in the regulation of Myc expression [24,25]. Inhibition of these transcription factors suppresses can-
cer cell survival in part by decreasing Myc expression. Most studies regarding IKKs are actually focused on
their downstream molecule, NF-B, and the thinking
that IKKs might be therapeutic targets is trying to indir-
ectly suppress NF-B activation [1,9]. However, accumu-
lating evidence has indicated that IKKs have NF-B-
independent effects on multiple proteins [1,10]. For
example, IKKb phosphorylates tumor suppressor The Myc protein level is further regulated by control
of protein stability, which is determined by a compli-
cated
protein
kinase/phosphatase
system. * Correspondence: alcheng@ntu.edu.tw
1Department of Oncology, National Taiwan University Hospital, No. 7,
Chung-Shan South Road, Taipei, 100, Taiwan
Full list of author information is available at the end of the article Page 2 of 12 Yeh et al. Molecular Cancer 2011, 10:53
http://www.molecular-cancer.com/content/10/1/53 Yeh et al. Molecular Cancer 2011, 10:53
http://www.molecular-cancer.com/content/10/1/53 Yeh et al. Molecular Cancer 2011, 10:53
http://www.molecular-cancer.com/content/10/1/53 Phosphorylation of Myc at Ser62 increases protein stabi-
lity. The kinases ERK (extracellular signal-regulated
kinase), JNK (c-Jun N-terminal kinase) and cdk1 (cyclin-
dependent kinase 1) have been identified to phosphory-
late Myc at Ser62 [16,26,27]. The Ser62 phosphorylated
Myc is further phosphorylated at Thr58 by glycogen
synthase kinase 3b. The Thr58/Ser62 dual phosphory-
lated Myc is acted on by protein phosphatase 2A
[PP2A] to dephosphorylate Ser62. Then, monopho-
sphorylated Myc (at Thr58) is degraded by ubiquitin/
proteosome system. Materials and methods
Patients The specimens were acquired between 2009 and 2010
from patients with infiltrating ductal carcinoma of the
breast, prior to chemotherapy without adjuvant, and
were kindly provided by the Department of Pathology,
National Taiwan University Hospital, on the basis of
their availability. Use of these tissue materials followed
the regulations of the research ethics committee of the
National Taiwan University Hospital. Background A cellular PP2A inhibitor cip2A
which is overexpressed in several cancers has been
shown to increase Myc levels via suppression of PP2A
activity [16,28,29]. Given the fact that numerous intra-
and extra-cellular stimuli regulate the activation of Myc,
it is expected that other unidentified kinases may be
also involved. streptavidin-biotin-peroxidase kit(Vectastain Universal
Quick Kit; Vector Laboratories, Burlingame, CA, USA)
according to the manufacturer’s instructions. DAB/chro-
mogen system (Dako Northern America, Inc.)was used
to develop the image. The antibodies, including anti-
IKKa (sc-7183), anti-IKKb (sc-7329), anti-NF-B p65
(sc-372) and anti-Myc (sc-40), were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA, USA) and
were used at a 1:50 dilution. The staining was judged
and counted by a researcher who was blinded regarding
the corresponding patient. Cells and reagents
ll Breast cancer cell line MCF7 was purchased from the
American Type Culture Collection. The cells were cul-
tured in Dulbecco’s Modified Eagle Medium supplemen-
ted with 10% FCS and incubated in 37°C with 5% CO2. All chemicals and reagents used were purchased from
Calbiochem or Sigma-Aldrich. In this study, we investigated the association of Myc
and IKK/NF-B in breast cancer. Interestingly, IHC
staining of breast cancer specimens showed that the
expression of Myc was closely associated with that of
IKKs but not with NF-B p65. We demonstrated that
IKKa and IKKb increased Myc protein levels by
prolonging protein stability, and this consequently pro-
moted the tumorigenic and invasive activity of breast
cancer cells. Our results also indicated that IKKa but
not IKKb directly interacted with Myc. In addition, we
showed that a conventional anti-cancer drug, doxorubi-
cin, activated the IKKs-Myc pathway which might
enhance tumor progression. Together, our study indi-
cated that suppression of IKKa and IKKb may decrease
basal and stress-induced Myc protein levels. The latter
suggested that inhibition of IKKs may be used to block
treatment-induced tumor progression. Plasmid and transfection The IKKa and IKKb (both wild-type and kinase-dead
mutant) expression vectors were kindly provided by Pro-
fessor WC Greene (Gladstone Institute of Virology and
Immunology, University of California, San Francisco),
and were used to transfect MCF7 cells using Lipofecta-
mine 2000 (Invitrogen, Carlsbad, CA, USA). The trans-
fected cells were selected and maintained with complete
medium containing 500 μg/ml G418. Coimmunoprecipitation and Western blot analysis p
p
y
Whole cell lysates were prepared in RIPA solution con-
taining a cocktail of protease and phosphatase inhibitors,
or the cells were fractionated into cytoplasmic and
nuclear fractions. For cellular fractionation, the cells were
harvested and resuspended in hypotonic solution (Buffer
1; 1 mM KCl, 0.2 mM MgCl2, 4 mM Tris, pH7.6, con-
taining a cocktail of protease inhibitors) for 20 minutes
on ice. The cells were then lysed by adding lysis buffer
(Buffer 1 containing 1% Triton X-100) and vigorously
vortexed for 20 seconds, and then centrifuged at 1500
rpm for 5 minutes. The supernatant was collected as
cytoplasmic fraction, and the pellet was washed once
with PBS buffer and then lysed by RIPA buffer as nuclear
fraction. After determination of protein concentration of
each lysates, the aliquots (15 μg) were subjected to Wes-
tern blot analysis. For coimmunoprecipitation, target
proteins were immunoprecipitated from whole cell
lysates (500 μg) by adding 2 μg antibody at 4°C overnight
followed by protein A/G agarose adsorption(Santa Cruz
Biotechnology, Santa Cruz, CA, USA). The complex was
washed once with RIPA buffer containing 500 mM NaCl,
once with RIPA buffer containing 250 mM NaCl, and
two times with RIPA buffer. The washed complex was Statistical analysis RNA was extracted using Trizol reagent (Invitrogen). cDNAs were synthesized by a reverse transcription reac-
tion. The expression of gene was quantified using SYBR
Green PCR Master Mix on an ABI PRISM 7900 system
(Applied Biosystems). The primers used were 5’-
TCGACTACGACTCGGTGCAG (forward), 5’-TGG
GCAGCAGCTCGAATTTC (reverse) for Myc; and 5’-
TCGGAGTCAACGGATTTGG(forward),
5’-GAAT
TTGCCATGGGTGGAAT (reverse) for GAPDH. The
expression level of GAPDH was used a control. The
PCR reaction was performed with the following pro-
gram: 95°C for 10 minutes, and then 40 cycles of 95°C
for 15 seconds and 60°C for 1 minute. The relative
expression level of the target gene was calculated using
the ΔCt (threshold cycle) method: relative expression =
2-ΔCt, where ΔCt = Ct (target gene) - Ct (control gene). The association of IKKa, IKKb, NF-B p65 and Myc
expression in a total of 21 breast cancer specimens was
analyzed with Fisher exact test (SPSS software for Win-
dows 11.0, SPSS, Inc., Chicago, IL). A probability of
error <5% was regarded as significant. Invasion assay An in vitro invasion assay was performed by analyzing
the ability of tumor cells to penetrate through Matrigel
(BD Biosciences). The cells (2 × 104) were seeded into
the chamber of a 24 trans-well plate preloaded with 0.1
ml Matrigel for 16 hours. The penetrated cells were
fixed with methanol, stained with Giemsa solution,
photographed, and counted. The expression of Myc is associated with IKKs but not
with NF-B in breast cancer To identify whether the expression of Myc was asso-
ciated with IKKs/NF-B expressions in vivo, IHC stain-
ing was used to identify the expression of IKKa, IKKb,
Myc and NF-B p65 in a total of 21 breast cancer spe-
cimens. The staining of more than 50% of the cells in
a single field of view and at least 5 fields in a specimen
were designated as positive [Figure 1]. The ratio of
positive staining was 67% (14/21) for IKKa, 57% (12/
21) for IKKb, 67% (14/21) for NF-B p65, and 62%
(13/21) for Myc. When the expression of Myc was
assessed based on nuclear staining, the ratio was 48%
(10/21), similar to the result of a previous study [30]. Statistical analysis showed that Myc expression was
correlated with IKKa and IKKb expression, whereas it
had no correlation with the expression of NF-B p65
(Figure 1). This result suggested that IKKs might regu-
late Myc expression through an NF-B-independent
pathway. Immunohistochemical study (IHC) The tumor tissue embedded in paraffin was cut in 5-μM
section, and then de-paraffinized in xylene and rehy-
drated. For antigen-retrieval, the sections were incubated
with 10 mM sodium citrate buffer (pH 6.0) in a boiling
water bath for 15 minutes. The slides were then incu-
bated with 3% H2O2 in methanol to block endogenous
peroxidase activity. IHC staining was performed using a Page 3 of 12 Yeh et al. Molecular Cancer 2011, 10:53
http://www.molecular-cancer.com/content/10/1/53 lysates (15 μg) were then subjected to Western blot ana-
lysis to identify the Myc and tubulin proteins. Protein
levels were quantified using VisionWorksLS version 7.0
software (UVP, Upland, CA, USA). then resolved in SDS-sample buffer and was subjected to
Western blot analysis. The antibodies used were as fol-
lows: anti-NF-B p65, anti- NF-B p50, anti-pMyc, anti-
Max, and anti-twist antibodies from Santa Cruz Biotech-
nology; anti-IKKa, anti-IKKb, anti-Myc, and anti-cyclin
D1 antibodies from Cell Signaling; anti-pS62 Myc anti-
body from Abnova (Taipei City, Taiwan); and anti-pT58
Myc antibody from Abgent(San Diego, CA, USA). Assay of growth rate and colony formation in soft agar
For proliferation assay, the cells (2000 cells/well) were
seeded into 96-well culture plate. The cell number was
evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT)-based semi-automated col-
orimetric assay. For soft agar colony-forming assay,
20000 cells in complete medium containing 0.3% Bacto-
agar were overlaid on 1% agar-complete medium in 60
mm culture dish for two weeks. The colony number
was counted under phase contrast microscopy at 4X
magnification. Confocal microscopy observation Breast cancer tissue slides previously identified as posi-
tive or negative staining by IHC analysis were used for
confocal microscopy observation. The process for de-
paraffin and antigen retrieval was as in the IHC staining
protocol. The slides were dual-stained using rabbit anti-
IKKa and mouse anti-Myc antibodies coupled with
FITC-conjugated goat anti-rabbit IgG and rodamine-
conjugated donkey anti-mouse IgG antibodies, or goat
anti-IKKb and mouse anti-Myc antibodies coupled with
FITC-conjugated donkey anti-goat IgG and rodamine-
conjugated donkey anti-mouse IgG antibodies. The
images were captured using a confocal microscope (TCS
SP2; Leica, Wetzlar, Germany) at the confocal micro-
scopy core-facilities of the National Taiwan University
Hospital. Determination of RNA and protein stability To determine the stability of mRNA, the cells were trea-
ted with 5 μg/ml actinomycin D to block new mRNA
synthesis. Total RNAs were extracted at different time
point after treatment. Random-primed reverse tran-
scribed cDNA was subjected to qPCR analysis to deter-
mine the relative expression levels of the Myc and
GAPDH genes. For protein stability analysis, the cells
were treated with 10 μM cycloheximide, and then whole
cell lysates were prepared after different durations. The Yeh et al. Molecular Cancer 2011, 10:53
http://www molecular cancer com/conten Yeh et al. Molecular Cancer 2011, 10:53
http://www.molecular-cancer.com/content/10/1/53 Page 4 of 12 http://www.molecular-cancer.com/content/10/1/53 Figure 1 Myc expression level is associated with IKKa and IKKb but not NF-B. IHC staining was used to identify the expression of IKKa,
IKKb, NF-B p65 and Myc in breast cancer specimens (upper panel). Representative pictures of positive and negative staining are shown. Statistic
analysis shows a positive association among IKKa and/or IKKb and Myc expression in 21 breast cancer specimens (lower panel). Figure 1 Myc expression level is associated with IKKa and IKKb but not NF-B. IHC staining was used to identify the expression of IKKa,
IKKb, NF-B p65 and Myc in breast cancer specimens (upper panel). Representative pictures of positive and negative staining are shown. Statistic
analysis shows a positive association among IKKa and/or IKKb and Myc expression in 21 breast cancer specimens (lower panel). (Figure 2D). Furthermore, we observed a reciprocal
upregulation of IKKa and IKKb (Figure 2D). (Figure 2D). Furthermore, we observed a reciprocal
upregulation of IKKa and IKKb (Figure 2D). Suppression of IKK activity decreases Myc protein levels
in MCF7 cells To explore the effects of the IKKs on Myc expression,
an IKK inhibitor Bay11-7082 [31,32] was used to treat
breast carcinoma MCF7 cells. Bay11-0782 blocked
TNFa-induced NF-B p65 nuclear translocation (Figure
2A), whereas it did not alter the basal level of cytoplas-
mic or nuclear NF-B p65 or p50, suggesting that
Bay11-7082 did not influence basal activity of NF-B. Bay11-7082 markedly decreased the level of phosphory-
lated and total Myc protein. The Myc-binding partner,
Max, was not affected by Bay11-7082 (Figure 2B). Suppression of either IKKa or IKKb slows the degradation
rate of Myc mRNA To identify whether IKKa or IKKb could increase the
transcription of Myc mRNA, qPCR was used to deter-
mine the relative levels of Myc mRNA in Bay11-7082
treated MCF7 cells and IKK-transfected cells (Figure
3A). Determination of RNA and protein stability Overexpression of either wild-type IKKa or IKKb
slightly increased the level of Myc mRNA. The induc-
tion level was less than two-fold of the control which is
the cut-point of most gene array analyses. Interestingly,
while Bay11-7082 only marginally reduced Myc mRNA
levels, a small increase of Myc mRNA (less than 1.5-fold
of control levels) was observed in both kinase-dead
IKKa- and IKKb-transfected cells. These results sug-
gested that IKKs could increase Myc protein levels in a
transcription-independent manner. To distinguish the role of IKKa and IKKb in the regu-
lation of Myc expression, MCF7 cells were transfected
with IKKa or IKKb [either wild-type or kinase-dead
mutant] expression vectors (Figure 2C). Wild-type IKKa
or IKKb increased Myc protein levels, on the other
hand, kinase-dead IKKa or IKKb decreased Myc expres-
sion. Further, Western blot analysis showed that while
the phosphorylation of Myc at Ser62 was increased in
wild-type IKKa- and IKKb-transfected cells and
decreased in kinase-dead mutant transfected cells, the
phosphorylation of Myc at Thr58 was not affected in
any of the transfected or control cells. The expression of
Max was not changed by the manipulation of IKKs To explore the mechanism which directs the increase
of Myc mRNA in kinase-dead IKKa- and IKKb -trans-
fected cells, we hypothesized that the degradation rate
of Myc mRNA might be prolonged in response to the
decreased Myc protein levels. We used actinomycin D
to block new mRNA synthesis and then determined the Yeh et al. Molecular Cancer 2011, 10:53
http://www molecular-cancer com/content/ Yeh et al. Molecular Cancer 2011, 10:53
http://www.molecular-cancer.com/content/10/1/53 Yeh et al. Molecular Cancer 2011, 10:53 Page 5 of 12 http://www.molecular-cancer.com/content/10/1/53 Figure 2 IKKa and IKKb increase Myc protein level. (A). MCF7 cells were treated with 10 μM Bay11-0782 for 12 hours and then were further
treated with 20 ng/ml TNF-a for 30 minutes. The cytoplasmic and nuclear extracts were subjected to Western blot analysis of NF-B p65
subcellular distribution. Stains of tubulin were used to represent the clearance of cellular fractionation and stains of actin were used as loading
control. (B). MCF7 cells were treated with 10 μM Bay11-0782 for 12 hours, and then the whole cell lysates, the cytoplasmic and nuclear extracts
were prepared. Equal amount of proteins (15 μg/lane) were subjected to Western blotting analysis. (C). MCF7 cells were transfected with IKKa
and IKKb (both wild-type and kinase-dead mutant). Determination of RNA and protein stability The transfected cells were analyzed by Western blotting of IKKa and IKKb.(D). The whole cell
lysates prepared from indicated cells were subjected to Western blot analysis. Figure 2 IKKa and IKKb increase Myc protein level. (A). MCF7 cells were treated with 10 μM Bay11-0782 for 12 hours and then were further
treated with 20 ng/ml TNF-a for 30 minutes. The cytoplasmic and nuclear extracts were subjected to Western blot analysis of NF-B p65
subcellular distribution. Stains of tubulin were used to represent the clearance of cellular fractionation and stains of actin were used as loading
control. (B). MCF7 cells were treated with 10 μM Bay11-0782 for 12 hours, and then the whole cell lysates, the cytoplasmic and nuclear extracts
were prepared. Equal amount of proteins (15 μg/lane) were subjected to Western blotting analysis. (C). MCF7 cells were transfected with IKKa
and IKKb (both wild-type and kinase-dead mutant). The transfected cells were analyzed by Western blotting of IKKa and IKKb.(D). The whole cell
lysates prepared from indicated cells were subjected to Western blot analysis. Figure 2 IKKa and IKKb increase Myc protein level. (A). MCF7 cells were treated with 10 μM Bay11-0782 for 12 hours and then were further
treated with 20 ng/ml TNF-a for 30 minutes. The cytoplasmic and nuclear extracts were subjected to Western blot analysis of NF-B p65
subcellular distribution. Stains of tubulin were used to represent the clearance of cellular fractionation and stains of actin were used as loading
control. (B). MCF7 cells were treated with 10 μM Bay11-0782 for 12 hours, and then the whole cell lysates, the cytoplasmic and nuclear extracts
were prepared. Equal amount of proteins (15 μg/lane) were subjected to Western blotting analysis. (C). MCF7 cells were transfected with IKKa
and IKKb (both wild-type and kinase-dead mutant). The transfected cells were analyzed by Western blotting of IKKa and IKKb.(D). The whole cell
lysates prepared from indicated cells were subjected to Western blot analysis. IKKa directly interacts with the Myc protein degradation rate of the Myc mRNA by qPCR. The decay
of Myc mRNA was at a comparable rate among Bay11-
7082 treated MCF7 cells and wild-type IKKa- and
IKKb-transfected cells, whereas a prolonged degradation
rate was observed in kinase-dead IKKa- and IKKb-
transfected cells (Figure 3B, C and 3D). y
y p
Next, we used reciprocal coimmunoprecipitation fol-
lowed by Western blot analysis to identify potential
interactions between IKKs and Myc. Determination of RNA and protein stability The result showed
that IKKa coimmunoprecitated with Myc, indicating a
direct interaction between IKKa and Myc. However, the
interaction between IKKb and Myc was barely detect-
able (Figure 4D). The interaction between IKKa and
Myc was not affected by loss of IKKa activity (Figure
4E), or by the presence of overexpressed wild-type or
kinase-dead IKKb (Figure 4F). This finding was further
supported by confocal microscopy observation. The spe-
cimens from a patient with breast cancer, which stained
positive for IKKa, IKKb and Myc, were dual-stained for
IKKa/Myc or IKKb/Myc. It was observed that IKKa
colocalized with Myc, whereas IKKb and Myc was not
found to be significantly colocalized (Figure 4G). IKKs increase Myc protein stability Next, we determined the degradation rate of the Myc
protein in Bay11-0782 treated MCF7 cells and in IKK-
transfected cells by Western blot analysis along a time
course after adding a protein synthesis inhibitor, cyclo-
heximide. Bay11-7082 induced a more rapid degradation
rate of Myc protein (Figure 4A). Wild-type IKKa or
IKKb increased the stability of Myc protein. On the
other hand, kinase-dead IKKa or IKKb enhanced Myc
protein degradation (Figure 4B and 4C). Yeh et al. Molecular Cancer 2011, 10:53
http://www.molecular-cancer.com/content/10/1/53 Page 6 of 12 Yeh et al. Molecular Cancer 2011, 10:53 http://www.molecular-cancer.com/content/10/1/53 Figure 3 IKKa and IKKb increase Myc in a transcription-independent manner. (A) MCF7 cells were treated with 10 μM Bay11-0782 for 12
hours. The RNA was extracted from indicated cells and random-primed reverse transcribed into cDNA. The relative expression of Myc was
determined by qPCR. The expression of GAPDH was used as an internal control. The specificity of the primer set for Myc and GAPDH was
demonstrated by agarose electrophoresis of PCR product (inserted figure) and by analysis of dissociation curve (data not shown). Each data
represents mean ± SD calculated from two independent experiments. To determine the degradation rate of Myc mRNA, the cDNAs were
prepared from (B) MCF7 cells were treated with 10 μM Bay11-0782 for 12 hours, (C) MCF7, wild-type IKKa and IKKb transfected cells, and (D)
MCF7, kinase-dead IKKa and IKKb transfected cells. The relative level of Myc mRNA was determined by qPCR and expressed along a time course
after adding actinimycin D. Each data represents mean ± SD calculated from two independent experiments. Figure 3 IKKa and IKKb increase Myc in a transcription-independent manner. (A) MCF7 cells were treated with 10 μM Bay11-0782 for 12
hours. The RNA was extracted from indicated cells and random-primed reverse transcribed into cDNA. The relative expression of Myc was
determined by qPCR. The expression of GAPDH was used as an internal control. The specificity of the primer set for Myc and GAPDH was
demonstrated by agarose electrophoresis of PCR product (inserted figure) and by analysis of dissociation curve (data not shown). Each data
represents mean ± SD calculated from two independent experiments. To determine the degradation rate of Myc mRNA, the cDNAs were
prepared from (B) MCF7 cells were treated with 10 μM Bay11-0782 for 12 hours, (C) MCF7, wild-type IKKa and IKKb transfected cells, and (D)
MCF7, kinase-dead IKKa and IKKb transfected cells. IKKs increase Myc protein stability The relative level of Myc mRNA was determined by qPCR and expressed along a time course
after adding actinimycin D. Each data represents mean ± SD calculated from two independent experiments. IKKs promote anchorage-independent growth and
invasiveness of MCF7 cells decreased in kinase-dead IKKa or IKKb-transfected cells
(Figure 5C). Because cyclin D1 and Twist are two
important down-stream effectors of Myc and their bio-
logical functions are associated with the invasive/tumori-
genic ability of cancer cells [20,33,34], cyclin D1 and
Twist protein levels were determined by Western blot
analysis. Consistently, the levels of these two proteins
were increased in wild-type IKKa- or IKKb- transfected
cells and decreased in kinase-dead IKKa-or IKKb-
transfected cells (Figure 5D). We next characterized the biological functions of IKKs
by analyzing IKK-transfected cells. Cells overexpressing
either wild-type or kinase-dead IKKa or IKKb showed
comparable growth rates (Figure 5A). However, wild-
type IKKa and IKKb increased cell growth in soft-agar,
a well-defined character of tumorigenesis, and kinase-
dead IKKa and IKKb suppressed this ability of the cells
(Figure 5B). Next, we assayed the ability of IKK-overex-
pressing cells to pass through matrigel, a well-estab-
lished method to determine the invasive activity of
cancer cells. Wild-type IKKa or IKKb overexpressing
cells showed a higher invasive ability than parental
MCF7 cells; by contrast, the invasive ability was KKs/Myc is a stress-inducible signaling pathway Because IKKs play an important role in tumor cells
response to various stresses, it was of interest to ask
whether common chemotherapeutic agents for breast Yeh et al. Molecular Cancer 2011, 10:53
http://www.molecular-cancer.com/content/10/1/53 Page 7 of 12 http://www.molecular-cancer.com/content/10/1/53 Figure 4 IKKa and IKKb increase Myc protein stability. The stability of Myc protein was analyzed by Western blot analysis of the whole cell
lysates prepared from (A) MCF7 with or without a 12 hours, 10 μM Bay11-0782 treatment (B) wild-type and kinase-dead IKKa transfected cells
(C) wild-type and kinase-dead IKKb transfected cells along a time course after adding cycloheximide. The stain of tubulin was used as loading
control. The reading of Myc density was normalized to the reading of tubulin density. Each data represents mean ± SD calculated from two
independent experiments. Whole cell lysates were prepared from (D) MCF7 cells, (E) wild-type and kinase-dead IKKa transfected cells, and (F)
wild-type and kinase-dead IKKb transfected cells were subjected to coimmuoprecipitation using indicated antibodies. The precipitated complex
was further Western blot analyzed the corresponding proteins shown in the figure. (G) Confocal microscopy observation. Breast cancer tissues
which were previously identified positive or negative for IKKa, IKKb and Myc expressions by IHC staining were used. The slides were dual stained
with rabbit anti-IKKa/mouse anti-Myc antibodies coupled with FITC-conjugated goat anti-rabbit IgG/Rodamine-conjugated donkey anti-mouse
IgG antibodies or goat anti-IKKb/mouse anti-Myc antibodies coupled with FITC-conjugated donkey anti-goat IgG/Rodamine-conjugated donkey
anti-mouse IgG antibodies, respectively. Figure 4 IKKa and IKKb increase Myc protein stability. The stability of Myc protein was analyzed by Western blot analysis of the whole cell
lysates prepared from (A) MCF7 with or without a 12 hours, 10 μM Bay11-0782 treatment (B) wild-type and kinase-dead IKKa transfected cells
(C) wild-type and kinase-dead IKKb transfected cells along a time course after adding cycloheximide. The stain of tubulin was used as loading
control. The reading of Myc density was normalized to the reading of tubulin density. Each data represents mean ± SD calculated from two
independent experiments. Whole cell lysates were prepared from (D) MCF7 cells, (E) wild-type and kinase-dead IKKa transfected cells, and (F)
wild-type and kinase-dead IKKb transfected cells were subjected to coimmuoprecipitation using indicated antibodies. The precipitated complex
was further Western blot analyzed the corresponding proteins shown in the figure. (G) Confocal microscopy observation. KKs/Myc is a stress-inducible signaling pathway Breast cancer tissues
which were previously identified positive or negative for IKKa, IKKb and Myc expressions by IHC staining were used. The slides were dual stained
with rabbit anti-IKKa/mouse anti-Myc antibodies coupled with FITC-conjugated goat anti-rabbit IgG/Rodamine-conjugated donkey anti-mouse
IgG antibodies or goat anti-IKKb/mouse anti-Myc antibodies coupled with FITC-conjugated donkey anti-goat IgG/Rodamine-conjugated donkey
anti-mouse IgG antibodies, respectively. Figure 4 IKKa and IKKb increase Myc protein stability. The stability of Myc protein was analyzed by Western blot analysis of the whole cell
lysates prepared from (A) MCF7 with or without a 12 hours, 10 μM Bay11-0782 treatment (B) wild-type and kinase-dead IKKa transfected cells
(C) wild-type and kinase-dead IKKb transfected cells along a time course after adding cycloheximide. The stain of tubulin was used as loading
control. The reading of Myc density was normalized to the reading of tubulin density. Each data represents mean ± SD calculated from two
independent experiments. Whole cell lysates were prepared from (D) MCF7 cells, (E) wild-type and kinase-dead IKKa transfected cells, and (F)
wild-type and kinase-dead IKKb transfected cells were subjected to coimmuoprecipitation using indicated antibodies. The precipitated complex
was further Western blot analyzed the corresponding proteins shown in the figure. (G) Confocal microscopy observation. Breast cancer tissues
which were previously identified positive or negative for IKKa, IKKb and Myc expressions by IHC staining were used. The slides were dual stained
with rabbit anti-IKKa/mouse anti-Myc antibodies coupled with FITC-conjugated goat anti-rabbit IgG/Rodamine-conjugated donkey anti-mouse
IgG antibodies or goat anti-IKKb/mouse anti-Myc antibodies coupled with FITC-conjugated donkey anti-goat IgG/Rodamine-conjugated donkey
anti-mouse IgG antibodies, respectively. Yeh et al. Molecular Cancer 2011, 10:53 Page 8 of 12 Yeh et al. Molecular Cancer 2011, 10:53
http://www.molecular-cancer.com/content/10/1/53 http://www.molecular-cancer.com/content/10/1/53 Figure 5 IKKa and IKKb increase tumorigenesis and invasiveness of MCF7 cells. (A) MTT assay was used to analyze the growth rate of
indicated cells. Each data represents mean ± SD calculated from three independent experiments, and there are four wells for each point in a
single experiment. (B) Assay of colony-forming ability in soft-agar. Each experiment have three 60 mm dishes and each data represents mean ±
SD calculated from two independent experiments. (C) Assay of invasive ability of indicated cells. Each experiment have three trans-wells and
each data represents mean ± SD calculated from two independent experiments. (D) Western blot analysis of the expression of cyclin D1 and
twist protein in the indicated cells. KKs/Myc is a stress-inducible signaling pathway Figure 5 IKKa and IKKb increase tumorigenesis and invasiveness of MCF7 cells. (A) MTT assay was used to analyze the growth rate of
indicated cells. Each data represents mean ± SD calculated from three independent experiments, and there are four wells for each point in a
single experiment. (B) Assay of colony-forming ability in soft-agar. Each experiment have three 60 mm dishes and each data represents mean ±
SD calculated from two independent experiments. (C) Assay of invasive ability of indicated cells. Each experiment have three trans-wells and
each data represents mean ± SD calculated from two independent experiments. (D) Western blot analysis of the expression of cyclin D1 and
twist protein in the indicated cells. cancer treatment could induce IKK and Myc activation. We used doxorubicin (200 nM; IC50 for MCF7 cells)
alone or combined with Bay11-0782 to treat MCF7
cells. Cells were exposed to doxorubicin with or without
Bay11-0782 for either 24-hour or for 3-hour followed by
a 24-hour release in the presence or absence of Bay11-
0782. Doxorubicin induced IKKs activation, with induc-
tion levels highest in cells released from a 3-houre dox-
orubicin treatment. When Bay11-0782 was re-added to
the cells released from a 3-hour treatment with doxoru-
bicin with or without Bay11-0782, decreased levels of
doxorubicin-induced IKKs activation were observed. IKK activity returned to control levels after a 24-hour
Bay11-0782 treatment. (Figure 6A). Consequently, the
phosphorylation of Myc at Ser 62 was increased by dox-
orubicin, and the highest level of induction was achieved
in cells released from a 3-hour doxorubicin treatment
with or without Bay11-0782 cotreatment. Re-addition of
Bay11-0782 blocked this induction. The phosphorylation
of Myc at Thr58 remained at a relatively constant level
following all treatments. The level of Myc protein was
changed with a consistent pattern as the status of phos-
phorylated Myc Ser62. Cyclin D1 and Twist levels were
also altered in a similar manner (Figure 6A). In addition,
Western blot analysis showed that NF-B was not
affected by these treatments (Figure 6B). IKKs/Myc activated by doxorubicin promotes
tumorigenesis and invasiveness of MCF7 cells cancer treatment could induce IKK and Myc activation. We used doxorubicin (200 nM; IC50 for MCF7 cells)
alone or combined with Bay11-0782 to treat MCF7
cells. Cells were exposed to doxorubicin with or without
Bay11-0782 for either 24-hour or for 3-hour followed by
a 24-hour release in the presence or absence of Bay11-
0782. KKs/Myc is a stress-inducible signaling pathway Doxorubicin induced IKKs activation, with induc-
tion levels highest in cells released from a 3-houre dox-
orubicin treatment. When Bay11-0782 was re-added to
the cells released from a 3-hour treatment with doxoru-
bicin with or without Bay11-0782, decreased levels of
doxorubicin-induced IKKs activation were observed. IKK activity returned to control levels after a 24-hour
Bay11-0782 treatment. (Figure 6A). Consequently, the
phosphorylation of Myc at Ser 62 was increased by dox-
orubicin, and the highest level of induction was achieved
in cells released from a 3-hour doxorubicin treatment
with or without Bay11-0782 cotreatment. Re-addition of
Bay11-0782 blocked this induction. The phosphorylation
of Myc at Thr58 remained at a relatively constant level
following all treatments. The level of Myc protein was
changed with a consistent pattern as the status of phos-
phorylated Myc Ser62. Cyclin D1 and Twist levels were
also altered in a similar manner (Figure 6A). In addition,
Western blot analysis showed that NF-B was not
affected by these treatments (Figure 6B). tumorigenesis and invasiveness of MCF7 cells
Because cyclin D1 and Twist proteins were increased in
MCF7 cells following a 3-hour doxorubicin exposure,
we characterized whether the tumorigenic and invasive
ability of MCF7 cells was subsequently enhanced. Indeed, the cells released from a 3-hour doxorubicin
treatment increased their ability to grow in soft-agar
and to pass through matrigel. Co-treatment with Bay11-
0782 partially suppressed these activities, and re-addi-
tion of Bay11-0782 after released from doxorubicin
markedly decreased these activities to lower than the
levels of the untreated control (Figure 6C and 6D). These results were further supported by analyzing the
effect of doxorubicin on IKK-transfected cells. Following
the same treatments as above, the protein levels of Myc,
cyclin D1, and Twist were increased in wild-type IKKa-
and IKKb-transfected cells (Figure 7A and 7C), but were
not changed in kinase-dead IKKa- and IKKb-transfected
cells (Figure 7B and 7D). Discussion In this study, we explored the relationship between IKKs
and Myc expression in breast cancers. IHC staining of
breast cancer specimens indicated that the expression of
Myc was associated with IKKa and IKKb, but was Yeh et al. Molecular Cancer 2011, 10:53
http://www molecular cancer com/content Yeh et al. Molecular Cancer 2011, 10:53
http://www.molecular-cancer.com/content/10/1/53 Page 9 of 12 http://www.molecular-cancer.com/content/10/1/53 Figure 6 IKKs-Myc pathway is inducible. (A) MCF7 cells were treated with 10 μM Bay11-0782 alone, or 200 nM doxorubicin in the presence or
absence of 10 μM Bay11-0782 for 24 hours (Dox, Bay, and Bay/Dox), or 3-hr short-term pulse followed by 24 hours release in the presence or
absence of Bay11-0782 (Dox3-R, Bay/Dox3-R, and Bay/Dox3-R/Bay). Whole cells lysates were subjected to Western blot analysis. (B) MCF7 cells
were treated 200 nM doxorubicin with or without 10 μM Bay11-0782 for 24 hours. The cytoplasmic and nuclear lysates were subjected to
Western blotting. (C) MCF7 cells were treated with 200 nM doxorubicin in the presence or absence of 10 μM Bay11-0782 for 3 hours. The cells
were then seeded into soft-agar in complete medium with or without 10 μM Bay11-0782 for two weeks. Each experiment have three 60 mm
dishes and each data represents mean ± SD calculated from two independent experiments. (D) MCF7 cells were treated as shown, and then the
ability of cells to penetrate matrigel was determined. Each experiment have three trans-wells and each data represents mean ± SD calculated
from two independent experiments. Figure 6 IKKs-Myc pathway is inducible. (A) MCF7 cells were treated with 10 μM Bay11-0782 alone, or 200 nM doxorubicin in the presence or
absence of 10 μM Bay11-0782 for 24 hours (Dox, Bay, and Bay/Dox), or 3-hr short-term pulse followed by 24 hours release in the presence or
absence of Bay11-0782 (Dox3-R, Bay/Dox3-R, and Bay/Dox3-R/Bay). Whole cells lysates were subjected to Western blot analysis. (B) MCF7 cells
were treated 200 nM doxorubicin with or without 10 μM Bay11-0782 for 24 hours. The cytoplasmic and nuclear lysates were subjected to
Western blotting. (C) MCF7 cells were treated with 200 nM doxorubicin in the presence or absence of 10 μM Bay11-0782 for 3 hours. The cells
were then seeded into soft-agar in complete medium with or without 10 μM Bay11-0782 for two weeks. Each experiment have three 60 mm
dishes and each data represents mean ± SD calculated from two independent experiments. Discussion (D) MCF7 cells were treated as shown, and then the
ability of cells to penetrate matrigel was determined. Each experiment have three trans-wells and each data represents mean ± SD calculated
from two independent experiments. It is well known that IKKs trigger IBa degradation
and subsequent activation of NF-B [10]. However, our
results showed that IKKa and IKKb regulated Myc
expression levels without altering NF-B activation. NF-
B activation is tightly auto-regulated by inducing the
expression of its natural inhibitor, IBa. For example, in
response to TNFa stimulation, IBa is degraded during
the first 15 minutes and quickly restored in one hour. Therefore, we hypothesized that transfection of wild-
type IKKa and IKKb may have transiently activate NF-
B, but that the activity of NF-B subsequently returned unrelated to that of NF-B. We demonstrated that IKKa
and IKKb did not enhance Myc transcription but
instead increased Myc protein stability. Importantly, we
also showed that the commonly used anticancer drug,
doxorubicin, activated IKKs, and thereby increased Myc
protein levels. Myc plays an important role in tumor
progression and is associated with metastasis and a poor
outcome of breast cancers [7,17,19]. However, there is
still no reliable drug which can effectively target Myc. Our study indicates that one possible way to block Myc
is by inhibition of IKKs. Page 10 of 12 Yeh et al. Molecular Cancer 2011, 10:53
http://www.molecular-cancer.com/content/10/1/53 Yeh et al. Molecular Cancer 2011, 10:53 http://www.molecular-cancer.com/content/10/1/53 Figure 7 IKK activity is necessary for doxorubicin to increase Myc, cyclin D1 and twist protein levels in MCF7 cells. The cells (A) MCF/
IKKa-WT, (B) MCF/IKKa-KD, (C) MCF/IKKb-WT, and (D) MCF/IKKb-KD were treated with 200 nM doxorubicin for 3hours, and then were released
with complete medium for further 24 hours. The cytoplasmic and nuclear extracts were subjected to Western blot analysis. yclin D1 and twist protein levels in MCF7 cells The cells (A) MCF/ Figure 7 IKK activity is necessary for doxorubicin to increase Myc, cyclin D1 and twist protein levels in MCF7 cells. The cells (A) MCF/
IKKa-WT, (B) MCF/IKKa-KD, (C) MCF/IKKb-WT, and (D) MCF/IKKb-KD were treated with 200 nM doxorubicin for 3hours, and then were released
with complete medium for further 24 hours. The cytoplasmic and nuclear extracts were subjected to Western blot analysis. to basal levels due to the balance between NF-B and
IBa. that IKKa and IKKb increased Myc protein stability by
regulating its phosphorylation status at Ser62. Discussion We
further demonstrated that IKKa but not IKKb directly
interacted with Myc. While the pathway connecting
IKKb to Myc remains to be identified, our study demon-
strated a reciprocal upregulation between IKKa and
IKKb, indicating that IKKb may indirectly increase Myc
through IKKa. However, it is of particular interest to
determine whether IKKb may also affect Myc through
regulation of related molecules, such as PP2A or cip2A. In this study, we showed that suppression of IKK
activity by transfection of kinase-dead IKKa or IKKb
decreased Myc protein levels, whereas slightly increased
the Myc mRNA levels. Our results demonstrated that
the change in Myc protein levels was not related to the
transcription of Myc, consistent with a previous study
performed in neuroblastoma [35]. Our study further
demonstrated that the turn-over rate of the Myc mRNA
was prolonged in kinase-dead IKKa- and IKKb-trans-
fected cells. Interestingly, Bay11-0782 did not show this
activity, suggesting that it may have other effect on
mRNA stability. While the underlying mechanism
remains unknown, our result may help explain the con-
flicting results in analyzing the relative levels of mRNA
and protein of certain genes. A comparable growth rate was observed among paren-
tal MCF7 cells and IKKs-transfected cells. Because
Bay11-0872 indeed decreased MCF cell growth [data
not shown], it is likely that un-identified pathways
which can compensate for Myc activity are developed
during the selection of kinase-dead IKKa- and IKKb-
transfected cells. In addition, our result indicated that
the invasive ability induced by Myc could be separated
from the growth potential of cancer cells and that Myc The stability of Myc is controlled by its phosphoryla-
tion at Ser62 and Thr58 [16]. In this study, we showed Yeh et al. Molecular Cancer 2011, 10:53
http://www.molecular-cancer.com/content/10/1/53 Yeh et al. Molecular Cancer 2011, 10:53
http://www.molecular-cancer.com/content/10/1/53 Page 11 of 12 that our finding may be applicable to other cancers. Taken together, our results suggested that IKKs/Myc
might be important therapeutic targets for breast can-
cer and provided a rationale for the use of IKK inhibi-
tors
following
chemotherapy
to
suppress
the
treatment-enhanced tumor progression. activity was indispensable for the enhanced invasiveness. Our results are consistent with the data from a previous
study using another breast cancer cell line MDA-MB
231 [17]. Based on gene array analysis, Myc has been shown to
regulate a set of gene signatures associated with metas-
tasis and poor-outcome of breast cancers [17]. References 1. Lee DF, Hung MC: Advances in targeting IKK and IKK-related kinases for
cancer therapy. Clin Cancer Res 2008, 14:5656-5662. 1. Lee DF, Hung MC: Advances in targeting IKK and IKK-related kinases for
cancer therapy. Clin Cancer Res 2008, 14:5656-5662. In this study, we provided evidence that doxorubicin
increased Myc protein levels probably through activat-
ing IKKa and IKKb. The cells that were released from
doxorubicin increased their invasive and tumorigenic
activities, and suppression of IKK activation blocked
these phenotypes. Myc regulates many downstream
gene expressions which share a similarity between
embryonic stem cells and cancer cells [37,38]. It is
important to determine whether doxorubicin stimu-
lates re-growth and progression of cancer cells in vivo. On the other hand, IKKs are major mediators linking
inflammation and cancer progression [10,39]. We have
not tested other stimulus, such as inflammation-related
cytokines, that may have similar effects on the IKK/
Myc pathway; however, it is possible that the death of
cancer cells caused by therapeutic treatment may trig-
ger an inflammation response, which activates IKKs
and Myc in the remaining cancer cells, and subse-
quently stimulates cancer progression. In addition,
IKKs and Myc are widely expressed in various cancers. For example, IKK and Myc have been separately
reported to be necessary for hepatocellular carcinoma
cell growth and invasiveness [2,8]; it is therefore likely 2. Cairo S, Wang Y, de Reyniès A, Duroure K, Dahan J, Redon MJ, Fabre M,
McClelland M, Wang XW, Croce CM, Buendia MA: Stem cell-like micro-RNA
signature driven by Myc in aggressive liver cancer. Proc Natl Acad Sci
USA 2010, 107:20471-20476. 3. Israë A: The IKK complex, a central regulator of NF-κB activation. Cold
Spring Harb Perspect Biol 2010, 2:a000158. 3. Israë A: The IKK complex, a central regulator of NF-κB activation. Cold
Spring Harb Perspect Biol 2010, 2:a000158. 4. Perkins ND: Integrating cell-signalling pathways with NF-κB and IKK
function. Nat Rev Mol Cell Biol 2007, 8:49-62. 5. Hayden MS, Ghosh S: Shared principles in NF-κB signaling. Cell 2008,
132:344-362. 6. Huber MA, Azoitei N, Baumann B, Grünert S, Sommer A, Pehamberger H,
Kraut N, Beug H, Wirth T: NF-κB is essential for epithelial-mesenchymal
transition and metastasis in a model of breast cancer progression. J Clin
Invest 2009, 114:569-581. 7. Acknowledgements
W
h
k P
f
W Acknowledgements
We thank Professor WC Greene for providing us wild-type and kinase-dead
IKKs expression vectors, JW Chen for help prepare tissue specimens, and the
confocal microscope core-facilities at the National Taiwan University Hospital
for microscopic observations. This study was supported by grants 99-2314-B-
002-032-MY3 from the National Science Council and DOH99-TD-C-111-001
from the Department of Health, Taiwan, R.O.C. Authors’ contributions PY designed and carried out the cellular and molecular studies, and wrote
the manuscript, YS carried out the clinical studies, DL carried out the gene
expression studies, and AL organized and supervised the whole study. All
authors read and approved the final manuscript. 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. The authors declare that they have no competing interests. Received: 8 March 2011 Accepted: 16 May 2011 Published: 16 May 2011 Received: 8 March 2011 Accepted: 16 May 2011 Published: 16 May 2011 Received: 8 March 2011 Accepted: 16 May 2011 Published: 16 May 2011 Author details
1 1Department of Oncology, National Taiwan University Hospital, No. 7,
Chung-Shan South Road, Taipei, 100, Taiwan. 2National Center of Excellence
for Clinical Trial and Research, College of Medicine, National Taiwan
University, No1, Jen Al Road Section1, Taipei, 100, Taiwan. 3Department of
Internal Medicine, National Taiwan University Hospital, No. 7, Chung-Shan
South Road, Taipei, Taipei, 100, Taiwan. 4Graduate Institute of Oncology,
College of Medicine, National Taiwan University, No1, Jen Al Road Section1,
Taipei, 100, Taiwan. Discussion Abnor-
mal expression of Myc promotes the epithelial to
mesenchymal transition and metastasis [14,18,36]. In
this study, we showed that Myc increased the tumori-
genic and invasive ability of MCF7 cells. We also identi-
fied that the levels of cyclin D1 and Twist were
consistently altered along with the Myc protein level. It
is reasonable to conclude that Myc enhances tumorigen-
esis, at least in part, through the upregulation of cyclin
D1 and Twist. However, overexpression of Myc in a
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does not promote its invasive ability, suggesting that
Myc is a necessary but not sufficient factor for cancer
cell invasiveness [22]. It is hypothesized that Myc should
cooperate with other factors to enhance the invasive
activity of the cells. In this study, Myc was increased
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and IKKb also regulate the expression of multiple genes
that are involved in cancer cell progression and metasta-
sis. Therefore, it is possible that a Myc-centered net-
work cooperates and/or merges with an IKK-centered
network to enhance the tumorigenic and invasive activ-
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http://www.molecular-cancer.com/content/10/1/53 Submit your next manuscript to BioMed Central
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and take full advantage of: Submit your next manuscript to BioMed Central
and take full advantage of: 32. Garcia M, Alaniz L, Lopes E, Blanco G, Hajos S, Alvarez E: Inhibition of NF-
κB activity by BAY 11-7082 increases apoptosis in multidrug resistant
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Eilers M: Stabilization of N-Myc is a critical function of Aurora A in
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Eilers M: Stabilization of N-Myc is a critical function of Aurora A in
human neuroblastoma. Cancer Cell 2009, 15:67-78.
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© The Author(s) 2020
F. Stjernfelt, A. M. Lauritzen, Your Post has been Removed,
https://doi.org/10.1007/978-3-030-25968-6_2 1 See Mchangama and Stjernfelt (2016) p. 135ff. 2 Our translation of “Déclaration des Droits de l’Homme et du Citoyen
de 1789” Conseil Constitutionnel. Last visited 08-04-18: conseil-constitu-
tionnel.fr/—Translations in the book are our own, except when citing
already translated works.
3 “First Amendment” Legal Information Institute.
4 “Immaturity” translates German “Unmündigkeit”, literally referring to
the nonage state of underage citizens without full autonomy and citizen
rights. Chapter 2
The Free Networks
of the Enlightenment In the seventeenth and eighteenth centuries, freedom of
expression emerged as a central theme of the dawning Age of
Enlightenment. This was caused not least by churches and
princes maintaining strict political control of expressions of
divergent opinions. The new Protestant state churches,
Lutheran as well as Calvinist, turned out not to offer more
freedom than the Catholic church. In fact, it was often less,
which meant that religious, philosophical and political dissi
dents regularly ran into serious problems when expressing
their ideas publicly. Thus, ideas of freedom of expression
began to surface. One of the first urgent calls for freedom of
expression came from Dutch-Jewish philosopher Baruch
Spinoza, who in his “Tractatus Theologico-Politicus” (1670)
called for libertas philosophandi—freedom of thought. As
stated by British historian Jonathan Israel, Spinoza was the
first major philosopher who was also a democrat. Spinoza
believed it important to distinguish politically between peo
ple’s actions and their views and expressions. Whereas the
former should be governed by legislators, the latter should
not. This would create a more free and peaceful society—and
if people had the possibility to influence the laws they were
subject to, they might be more inclined to respect them. 9 Chapter 2. The Free Networks of the Enlightenment 10
1
1 Chapter 2. The Free Networks of the Enlightenment 11 2
3 2
3 4 Chapter 2. The Free Networks of the Enlightenment 12
5 Historically, the “general public” Kant refers to had
emerged from the learned networks of the Enlightenment
Age as a self-organized communications forum with its own
media, outside of and across the narrow public spheres main
tained and controlled by churches and courts. In principle,
this general public, beyond the control of governments and
religions, now lends legitimacy to the new democratic states. The general public, as in the civil society and their networks,
organizations and the media which thrive off them, enables
enlightenment to take place. Mistaken ideas are corrected;
arguments are developed and contradicted; new ideas and
science are formed; viewpoints clash; criticism of and protests
against policies are articulated; political agreement and dis
agreement can be formulated, and last but not least: informed
elections can take place. 5 Kant (1784) pp. 484–85. 6 See the chapter on “Homo humanitatis” in Budtz Pedersen et al.
(2018). Chapter 2
The Free Networks
of the Enlightenment It is well known that the realization
of this ideal public sphere has shown its disadvantages, as
mapped by disciplines such as mass psychology and cultural
criticism: fads, seduction of the masses led by charismatic Chapter 2. The Free Networks of the Enlightenment 13 figures, the public impact of culture and pop industries, not to
mention what we today refer to as echo chambers and filter
bubbles. However, despite these built-in disadvantages, most
theories of democracy agree that free debate is a fundamen
tal condition for modern liberal democracies.
6 At the beginning of the nineteenth century, Jeremy
Bentham criticized this very idea of natural rights as “non
sense upon stilts”—to him the only legitimate rights were
those guaranteed by a political authority. This led to an alter
native, utilitarian justification of free speech that found its
classic articulation in John Stuart Mill’s On Liberty from
1859. To him the utility of freedom of expression is the mea
suring stick and the central argument is that if a given society
lacks freedom of expression, not all possible suggestions for
the solution of a given problem will be expressed, and it will
then not be possible to reach the best solution. Obviously, this
basic reason for having freedom of expression is completely
different from the one found in Kantian thought—but this
should not block our understanding that the two reasons are
oftentimes in agreement with each other, and that in most
concrete cases, they work well together. As it is notoriously
difficult to measure utility, at the end of the day the utilitarian
argument is hardly less speculative than the Kantian one. Is it
useful for a democratic society to accept anti-democratic
statements from Nazis, Communists and Islamists? At first Chapter 2. The Free Networks of the Enlightenment 14 glance, probably not. But the counter-argument goes: Such
acceptance might be useful after all, since the knowledge of
anti-democratic views may help immunize the public against
those very views. It is generally useful to have an uncensored
public sphere that assures people that others mean what they
say and are not forced to pretend or lie because of legislation. Unlike the Kantian definition, the utilitarian one emphasizes
the pragmatic, social benefits of free expression. Chapter 2
The Free Networks
of the Enlightenment In a sense,
this dimension complements a rights- and individual-based
definition so important for this book, which has as its core
topic the tech giants’ transformation of the public sphere. However, the emphasis on benefit to the public must always
be counterbalanced by freedom of expression as an individ
ual right. In cases where the two definitions clash, in our
opinion the latter should outweigh the former. glance, probably not. But the counter-argument goes: Such
acceptance might be useful after all, since the knowledge of
anti-democratic views may help immunize the public against
those very views. It is generally useful to have an uncensored
public sphere that assures people that others mean what they
say and are not forced to pretend or lie because of legislation. Both theories are compatible with the idea of freedom of
expression as a means of testing authorities and established
legislation—and ultimately breaking with them. Two of the
many examples from modern times are the decline of slavery
around 1800 and women’s right to vote around 1900—both
changes became possible through extensive public debate
prior to their realization. In this sense, a free public sphere
enables the articulation and breakthrough of new political
views and movements in a democracy. The abolition of censorship in most modern democratic
states took place from the 18th to the twentieth centuries and
has, as a tendency, gone hand in hand with a greater tolerance
of divergent views and opinions—be they religious, political,
ethnic, etc. As mentioned earlier, this does not mean that
freedom of expression is absolute. In a certain way, the ongo
ing negotiation of its boundaries is a central theme in modern
democratic politics, due to the idea of freedom of expression
as a fundamental right, which should only be limited in cases
where very convincing counter-arguments to do so are pres
ent. Threats, explicit incitement or planning of violence and
false personal defamation belong in this category. More
debated examples include “hate speech”, which is not a crime
in the US, but which has been criminalized in many European
countries in various ways. Other controversial examples are Chapter 2. The Free Networks of the Enlightenment 15 criticism of religion and blasphemy, which are no longer pro
hibited in most modern democracies since the Enlightenment. Chapter 2
The Free Networks
of the Enlightenment This is not the case in many Muslim countries, which are
working on enacting such laws internationally through the
UN and by formally or informally pressuring public opinion
in countries without such prohibitions. In a certain sense, freedom of expression is counterintui
tive—why not just silence abhorrent statements? The toler
ance that freedom of expression implies is not easily achieved. It includes the duty, both of the government and of the indi
vidual, to tolerate views, statements, pictures and books
which may be considered abominable and grotesque, but
which also have a right to reach the public. As has so often
been said, tolerating views one agrees with is the easy part. But the fact that also Nazis, Islamic extremists or Communists
should have the right to express their views on reality and the
future is something that many people need a certain degree
of self-reflection to accept. Something similar applies in the
case of “hate speech”, which is why some argue that it should
be tolerated and not banned. “Hate speech” is a notoriously
ill-defined category, and in the laws of many countries, it is
only described by simply listing a number of somewhat ran
domly selected groups of people—labelled for example reli
gious, ethnic, sexual, racial, etc. These groups cannot be
criticized beyond a certain limit, but it is a difficult limit to
define accurately—“insult”, “mockery”, “degradation”, etc. are
imprecise terms often used. Compared to threats, which are
usually covered in a separate clause, “hate speech” is less
clearly defined. “Hate speech” legislation is not just a collec
tive libel clause either. Most often, the definition of “hate
speech” differs from that of libel in that it does not involve
any assessment of the veracity of the statement (in the case
of libel, charges may be dropped if the allegations are proven
to be true, which is usually not the case with “hate speech”). Very often, “hate speech” legislation and verdicts end up
applying also to the political criticism of the behavior of such
groups. Such criticism is not necessarily untrue or politically
illegitimate; most political activity naturally includes the dis
cussion and changes of the general conditions of different Chapter 2. The Free Networks of the Enlightenment 16 groups in society (rich, poor, public employees, entrepre
neurs, refugees, retirees, etc.) who are therefore addressed in
general terms. Chapter 2
The Free Networks
of the Enlightenment It is therefore difficult and maybe even impos
sible to maintain “hate speech” legislation and at the same
time avoiding its misuse to silence legitimate political stand
points and even true statements about problems concerning
different groups in society. As Professor at Law Nadine
Strossen remarks, the introduction of “hate speech” legisla
tion very often results in its use against those marshaling it,
because different governments may use it to try to silence
their opponents, once a “hate speech” law is accepted. However, it is a defining feature of modern, liberal democ
racies that the very discussion itself of such boundaries must
take place in full public view. Such discussion makes use of
freedom of expression, which guarantees people the ability to
cite examples of prohibited content or of what others would
like to see prohibited. This is unlike what happens in dictator
ships or absolutist states. Here, both the general boundaries
of statements and their translation into individual decisions
and decrees may be decided secretly in the government appa
ratus without legal trial or public insight or discussion. In this
sense, the limits of freedom of expression are and should be
the subject of ongoing, public and free debate. Open Access This chapter is licensed under the terms of the Creative
Commons Attribution 4.0 International License (http://creativecom-
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link to the Creative Commons license and indicate if changes were
made. The images or other third party material in this chapter are included in
the chapter’s Creative Commons license, unless indicated otherwise in a
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Creative Commons license and your intended use is not permitted by
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Developing and validating a dynamic model of water production by direct-contact membrane distillation
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PloS one
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cc-by
| 12,076
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Developing and validating a dynamic model of
water production by direct-contact
membrane distillation Emad AliID1☯*, Jamel Orfi2☯, Abdullah Najib2☯
1 Department of Chemical Engineering, King Saud University, Riyadh, Saudi Arabia, 2 Department of
Mechanical Engineering, King Saud University, Riyadh, Saudi Arabia ☯These authors contributed equally to this work. * amkamal@ksu.edu.sa OPEN ACCESS OPEN ACCESS
Citation: Ali E, Orfi J, Najib A (2020) Developing
and validating a dynamic model of water
production by direct-contact membrane distillation. PLoS ONE 15(3): e0230207. https://doi.org/
10.1371/journal.pone.0230207
Editor: Zuwei Liao, UNILAB Research Center for
Chemical Reaction Engineering, CHINA
Received: November 7, 2019
Accepted: February 24, 2020
Published: March 24, 2020 Citation: Ali E, Orfi J, Najib A (2020) Developing
and validating a dynamic model of water
production by direct-contact membrane distillation. PLoS ONE 15(3): e0230207. https://doi.org/
10.1371/journal.pone.0230207 Citation: Ali E, Orfi J, Najib A (2020) Developing
and validating a dynamic model of water
production by direct-contact membrane distillation. PLoS ONE 15(3): e0230207. https://doi.org/
10.1371/journal.pone.0230207 Editor: Zuwei Liao, UNILAB Research Center for
Chemical Reaction Engineering, CHINA
Received: November 7, 2019
Accepted: February 24, 2020
Published: March 24, 2020 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.0230207 PLOS ONE RESEARCH ARTICLE Abstract We consider the development and fitting of a dynamic model for desalinated water produc-
tion by a direct-contact membrane distillation (DCMD) unit. Two types of dynamic-model
structures, namely, lumped parameter and spatial, were evaluated. Both the models were
validated using experimental response data generated by step testing the inlet hot stream
temperature of a DCMD pilot plant. Both the model structures failed to follow the dynamic
response adequately. However, a modification of the model by adding a heat loss term
resulted in enhanced predictions for both model structures. The overall relative error in the
model–plant mismatch was approximately 3%. This is reasonable considering the random
uncertainties associated with the plant operation. This observation also improves our under-
standing of the importance of using better correlations for heat-transfer coefficients, to
develop a more reliable and accurate predictive model for a wide range of operating
conditions. OPEN ACCESS
Citation: Ali E, Orfi J, Najib A (2020) Developing
and validating a dynamic model of water
production by direct-contact membrane distillation. PLoS ONE 15(3): e0230207. https://doi.org/
10.1371/journal.pone.0230207
Editor: Zuwei Liao, UNILAB Research Center for
Chemical Reaction Engineering, CHINA
Received: November 7, 2019
Accepted: February 24, 2020
Published: March 24, 2020 PLOS ONE PLOS ONE Introduction The shortage of potable water is a major problem persisting in several regions worldwide. The
growth and development of the desalination industry are remarkable. In 2016, global desalina-
tion production attained approximately 85 × 106 m3/day [1]. The conventional desalination
technologies, namely, multi-stage flash, multi-effect evaporation, and reverse osmosis, are
known for being energy intensive. Among the most innovative and potential desalination tech-
nologies, membrane distillation (MD) is gaining interest because of its advantages compared
to conventional technologies. The direct-contact membrane distillation (DCMD) configura-
tion, which is a widely employed MD configuration, is known for its attractive characteristics
such as its requirement of low operating temperature and hydrostatic pressure. It can achieve
approximately 100% rejection of salt ions. Moreover, its permeate quality is marginally affected
by the feed concentration. Furthermore, it has a compact and flexible structure [2–5]. Notwith-
standing the appealing features of the MD technology, its industrial commercialization is hin-
dered by certain technical barriers and deficiencies, such as low recovery ratio; high specific Copyright: © 2020 Ali et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in
any medium, provided the original author and
source are credited. Data Availability Statement: All relevant data are
within the manuscript and Supporting Information
files. Funding: The research is funded by the Deanship
of Scientific Research, King Saud University PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 1 / 24 PLOS ONE Transient model for mass flux in membrane distillation through Research Group no (RG- VPP 091). The
funder had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript. energy requirements; and fouling and scaling, which eventually deteriorate the permeate flux
and membrane pore wetting [2,6–8]. Therefore, several investigations were performed during
the past years to enhance the feasibility of the MD technology as a commercial desalination
technology [9–11]. For example, many researchers focused on improving the MD perfor-
mance by performing reconfiguration or retrofitting such as incorporation of heat-recovery
systems [10,12], recycling of the discharged brine to the inlet stream [13,14], and use of multi-
staging [10,15]. Integration of the MD technology with low-grade energy sources such as waste
heat, solar, and geothermal energy has been addressed [16–18]. Introduction To address science and engi-
neering issues that limit the development and commercialization of the MD technology, theo-
retical investigations concerning the design and optimization of the MD process have been
performed [3,19–26]. These theoretical investigations, which modeled several physical princi-
ples integral to the MD process, are subject to the following limiting assumptions: Competing interests: The authors have declared
that no competing interests exist. • Unlike the studies on steady-state regime, those on transient conditions are very few [21,27]. • Heat and mass transfers are generally space independent (using lumped approach) or one
dimensional [5,20]. Only a few works have been concerned with refined multidimensional
aspects [28–30]. • The transfer coefficients are generally evaluated using inappropriately developed correla-
tions, e.g., for impermeable interfaces and not for permeable ones [31,32]. A deeper percep-
tion of the heat- and mass-transfer phenomena should be addressed. • It has been reported that the uncertainty of membrane properties causes inadequate mass
fluxes. Camacho et al. [33] reported that membrane properties such as porosity, thickness,
and pore size may change because of the membrane compression caused by the hydrody-
namic pressure associated with the circulating feed and permeate flow rates. As mentioned
by Andrjesdo´ttir et al. [9], the simultaneous fitting of heat and mass data to a first-principles
model can aid the understanding of the underlying physics of the MD process and, thereby,
highlight the deficiencies of similar physical models. Irrespective of the effectiveness and complexity of these models, they are limited and cannot
be utilized for applications such as automation and control implementation because they are
stationary. In addition, these models cannot be integrated with time-varying energy resources
such as solar and wind energies, which are characterized by intermittent energy outputs. Therefore, it is important to develop dynamic models that consider the fluctuation or abrupt
variations in energy supply. Charfi et al. [28] undertook one of the earliest efforts to address
the dynamics modeling of the MD processes. Hassan et al. [34] developed a dynamic, spatial
finite-difference model to investigate the transient performance of a vacuum MD when hot
feed parameters such as mass-flow rate, temperature, and concentration distributions undergo
variations in steps. Recently, Eliewi et al. and Karam et al. [21,27] proposed DCMD-based dynamic models
that consider heat and mass variations both in one- and two-dimensional spaces. These space-
dependent models are suitable for exploring the impact of hidden unmeasured parameters. PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 Introduction However, for control applications and/or integrating the MD technology with fluctuating
energy sources, the dynamics of the external inputs and outputs are important. Moreover, in
the works of Eliewi et al. and Karam et al. [21,27], the dynamics of a single-process output, i.e.,
the permeate outlet temperature, is validated, whereas those for the brine outlet temperature is
omitted. Furthermore, the validation is based on the ramp variations in the temperature. Fur-
thermore, this type of dynamics is not common for the MD process, and common dynamic
capturing should be based on step testing because it defines both the transient and stationary PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 2 / 24 PLOS ONE Transient model for mass flux in membrane distillation behaviors of the MD process. Recently, Ali [35] developed a dynamic model for the outlet cold
and hot temperatures in a DCMD. The model is converted to a transfer function (TF) to ana-
lyze the dynamic characteristics of the process outputs. This study is a continuation of a previous work [35] by performing dynamic analysis and
modeling of the DCMD process. Specifically, the dynamics of mass production is modeled,
because it is the main product of the process. To the authors’ knowledge, no detailed deriva-
tion or validation of dynamic model of mass flux in DCMD has been published. All the pub-
lished studies address the modeling of only the dynamics of the outlet temperatures of the MD
process. Furthermore, a detailed derivation of the explicit formulation of the mass flux dynam-
ics is presented. The generated model is based on first principles that include the internal phys-
ics of the MD process. The measured dynamic data of the mass accumulation in the DCMD
plant for water desalination are analyzed and correlated to obtain a typical transient behavior. Standard reaction-curve methods [36] are utilized to infer the dynamic characteristics of the
mass production response from the correlated transient responses. Moreover, the correlated
transient behavior obtained is used to validate the theoretical dynamics model. The analysis
here is limited to the DCMD module using a spiral-wound membrane made of polyethylene
tetrafluoride. The analysis covers a wide range of operating conditions for the mass-flow rate
and inlet hot temperature. Experimental setup description The development and validation of the model are based on the experimental data generated
from an MD pilot plant. The pilot plant was developed by SolarSpring [37] and equipped with
a DCMD module with an effective membrane-area of 10 m2, membrane thickness of 230 μm,
channel length of 14 m, channel height of 0.7 m, pore diameter of 0.2 μm, and channel gap of 2
mm. The membrane porosity is 0.8, and the water-entry pressure is 4.1 bar. A schematic of the
MD process is depicted in Fig 1. A data-acquisition system is employed for data logging and
for regulating various instruments. An external electrical heater (H1) is used to heat the evapo-
rator circuit, i.e., the membrane’s hot feed stream. The hot feed temperature is controlled effec-
tively using a programmable logic controller. An external cooler (H2) is also incorporated to
adjust the temperature of the MD inlet cold stream. The temperature of the inlet cold stream is
regulated manually. Hence, the inlet cold temperature undergoes fluctuation and disruptions
because of the fuzziness of manual control. The desalinated water is separated through an
overflow, accumulated in the storage tank T3, and measured using an electronic balance. Further details of the experimental setup and procedure are available in previous works
[32,35,38,39]. In the previous works, the experimental device was used to generate data to be
used for calibrating a steady state MD model, conducting energy and exergy analysis, and
validating a dynamic model of the MD outlet temperatures. In this work, the time-evolution
measurement of the mass flux is used to validate the proposed dynamic model for mass pro-
duction. Note that in all the experiments, the mass-flow rates both on the hot and cold side are
maintained equal. This is because the experimental module does not allow for non-equal flow
rates on the two sides, to avoid membrane sheet deformation. Mass-flux dynamic model The water distillate is the principal output of the process. The distillate production undergoes
transient behavior during the startup or stepped variations in the process inputs such as feed
temperature and/or feed-flow rate. Hence, we intend to develop a theoretical model that cap-
tures the dynamic behavior of mass production. We assume an absence of heat loss to the envi-
ronment, as well as stable membrane properties such as thickness, tortuosity, porosity, and 3 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 PLOS ONE Transient model for mass flux in membrane distillation Fig 1. Module test facility (flowsheet). https://doi.org/10.1371/journal.pone.0230207.g001 Fig 1. Module test facility (flowsheet). https://doi.org/10.1371/journal.pone.0230207.g001 https://doi.org/10.1371/journal.pone.0230207.g001 pore size. In addition, we assume the total pressure drop across the membrane to be negligible. Furthermore, the physical properties of water such as density, heat capacity, thermal conduc-
tivity, viscosity, and heat-transfer coefficient are functions of temperature. The dynamic model
for mass production of water in the lumped-parameter case is expressed by the following ini-
tial-value problem (IVP): pore size. In addition, we assume the total pressure drop across the membrane to be negligible. Furthermore, the physical properties of water such as density, heat capacity, thermal conduc-
tivity, viscosity, and heat-transfer coefficient are functions of temperature. The dynamic model
for mass production of water in the lumped-parameter case is expressed by the following ini-
tial-value problem (IVP):fififififififififififififififififi dmw
dt ¼
CmA
ð1 þ CmbhchHv þ CmbcccHvÞ
bhah bcbh
ð
Þ 1
2
ffiffiffiffiffiffiffiffiffi
Thin
Thout
s
dThout
dt
þ bhac bcbc
ð
Þ 1
2
ffiffiffiffiffiffiffiffi
Tcin
Tcout
s
dTcout
dt
! ð1Þ
vrCp dThout
dt
¼ mhinCp Thin Trf
mhoutCp Thout Trf
hmA Thm Tcm
jwAHv
ð2Þ
vrCp dTcout
dt
¼ mcinCp Tcin Trf
mcoutCp Tcout Trf
þ hmA Thm Tcm
þ jwAHv
ð3Þ The derivation of the mass production dynamic (Eq 1) and the definitions of the underlying
parameters are provided in Appendix A. The thermal Eqs (2) and (3) are adopted from Karam
et al. [27] and presented and discussed in another work [40]. The thermal equations are devel-
oped by applying the energy conservation law on the entire MD module (S1 Fig) both on the
feed and permeate sides. PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 Mass-flux dynamic model Hence, the dynamic model of the MD (Eqs (1)–(3)) can be written for each
control element (S1 Fig) (denoted by subscript i) along the membrane length, using the
lumped-capacitance method as follows: v
n
rCp dThi
dt ¼ mhi 1Cp Thi 1 Trf
mhiCp Thi Trf
hmi D xh Thm;i Tcm;i
jwi D xhHvð4Þ
v
n
rCp dTci
dt ¼ mciþ1 Cp Tciþ1 Trf
mciCp Tci Trf
þ hmi D xh Thm;i Tcm;i
þ jwi D xhHvð5Þ
dmwi
dt
¼
CmiA
ð1 þ CmibhichiHvi þ CmibcicciHviÞ
bhiahi bcibhi
dThi
dt þ
bhiaci bcibci
dTci
dt
ð6Þ
i = 1, , n
f
i
1
T
T
T
T v
n
rCp dThi
dt ¼ mhi 1Cp Thi 1 Trf
mhiCp Thi Trf
hmi D xh Thm;i Tcm;i
jwi D xhHvð4Þ
v
n
rCp dTci
dt ¼ mciþ1 Cp Tciþ1 Trf
mciCp Tci Trf
þ hmi D xh Thm;i Tcm;i
þ jwi D xhHvð5Þ dmwi
dt
¼
CmiA
ð1 þ CmibhichiHvi þ CmibcicciHviÞ
bhiahi bcibhi
dThi
dt þ
bhiaci bcibci
dTci
dt
ð6Þ for i ¼ 1 ! Thi 1 Thin ; Tci Tcout
for i ¼ n ! Thi Thout ; Tciþ1 Tcin for i ¼ 1 ! Thi 1 Thin ; Tci Tcout for i ¼ n ! Thi Thout ; Tciþ1 Tcin The above-mentioned equations are written repeatedly starting from the time when the hot
stream is fed until the time that the cold stream is fed. At the boundary of the module, the spec-
ified terminal values (Thin; Tcin) are used such that the degree of freedom is zero. The size of
the system of ordinary differential equations (ODEs) depends on the number of divisions of
the module length. Note that the total distillate production is the average of the mass produc-
tion of each control volume. The above Eqs (4) and (5) are originally partial differential equa-
tions (PDEs) with advective-conduction terms in one dimension, which is the axial direction
(direction of the flow). Mass-flux dynamic model In due course, the accumulation of energy within the hot channel
becomes equal to the difference between the heat loss from the hot fluid as sensible heat and
the heat transferred to the cold side. Within the cold side, the accumulation of energy is equal PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 4 / 24 PLOS ONE Transient model for mass flux in membrane distillation to the difference between the heat gained by the permeate fluid and the heat transferred from
the hot side. The heat transfer between the two sides occurs through two mechanisms: conduc-
tion through the membrane material and latent heat of vaporization. The initial conditions for
the IVP are that the mass production is zero, and the bulk temperature is equal to the room
temperature. The reference temperature (Trf) is also considered to be equal to the room tem-
perature. The IVP is solved simultaneously to obtain the time evolution of mass production. Although the above-mentioned dynamic model appears linear in its structure, nonlinearity
arises from the membrane heat-transfer coefficient (hm), membrane coefficient (Cm), mem-
brane surface temperature (Thm; TcmÞ, and underlying parameters (a’s, b’s, and β’s). These
coefficients and parameters are implicitly functions of the bulk temperature, membrane inter-
face temperatures, and mass flux. Hence, these variables are calculated using an iterative proce-
dure assuming the pseudo-steady state specified in Appendix B (see Algorithm S1). Notably,
the thermal behavior of the process is represented by the boundary temperatures, which are
measured and therefore, verifiable. It is apparent from Eqs (1)–(3) that the dynamic of the
mass production follows that of the outlet temperatures. Note that an equal inlet feed-flow rate
(mhin ¼ mcinÞ is used for the hot and cold sides, which is imposed by the experimental setup. For long membrane modules, the lumped-parameter model is limited because it uses a con-
stant value for the temperature profile along the membrane length. A better representation of
the process behavior can be achieved by using spatial model formulation. In this case, the pro-
cess variables such as the bulk temperature and mass flux are permitted to vary over the mem-
brane length. PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 Mass-flux dynamic model In this case, the advective-conduction terms are being upwind discre-
tized, whereby the PDEs are reduced to a system of IVPs. The initial values of the state vari-
ables are equal to those mentioned before. PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 5 / 24 PLOS ONE Transient model for mass flux in membrane distillation Fig 2. Time evolution of mass production at mhin ¼ mcin ¼ 50 kg=h. https://doi.org/10.1371/journal.pone.0230207.g002 Fig 2. Time evolution of mass production at mhin ¼ mcin ¼ 50 kg=h. https://doi org/10 1371/journal pone 0230207 g002 Fig 2. Time evolution of mass production at mhin ¼ mcin ¼ 50 kg=h. https://doi.org/10.1371/journal.pone.0230207.g002 Fig 2. Time evolution of mass production at mhin ¼ mcin ¼ 50 kg=h. PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 Results and discussion The transient behavior of the accumulated mass of purified water for the selected operating
conditions is depicted in Figs 2 and 3. The results for flow rates of 50 and 300 L/h are shown in
the figure, but those of 100 and 200 L/h are not shown. Moreover, the trends of the responses
(specified and missing) are similar albeit with different extents. The inlet cold stream tempera-
ture is maintained at 25 ˚C, and the inlet water salinity at 0.5% for all the experiments. The
water mass is represented by a straight line because it is the measured mass of water in the Fig 3. Time evolution of mass production at mhin ¼ mcin ¼ 300 kg=h. https://doi.org/10.1371/journal.pone.0230207.g003 Fig 3. Time evolution of mass production at mhin ¼ mcin ¼ 300 kg=h. https://doi.org/10.1371/journal.pone.0230207.g003 Fig 3. Time evolution of mass production at mhin ¼ mcin ¼ 300 kg=h. https://doi.org/10.1371/journal.pone.0230207.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 6 / 24 PLOS ONE Transient model for mass flux in membrane distillation collection tank at each sampling time. The error bars in the figures denote the uncertainty in
the accumulated mass. The uncertainty is estimated as the standard deviation of several experi-
mental results under identical operating conditions. Note that the error bars are placed on spe-
cific samples because their inclusion at each sampling time overfills the figure, resulting in
degraded visibility. The sampling time is 10 s. The accumulated mass was transformed into the
production rate and is depicted in these figures by the dotted curve. The production rate was
obtained by dividing the accumulated mass by the corresponding time. The production rate is
a better representation of distillate production because it clearly reveals the dynamic response. It also assists in the evaluation of the steady-state production rate. However, for certain specific
operating conditions, the production rate does not attain a completely steady state. This is
attributed to the fact that the experiment is terminated prematurely. The reason for the prema-
ture termination is the limited capacity of the collection tank (T3). The tank can accumulate a
maximum of 25 kg of water. Beyond this, it must be emptied for sustaining continuous opera-
tion. This interruption disrupts the calculation of the mass flux over a long period. Therefore,
to estimate the steady-state production rate, the latter must be extrapolated for a prolonged
time. PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 Results and discussion To achieve this, the measured water mass is fitted to a correlation with time. The mass
accumulation at each operating condition is fitted as a straight line. The correlated mass accu-
mulation is demonstrated using dashed lines in Figs 2 and 3. Note that the correlated collected
mass is unique for each specific operating condition. That is, the collected mass for an operat-
ing condition cannot be extended for another operating condition. The aim is to obtain the
perfect match of the experimental results, as evidently illustrated. The resulting correlation is then extrapolated and transformed into the rate of change to
obtain the monotone function depicted in Fig 4, for all the tested flow rates and feed tempera-
tures. The extrapolation ensures that the mass-flux rate attains a steady state. Hence, the
dynamic behavior can be analyzed conveniently, and the steady-state production rate can be
estimated accurately. The process of correlation, extrapolation, and transformation into a
rate of change in mass accumulation is a crucial step in this analysis. The generated asymptotic
rate of mass production facilitates the dynamic analysis aimed at in this study, rather than Fig 4. Extrapolated response of mass production by using model correlation. https://doi.org/10.1371/journal.pone.0230207.g004 Fig 4. Extrapolated response of mass production by using model correlation. https://doi.org/10.1371/journal.pone.0230207.g004 PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 7 / 24 PLOS ONE Transient model for mass flux in membrane distillation integrating mass accumulation. First, the generated monotone response of the mass produc-
tion can be used to determine the dynamic characteristics by using the standard reaction-
curve method. Second, the generated monotone time response of the mass production can be
used to validate the theoretical model given by Eqs (1)–(6). It should be noted that the dynamic
model in Eqs (1–6) generates a mass production rate that exhibits an asymptotic response
unlike the measured mass production, which is presented as mass accumulation. It is apparent from Fig 4 that at each feed-flow rate, the production capacity increases pro-
portional to the feed temperature. This observation is anticipated and reported in earlier
works [7,9,41–44] because the increment in the feed temperature escalates the sensible heat of
the hot stream, which results in improved heat transfer from the hot bulk side to the mem-
brane surface. Similarly, at any operating feed temperature, the corresponding mass produc-
tion rate increases with the feed-flow rate. PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 Results and discussion https://doi.org/10.1371/journal.pone.0230207.g005 PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 8 / 24 PLOS ONE Transient model for mass flux in membrane distillation Fig 6. Steady-state gain for mw at selected operating conditions. https://doi.org/10.1371/journal.pone.0230207.g006 Fig 6. Steady-state gain for mw at selected operating conditions. https://doi.org/10.1371/journal.pone.0230207.g006 https://doi.org/10.1371/journal.pone.0230207.g006 illustrated in Fig 6. These values are directly proportional to the feed-flow rate and feed tem-
perature, as discussed previously. The static gain provides information on the effectiveness of
the operating conditions. For the minimum flow rate, the static gain is almost invariant with
the feed temperature. This indicates that increasing the feed temperature is ineffective for this
case. Similarly, at the flow rate of 100 L/h, the static gain varies insignificantly. However, at
higher flow rates, the favorable effect of the static gain on the operation is apparent. Appar-
ently, the static gain and time constant vary considerably with operating conditions. Therefore,
presentation of the mass production dynamics by a simple transfer function is not recom-
mended. It is more effective to capture the mass production dynamics using the entire system
of nonlinear ODEs, as discussed in the following section. Further, we seek to validate the dynamic model based on the first principles expressed as
Eqs (1)–(6), using the experimental data. It is to be remembered that the theoretical model dif-
fers from the straight-line correlation used previously for extrapolating the experiment data. This model is universal in that it is applicable to different operating conditions. Moreover, it is
comprehensive, i.e., it explicitly includes the process parameters and other design parameters. In addition, the resultant model is a standard ODE, which is typical for dynamic and control
analysis and application. The ODE model is simulated by stepping Thin while keeping Tcin con-
stant at 25 ˚C. The mass-flow rate is considered to be a stepwise constant, i.e., it remains con-
stant while the IVP is solved. The IVP is solved using Euler’s method with a step size of 1 s. This step size was observed to be adequate for providing a stable numerical solution. In each
step of Euler’s method, the intermediate parameters are determined by following the algorithm
provided in Appendix B. In Fig 7, the result of the simulation is depicted and is compared to
the time response of the plant. Results and discussion As the feed-flow rate increases, the turbulence on
the membrane surface also increases. This phenomenon improves the heat- and mass-transfer
mechanisms, resulting in higher production rates [7,9,11,41–44] It is apparent that the transient response of mw in Fig 4 resembles the reaction curve of a
typical first-order system in response to stepped variations. Therefore, the linear system theory
can be used to determine the dynamic characteristics, i.e., time constant and static gain, of the
mass production, mw. In this case, the dynamic characteristics of mw in response to the stepped
variations in Thin are estimated. The estimated time constant for the reaction curves in Fig 4 is
presented in Fig 5. The measured time constant varies notably with respect to the operating
conditions, indicating the nonlinearity of the process. In general, the time constant can vary
between 5 and 15 min. For the lowest value of the operating feed temperature, the time con-
stant decreases linearly as the flow rate increases. This implies that the process response
becomes faster as the flow rate increases. For higher feed temperatures, the time constant
decreases as the flow rate increases up to 200 L/h. Thereafter, it increases with further increases
in the flow rate. The deceleration of the process dynamic at higher flow rates, specifically at
300 L/h, is not related to the fundamental mass- and heat-transfer operation inside the mem-
brane. This behavior is ascribed to the dynamics of the external heater (H1). The reaction-
curve method requires the output response to the instantaneous stepped variations in the
input. However, in our experiments, the stepped variations in Thin exhibits a certain time lag
induced by the capacitance of the external heater. This underlying dynamic is marginal at
small flow rates. However, it increases substantially at high flow rates and temperatures [23]. This behavior is reflected by the response speed of mw at high operating conditions. The static mass production gain at different operating conditions is extracted from the reac-
tion curves in Fig 4 by using the reaction-curve method. The results of the extraction are Fig 5. Time constant for mass production in response to a stepped variation in Thin. https://doi.org/10.1371/journal.pone.0230207.g005 Fig 5. Time constant for mass production in response to a stepped variation in Thin Fig 5. Time constant for mass production in response to a stepped variation in Thin. PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 Results and discussion The figure depicts the simulation of the lumped model and spa-
tial model with n = 10 for a wide range of operating conditions. The performance of the
lumped and spatial models exhibits a notable model–plant mismatch, particularly in terms of
the static gain, except at the lowest flow rate and feed temperature (50 L/h, 50 ˚C). The mis-
match increases notably with flow rate and to a certain extent, with feed temperature. Whereas
the lumped and spatial models delivered almost similar performance at low operating flow
rates, they exhibited divergent behaviors at high flow rates. It is noteworthy that the lumped
model outperformed the spatial model in terms of the overall average error. Numerically, the
overall relative error for the lumped model is less than that for the spatial model, as presented
in Table 1. Note that for the lumped model, the geometric average bulk temperature is used in
Algorithm S1. When terminal temperatures are used as approximations of the bulk tempera-
ture in the limped model, the performance deteriorates as the overall average error attains PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 9 / 24 PLOS ONE Transient model for mass flux in membrane distillation Fig 7. Mass production response for model and plant to stepped variation; solid: Plant, dashed: Lumped model,
dotted: Spatial model. https://doi org/10 1371/journal pone 0230207 g007 Fig 7. Mass production response for model and plant to stepped variation; solid: Plant, dashed: Lumped model,
dotted: Spatial model. https://doi org/10 1371/journal pone 0230207 g007 Fig 7. Mass production response for model and plant to stepped variation; solid: Plant, dashed: Lumped model
dotted: Spatial model. https://doi.org/10.1371/journal.pone.0230207.g007 https://doi.org/10.1371/journal.pone.0230207.g007 51%, as listed in Table 1. The relative error presented in Table 1 is computed using the follow-
ing formula: 51%, as listed in Table 1. The relative error presented in Table 1 is computed using the follow-
ing formula: Er ¼
X
80
T¼50
X
nt
i¼1
mm
wðtiÞ me
wðtiÞ
me
wðtiÞ
! T
100
ð7Þ ð7Þ where mm
w and me
w denote the model response and plant response, respectively, for the mass
production. nt denotes the simulation time. Er is computed for each flow rate, and the overall
error is the mean value across all the flow rates. The inferiority of both the model structures can be attributed to the omission of the heat
losses to the surrounding and to modeling error. https://doi.org/10.1371/journal.pone.0230207.t001 Results and discussion As a remedy, we propose the following model modification to incorporate the
effect of heat losses to the surrounding:
For lumped case: vrCp dThout
dt
¼ mhinCp Thin Trf
mhoutCp Thout Trf
hmA Thm Tcm
jwAHv flosmhinCp Thin Thout
ð8Þ For spatial case: Trf
mhiCp Thi Trf
hmi D xh Thm;i Tcm;i
jwi D xhHv flosmhi 1Cp Thi 1 Thi
ð9Þ v
n
rCp dThi
dt ¼ mhi 1Cp Thi 1 Trf
mhiCp Thi Trf
hmi D xh Thm;i Tcm;i
jwi D xhHv flosmhi 1Cp Thi 1 Thi
ð9Þ Note that the heat loss term is incorporated in the thermal equation only for the hot chan-
nel. We assume that a portion of the sensible heat of the hot side is lost to the ambient. This
implies that only a fraction of the hot sensible heat is available for heat transfer to the cold bulk
side. The heat loss (correction term) becomes proportional to the operating temperature with
the incorporation of the heat loss as a fraction of the sensible heat. This formulation mimics
the modeling error observed in Fig 7, which increases with the operating temperature. To
make the correction term increase with the flow rate, the fraction parameter (flos) is set propor-
tional to the flow rate, as presented in Table 2. The values of flos in Table 2 are determined by
trial and error and heuristics. Fig 8 depicts the model performance when the proposed tuning strategy is applied. It is
highly evident that the model’s effectiveness for both the structures is enhanced substantially. Specifically, the overall relative error is reduced to approximately 3%, as presented in Table 1. It is equally evident that the spatial model is not more advantageous than the lumped one
because both structures provided remarkable fitting of the plant data when they were effec-
tively tuned. A few marginal discrepancies are present in the time response, which are dis-
cussed in the final paragraphs. Note that the correction term in Eqs (8) and (9) is maintained Fig 8. Results and discussion Heat loss to the surroundings is reported by
other works [38,44]. In general, heat losses increase with feed temperature (as manifested by
the marginally increasing mismatch in Fig 7) because the difference between the module and
ambient temperatures also increases. However, the increment in heat loss/modeling error with
flow rate is unclear. Nevertheless, we can refer to the nonlinearity of the process observed in
[32,38]. As the flow rate increases, the heat-transfer coefficients enhance the heat transfer,
resulting in a higher temperature at the permeate bulk side. As the flow rate continues to
increase, the heat transfer becomes asymptotic because the driving force (temperature differ-
ence between the bulk hot side and bulk cold side) decreases. Simultaneously, the temperature
difference at the membrane interface increases, resulting in improved mass flux. This behavior
is reflected on both the model performances when a flow rate of 200 L/h is implemented. Unlike the other cases, the model-plane discrepancy decreases with feed temperature for the Table 1. Percentage relative error of model–plant mismatch. mf
Without tuning
With tuning
Without tuning and using terminal temperature
Lumped
Spatial
Lumped
Spatial
Lumped
50 L/h
7.78
14.00
3.08
3.04
49.08
100 L/h
10.01
15.97
3.78
2.90
46.46
200 L/h
9.31
18.92
3.29
3.54
53.19
300 L/h
16.99
25.19
2.71
3.51
56.51
Overall
11.02
18.52
3.22
3.25
51.31 Table 1. Percentage relative error of model–plant mismatch. PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020
10 / 24 10 / 24 PLOS ONE Transient model for mass flux in membrane distillation Table 2. Tuning parameter for adjusting heat loss (flos). 50 L/h
100 L/h
200 L/h
300 L/h
n = 10
0.15
0.2
0.22
0.26
n = 1
0.077
0.11
0.1
0.17
https://doi.org/10.1371/journal.pone.0230207.t002 Table 2. Tuning parameter for adjusting heat loss (flos). case of 200 L/h. As a remedy, we propose the following model modification to incorporate the
effect of heat losses to the surrounding:
For lumped case: case of 200 L/h. As a remedy, we propose the following model modification to incorporate the
effect of heat losses to the surrounding:
For lumped case: case of 200 L/h. Results and discussion Mass production response of model and plant to stepped variation with tuned U; solid: Plant, dashed:
Lumped model, dotted: Spatial model, dash-and-dot: Independently tuned lumped model. https://doi.org/10.1371/journal.pone.0230207.g008 Fig 8. Mass production response of model and plant to stepped variation with tuned U; solid: Plant, dashed:
Lumped model, dotted: Spatial model, dash-and-dot: Independently tuned lumped model. Fig 8. Mass production response of model and plant to stepped variation with tuned U; solid: Plant, dashed:
Lumped model, dotted: Spatial model, dash-and-dot: Independently tuned lumped model. https://doi.org/10.1371/journal.pone.0230207.g008 11 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 PLOS ONE Transient model for mass flux in membrane distillation as simple as possible to maintain the model simplicity and predictiveness. Although the correc-
tion term is denoted as heat losses, it appears to account for both the heat losses and other
uncertain modeling errors. According to the values of flos in Table 2, the lumped modeling
errors are 7%–17% in the lumped model and 15%–26% in the spatial model. An enhancement
of the lumped model that uses terminal temperature as bulk temperature is not presented here. Our investigation revealed that tuning such a model mandates an ambiguously large correc-
tion factor (flos). To understand the effect of omitting the effect of heat losses on the model performance, the
temperature difference at the membrane interface (DTm ¼ Thm Tcm) is plotted as shown
in Fig 9. This result corresponds to the case where the correction term is not incorporated in
Eq (9), i.e., it belongs to the results shown in Fig 7. The interface temperature difference is rela-
tively high compared to that shown in Fig 10, which corresponds to the case where the correc-
tion term is employed in the model. This implies that the model predicted a ΔTm value that is
larger than its true values, causing the mass production predicted by the model to inflate as
shown in Fig 7. When the heat losses are omitted, the entire available sensible heat is trans-
ferred from the hot side to the cold side, whereby the permeate outlet temperature is increased. As a result, the bulk temperature difference as well as the associated interface temperature dif-
ference increase. When the heat losses term is involved, the model predicts smaller interface
temperature differences, as illustrated in Fig 10. PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 Results and discussion In this case, the model considers less amount
of sensible heat to be transferred, resulting in a lower permeate outlet temperature and hence,
smaller bulk and interface temperature differences. The smaller temperature differences gener-
ated better estimates of the mass fluxes, as depicted in Fig 8. It should be noted that the
responses of ΔTm shown in Figs 9 and 10 are for the spatial model case, where the average
value of ΔTm over the module length is plotted. A similar trend, albeit with a different extent,
is obtained for the lumped model case. The results are not shown here because Figs 9 and 10
serve the purpose. Regardless of the model structure used, the trend of ΔTm response coincides Fig 9. Average difference between hot and cold membrane interface temperatures omitting heat losses. https://doi.org/10.1371/journal.pone.0230207.g009 Fig 9. Average difference between hot and cold membrane interface temperatures omitting heat losses. https://doi.org/10.1371/journal.pone.0230207.g009 PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 12 / 24 PLOS ONE Transient model for mass flux in membrane distillation Fig 10. Average difference between hot and cold membrane interface temperatures considering heat losses. https://doi.org/10.1371/journal.pone.0230207.g010 Fig 10. Average difference between hot and cold membrane interface temperatures considering heat losses. https://doi.org/10.1371/journal.pone.0230207.g010 https://doi.org/10.1371/journal.pone.0230207.g010 with that of the mass production shown in Fig 4, wherein it is proportional to the hot feed tem-
perature and operating flow rate. The proposition of heat losses to the surroundings is reasonable and has been observed by
others as mentioned earlier. However, the proportionality of the heat losses to the flow rate is
unclear. Another method to improve the model performance through a better estimation of
the heat transfer mechanism is also valid. Figs 7 and 9 show that the values of the mass flux
predicted by the model are higher than the measured ones because of the erroneous estimation
of the interface temperatures. The latter are estimated by Eqs (B.12) and (B.13). Hence, the
erroneous values of the interface temperatures could be owing to the limitation of the model
in adapting the values of hp and hf or U to variations in the operating conditions. Therefore, a
better estimation of the interface temperatures and consequently the mass flux can be achieved
by penalizing U directly or using better Nusselt correlation. Investigation on this by the
authors is underway. PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 Results and discussion It should be reminded that Algorithm S1 is still applied in the numerical solution of the
IVP (Eqs (1)–(6)) because the algorithm estimates internal parameters of the model, such as
the membrane coefficient, Cm, membrane-surface temperatures, Thm; Tcm, and local heat-
transfer coefficients, hf, hp. These parameters are embedded in the IVP problem. Nevertheless,
the origin of the model–plant mismatch exhibited in the model performance should be
highlighted here. This versatile mismatch is the result of several factors such as uncertainty in
process measurements, uncertainty owing to the modeling simplifications, limitation of the
process design, and nonlinearity of the process. The model should not be constrained to
match the experimental data because the measured data contain certain uncertainties. The
plant experiments exhibited both measurement and random errors, as illustrated in Figs 2 and
3. The models were simulated using identical initial values, whereas the actual initial tempera-
tures in the experiments varied across the tests by ±1–3˚ depending upon the room tempera-
ture. The stepped variations in Thin in the real plant are not instantaneous and rather exhibit a
dynamic behavior. Tcin is not constant during the experiments. It exhibits randomness owing PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 13 / 24 PLOS ONE Transient model for mass flux in membrane distillation to the manual-control system. This issue is particularly discussed in a previously published
work [32]. to the manual-control system. This issue is particularly discussed in a previously published
work [32]. Conclusions In this study, a first-principles dynamic model for purified water production using the DCMD
process is developed. Specifically, the structures of both the lumped-parameter model and spa-
tial model were constructed. The accuracy of both the models in predicting the distillate rate
was tested against experimental data generated by step testing a DCMD pilot plant. The accu-
racy of both the model structures was low, with the lumped-model structure being marginally
superior to the spatial model. Although the underlying parameters of both the models were
calculated at each sampling time by solving combined mass and energy balances, the models
could not effectively track the mass production response over a wide range of operating condi-
tions. An adjustment of the model by incorporating a heat loss term improved their perfor-
mances: the overall relative error reduced to approximately 3%. Alternatively, the heat transfer
correlation could be revised for enhanced model performance. PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 Appendix A Therefore, from the left
side of the last two equations, i.e., by equating the membrane conduction plus latent heat
terms to the convection from the bulk to the membrane surface, we obtain Thm ¼
hfðhm þ hpÞ
hphf þ hmðhp þ hfÞ Thb þ
hmhp
hphf þ hmðhp þ hfÞ Tcb ð
hp
hphf þ hmðhp þ hfÞÞ jwHv
ðA:6Þ
Tcm ¼
hfhm
hphf þ hmðhp þ hfÞ Thb þ
hpðhm þ hfÞ
hphf þ hmðhp þ hfÞ Tcb þ
hf
hphf þ hmðhp þ hfÞ jwHv
ðA:7Þ PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 14 / 24 PLOS ONE Transient model for mass flux in membrane distillation or in a compact form as or in a compact form as Thm ¼ ah Thb þ ac Tcb ch jwHv
ðA:6aÞ
Tcm ¼ bhThb þ bcTcb þ cc jwHv
ðA:7aÞ Thm ¼ ah Thb þ ac Tcb ch jwHv
ðA:6aÞ Thm ¼ ah Thb þ ac Tcb ch jwHv ðA:6aÞ Tcm ¼ bhThb þ bcTcb þ cc jwHv
ðA:7aÞ ðA:7aÞ ðA:8Þ ah
hphf þ hmðhp þ hfÞ
ðA:8Þ
ac ¼
hphm
hphf þ hmðhp þ hfÞ
ðA:9Þ
bh ¼
hfhm
hphf þ hmðhp þ hfÞ
ðA:10Þ
bc ¼
hpðhm þ hfÞ
hphf þ hmðhp þ hfÞ
ðA:11Þ
ch ¼
hp
hphf þ hmðhp þ hfÞ
"
#
ðA:12Þ
cc ¼
hf
hphf þ hmðhp þ hfÞ
"
#
ðA:13Þ ðA:9Þ ðA:10Þ bc ¼
hpðhm þ hfÞ
hphf þ hmðhp þ hfÞ
ðA:11Þ ðA:11Þ ðA:12Þ cc ¼
hf
hphf þ hmðhp þ hfÞ
"
#
ðA:13Þ ðA:13Þ Cm is the MD coefficient and is considered a weak function of temperature while deriving
the dynamic model. Further details on computing Cm are provided in Appendix B. Similarly,
the physical properties of water are a function of temperature. However, we omit their deriva-
tive with respect to temperature. Hence, the derivative of (A.1) with respect to time is dmw
dt ¼ CmA d
dt P1 P2
ð
Þ:
ðA:14Þ ðA:14Þ The above equation was presented by Hassan et al. [34]. However, they did not consider cal-
culating the derivative of the vapor pressures. Appendix A Considering the whole MD unit as a lumped-parameter system as shown in S1 Fig, the steady
state mass production rate of water is expressed as follows [32]: mw ¼ CmðP1 P2ÞA
ðA:1Þ ðA:1Þ P1 and P2 are the partial pressures of water vapor estimated at the membrane surface tem-
peratures Thm and Tcm, respectively. These can be calculated using the Antoine equation [6,7]: P1 ¼ expða b
Thm cÞ
ðA:2Þ
P2 ¼ expða b
Tcm cÞ
ðA:3Þ P1 ¼ expða b
Thm cÞ
ðA:2Þ ðA:2Þ P2 ¼ expða b
Tcm cÞ
ðA:3Þ ðA:3Þ The membrane interface temperatures can be related to the membrane bulk temperature
when the convection and conduction heat transfer attain equilibrium [8]: The membrane interface temperatures can be related to the membrane bulk temperature
when the convection and conduction heat transfer attain equilibrium [8]: hmðThm TcmÞ þ jwHv ¼ hfðThb ThmÞ ¼ UðThb TcbÞ
ðA:4Þ
hmðThm TcmÞ þ jwHv ¼ hpðTcm TcbÞ ¼ UðThb TcbÞ
ðA:5Þ ðA:4Þ ðA:5Þ Note that the equalities (A.4) and (A.5) hold at steady state. Because we are developing a
dynamic model, we assume that they hold at a pseudo-steady state. PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 Appendix A In the following, we present the derivation of the
partial derivatives to obtain a complete expression for the time evolution of the transmem-
brane flux: @P1
@Thm
@Thm
@Tcb
dTcb
dt þ @P1
@Thm
@Thm
@mw
dmw
dt @P2
@Tcm
@Tcm
@Thb
dThb
dt @P2
@Tpm
@Tcm
@Tcb
dTcb
dt
@P2
@Tcm
@Tcm
@mw
dmw
dt
! ðA:14aÞ The partial derivatives in Eq (A.14) can be obtained as follows. The derivatives of the partial
pressure given in Eqs (A.2) and (A.3), with respect to the membrane interface temperatures The partial derivatives in Eq (A.14) can be obtained as follows. The derivatives of the partial
pressure given in Eqs (A.2) and (A.3), with respect to the membrane interface temperatures PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 15 / 24 PLOS ONE Transient model for mass flux in membrane distillation are @P1
@Thm
¼
b
ðThm cÞ
2 exp a b
Thm c
! bh
ðA:15Þ @P1
@Thm
¼
b
ðThm cÞ
2 exp a b
Thm c
! bh
ðA:15Þ
@P2
@Tcm
¼
b
ðTcm cÞ
2 exp a b
Tcm c
! bc
ðA:16Þ ðA:15Þ @P2
@Tcm
¼
b
ðTcm cÞ
2 exp a b
Tcm c
! bc
ðA:16Þ ðA:16Þ The derivatives of the membrane interface temperature given in Eqs (A.6a) and (A.7a),
with respect to the bulk temperature and mass flux are The derivatives of the membrane interface temperature given in Eqs (A.6a) and (A.7a),
with respect to the bulk temperature and mass flux are @Thm
@Thb
¼ ah; @Thm
@Tcb
¼ ac ; @Thm
@mw
¼ chHv=A
ðA:17Þ
@Tcm
@Thb
¼ bh; @Tcm
@Tcb
¼ bc ; @Tcm
@mw
¼ ccHv=A
ðA:18Þ @Thm
@Thb
¼ ah; @Thm
@Tcb
¼ ac ; @Thm
@mw
¼ chHv=A
ðA:17Þ ðA:17Þ @Tcm
@Thb
¼ bh; @Tcm
@Tcb
¼ bc ; @Tcm
@mw
¼ ccHv=A
ðA:18Þ ðA:18Þ Substituting Eqs (A.15)–(A.18) into Eq (A.14) yields Substituting Eqs (A.15)–(A.18) into Eq (A.14) yields Substituting Eqs (A.15)–(A.18) into Eq (A.14) yields dmw
dt ¼
CmA
ð1 þ CmbhchHv þ CmbcccHvÞ
bhah bcbh
ð
Þ dThb
dt þ bhac bcbc
ð
Þ dTcb
dt
ðA:19Þ ðA:19Þ The dynamic model for the mass production in Eq (A.19) depends explicitly on the dynam-
ics of the bulk temperature in the hot and cold sides. Appendix A Hence, to completely specify the IVPs, i.e., link the three state variables, we assume
the following relationship between the bulk and boundary temperatures: Thb ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ThinThout
q
; Tcb ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
TcinTcout
q
ðA:24Þ ðA:24Þ 16 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 PLOS ONE Transient model for mass flux in membrane distillation The derivatives of the last equations with respect to time are dThb
dt ¼ 1
2
ffiffiffiffiffiffiffiffiffi
Thin
Thout
s
dThout
dt
ðA:25Þ ðA:25Þ dTcb
dt ¼ 1
2
ffiffiffiffiffiffiffiffi
Tcin
Tcout
s
dTcout
dt
ðA:26Þ ðA:26Þ Inserting Eqs (A.25) and (A.26) into Eq (A.19) yields Inserting Eqs (A.25) and (A.26) into Eq (A.19) yields Inserting Eqs (A.25) and (A.26) into Eq (A.19) yields dmw
dt ¼
CmA
ð1 þ CmbhchHv þ CmbcccHvÞ
bhah bcbh
ð
Þ 1
2
ffiffiffiffiffiffiffiffiffi
Thin
Thout
s
dThout
dt
þ bhac bcbc
ð
Þ 1
2
ffiffiffiffiffiffiffiffi
Tcin
Tcout
s
dTcout
dt
! ðA:27Þ Hence, Eqs (A.20)–(A.23) and (A.27) define the complete dynamic model for the mass pro-
duction in the lumped form. In this study, the inlet flow rates for the cold and hot sides are
equal, i.e., mcin ¼ mhin. The operation is limited by the experimental procedure. Note that the
membrane surface temperatures,Thm; Tcm, internal heat transfer coefficients, hf hp, hm, and
intermediate variables, jw, Hv are obtained by solving the combined mass and heat transfer
equations as described in Appendix B. These parameters are determined at each time-instant
during the numerical solution of the IVP (Eqs A.20, A.21 and A.27). Appendix A The dynamic of the thermal behavior of
the MD was developed by [32] and is as follows: The dynamic model for the mass production in Eq (A.19) depends explicitly on the dynam-
ics of the bulk temperature in the hot and cold sides. The dynamic of the thermal behavior of
the MD was developed by [32] and is as follows: rCp dThout
dt
¼ mhinCp Thin Trf
mhoutCp Thout Trf
hmA Thm Tcm
jwAHv ðA:20Þ
vrCp dTcout
dt
¼ mcinCp Tcin Trf
mcoutCp Tcout Trf
þ hmA Thm Tcm
þ jwAHv ðA:21Þ rCp dThout
dt
¼ mhinCp Thin Trf
mhoutCp Thout Trf
hmA Thm Tcm
jwAHv ðA:20Þ vrCp dTcout
dt
¼ mcinCp Tcin Trf
mcoutCp Tcout Trf
þ hmA Thm Tcm
þ jwAHv ðA:21Þ The mass balance equations are as follows: The mass balance equations are as follows: The mass balance equations are as follows: mhout ¼ mhin mw
ðA:22Þ
mcout ¼ mcin þ mw
ðA:23Þ mhout ¼ mhin mw
ðA:22Þ mhout ¼ mhin mw
ðA:22Þ ðA:22Þ ðA:23Þ mcout ¼ mcin þ mw
ðA:23Þ For the lumped-parameter model, Eq (A.19) requires the time-derivative of the bulk tem-
perature, whereas the thermal Eqs (A.20) and (A.21) are defined in terms of the terminal tem-
perature. Extension of lumped dynamic model to one-dimensional formulation To develop the axial dynamic model, the thermal balance represented by Eqs (A.20) and
(A.21) can be written for a specific control volume of the membrane, as shown in S1 Fig. Hence, by discretizing Eqs (A.20) and (A.21), the resulting system of IVPs is expressed as fol-
lows: v
n
rCp dThi
dt ¼ mhi 1Cp Thi 1 Trf
mhiCp Thi Trf
hmi D xh Thm;i Tcm;i
jwi D xhHvðA:28Þ
v
n
rCp dTci
dt ¼ mciþ1 Cp Tciþ1 Trf
mciCp Tci Trf
þ hmi D xh Thm;i Tcm;i
þ jwi D xhHvðA:29Þ v
n
rCp dThi
dt ¼ mhi 1Cp Thi 1 Trf
mhiCp Thi Trf
hmi D xh Thm;i Tcm;i
jwi D xhHvðA:28Þ
v
dTci
T
T
T
T
h
D h T
T
D h
ð
Þ p dThi
dt ¼ mhi 1Cp Thi 1 Trf
mhiCp Thi Trf
hmi D xh Thm;i Tcm;i
jwi D xhHvðA:28Þ n
dt
v
n
rCp dTci
dt ¼ mciþ1 Cp Tciþ1 Trf
mciCp Tci Trf
þ hmi D xh Thm;i Tcm;i
þ jwi D xhHvðA:29Þ v
n
rCp dTci
dt ¼ mciþ1 Cp Tciþ1 Trf
mciCp Tci Trf
þ hmi D xh Thm;i Tcm;i
þ jwi D xhHvðA:29Þ i = 1, , n for i ¼ 1 ! Thi 1 Thin ; Tci Tcout
for i ¼ n ! Thi Thout ; Tciþ1 Tcin for i ¼ 1 ! Thi 1 Thin ; Tci Tcout for i ¼ n ! Extension of lumped dynamic model to one-dimensional formulation Thi Thout ; Tciþ1 Tcin The corresponding infinitesimal dynamic model for the mass production (A.19) can be
expressed as follows: dmwi
dt
¼
CmiA
ð1 þ CmibhichiHvi þ CmibcicciHviÞ
bhiahi bcibhi
dThi
dt þ
bhiaci bcibci
dTci
dt
ðA:30Þ PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020
17 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 17 / 24 17 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 PLOS ONE Transient model for mass flux in membrane distillation where
bhi ¼
b
ðThm;i cÞ
2 exp a b
Thm;i c
! ðA:31Þ
bci ¼
b
ðTcm;i cÞ
2 exp a b
Tcm;i c
! ðA:32Þ
ahi ¼
hfiðhmi þ hpiÞ
hpihfi þ hmiðhpi þ hfiÞ
ðA:33Þ
aci ¼
hpihmi
hpihfi þ hmiðhpi þ hfiÞ
ðA:34Þ
bhi ¼
hfihmi
hpihfi þ hmiðhpi þ hfiÞ
ðA:35Þ
bci ¼
hpiðhmi þ hfiÞ
hpihfi þ hmiðhpi þ hfiÞ
ðA:36Þ
chi ¼
hpi
hpihfi þ hmiðhpi þ hfiÞ
"
#
ðA:37Þ
cci ¼
hfi
hpihfi þ hmiðhpi þ hfiÞ
"
#
ðA:38Þ bhi ¼
b
ðThm;i cÞ
2 exp a b
Thm;i c
! ðA:31Þ
bci ¼
b
ðTcm;i cÞ
2 exp a b
Tcm;i c
! ðA:32Þ ðA:31Þ ðA:32Þ ðA:33Þ ðA:33Þ ðA:34Þ ðA:38Þ whereas hf i; hpi; hmi; Cmi are calculated from (B.1, B.11, B.4–B.6) using the bulk temperature
for each ith control volume. whereas hf i; hpi; hmi; Cmi are calculated from (B.1, B.11, B.4–B.6) using the bulk temperature
for each ith control volume. whereas hf i; hpi; hmi; Cmi are calculated from (B.1, B.11, B.4–B.6) using the bulk temperature
for each ith control volume. PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 Appendix B The overall heat-transfer coefficient, membrane interface temperatures, and membrane coeffi-
cient are calculated by the following algorithm (S1). The following formulation is the conclu-
sion of our previously conducted work on MD modeling and analysis [32,45–47]. 1. Given the bulk temperatures at both sides of the MD membrane, Thb ; Tcb, the local heat-
transfer coefficients, hf, hp, are calculated from the Nusselt number as follows (Alkhudairi
et al., 2012): Nu ¼ 0:298Ren1Prn2
ðB:1Þ ðB:1Þ where Re denotes the Reynolds number, Pr denotes the Prandtl number, n1 = 0.646, and
n2 = 0.316. where Re denotes the Reynolds number, Pr denotes the Prandtl number, n1 = 0.646, and
n2 = 0.316. For the lumped-parameter model, the inlet temperature of the hot stream (Thin) is generally
approximated as the bulk temperature of the hot stream, whereas the outlet temperature of
the cold stream (Tcout) is approximated as that of the cold stream. The use of boundary For the lumped-parameter model, the inlet temperature of the hot stream (Thin) is generally
approximated as the bulk temperature of the hot stream, whereas the outlet temperature of
the cold stream (Tcout) is approximated as that of the cold stream. The use of boundary PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 18 / 24 PLOS ONE Transient model for mass flux in membrane distillation temperatures for the bulk results in higher values of mw in the steady state [32]. Alterna-
tively, the geometric mean of the boundary temperatures (A.25) and (A.26) can be used. For the spatial model, the bulk temperatures are approximated as the bulk temperature of
each control volume. 2. Set T0
hm ¼ Thb and T0
c m ¼ Tcb 2. Set T0
hm ¼ Thb and T0
c m ¼ Tcb 3. Calculate the vapor pressure at the membrane interface using the following [6]: P1 ¼ expð23:238 3841
Thm 45Þ 1 Cs
ð
Þ 1 0:5Cs 10C2
s
ðB:2Þ ðB:2Þ P2 ¼ expð23:238 3841
Tcm 45Þ
ðB:3Þ ðB:3Þ 4. Appendix B The membrane interface temperatures are computed using the right-
side equality of Eqs (A.6) and (A.7): 8. At equilibrium, all the heat-transfer mechanisms within the MD are equivalent. Hence, Eqs
(A.4) and (A.5) hold. The membrane interface temperatures are computed using the right-
side equality of Eqs (A.6) and (A.7): 8. At equilibrium, all the heat-transfer mechanisms within the MD are equivalent. Hence, Eqs
(A.4) and (A.5) hold. The membrane interface temperatures are computed using the right-
side equality of Eqs (A.6) and (A.7): hfðThb ThmÞ ¼ UðThb TcbÞ
ðB:12Þ
hpðTcm TcbÞ ¼ UðThb TcbÞ
ðB:13Þ ðB:12Þ ðB:13Þ 9. If Thm ¼ T0
h m and Tcm ¼ T0
c m; stop the iteration; otherwise, set T0
h m ¼ Thm and T0
c m ¼ Tcm. Then, return to Step 3. 9. If Thm ¼ T0
h m and Tcm ¼ T0
c m; stop the iteration; otherwise, set T0
h m ¼ Thm and T0
c m ¼ Tcm. Then, return to Step 3. The above-mentioned algorithm is terminated using a termination tolerance of 1 × 10−7. Note that the algorithm is applicable to both the lumped and spatial versions of the model. Furthermore, for both models, the above-mentioned algorithm is solved assuming quasi-
steady-state conditions. Appendix B After determining the membrane characteristics and average membrane temperature, i.e.,
T ¼
Thm þTcm
2
, the membrane coefficient Cm can be estimated using the correlation provided
in [8] according to the designated mechanism as follows: • Knudson-flow mechanism, kn > 1: Ck
m ¼ 2εr
3td
8Mw
pRT
1=2
ðB:4Þ ðB:4Þ • Molecular-diffusion mechanism, kn < 0.01: • Molecular-diffusion mechanism, kn < 0.01: • Molecular-diffusion mechanism, kn < 0.01: CD
m ¼ ε
td
PD
Pa
Mw
RT
ðB:5Þ ðB:5Þ • Knudsen molecular-diffusion transition mechanism, 0.01 < kn < 1: • Knudsen molecular-diffusion transition mechanism, 0.01 < kn < 1: • Knudsen molecular-diffusion transition mechanism, 0.01 < kn < 1: CC
m ¼
3
2
td
εr
pRT
8Mw
1=2
þ td
ε
Pa
PD
RT
Mw
"
# 1
ðB:6Þ ðB:6Þ where the Knudsen number is defined as kn ¼ l
d. λ is the mean free path of water molecules
and is expressed as [4]: where the Knudsen number is defined as kn ¼ l
d. λ is the mean free path of water molecules
and is expressed as [4]: l ¼
kBT
ffiffiffi
2
p
pPde
2
ðB:7Þ ðB:7Þ 5. Calculate the latent heat of vaporization at the average membrane temperature as 5. Calculate the latent heat of vaporization at the average membrane temperature as HvðTÞ ¼ 1850:7 þ 2:8273T 1:6 10 3T2
ðB:8Þ ðB:8Þ PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 19 / 24 PLOS ONE Transient model for mass flux in membrane distillation 6. Calculate the mass flux as 6. Calculate the mass flux as 6. Calculate the mass flux as jw ¼ CmðP1 P2Þ
ðB:9Þ ðB:9Þ 7. Compute the overall heat-transfer coefficient using [8] 7. Compute the overall heat-transfer coefficient using [8] 7. Compute the overall heat-transfer coefficient using [8] U ¼
1
hf
þ
1
hm þ
JHv
Thm Tcm
þ 1
hp
"
# 1
ðB:10Þ ðB:10Þ The membrane heat-transfer coefficient (hm) represents the heat resistance owing to con-
duction and can be estimated using [11] The membrane heat-transfer coefficient (hm) represents the heat resistance owing to con-
duction and can be estimated using [11] hm ¼ km
d ¼ ð1 εÞks þ εkg
d
ðB:11Þ ðB:11Þ 8. At equilibrium, all the heat-transfer mechanisms within the MD are equivalent. Hence, Eqs
(A.4) and (A.5) hold. PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 Nomenclature A
Cross-sectional area, m2
A
Antione equation constant
b
Antione equation constant
c
Antione equation constant
Cm
Permeability coefficient, kg/m2sPa
Ck
m
Knudsen mass-flux coefficient, kg/m2sPa
Cd
m
Molecular diffusion mass-flux coefficient, kg/m2sPa
CC
m
Transition mass-flux coefficient, kg/m2sPa
Cp
Heat capacity, J/kgK
Cs
Salt concentration, %
Er
Percentage relative error for model–plant mismatch
Hv
Latent heat of vaporization, J/kg
(Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
March 24, 2020 20 / 24 PLOS ONE Transient model for mass flux in membrane distillation Supporting information
S1 Fig. Schematic of MD process: (a) lumped module, (b) ith control volume of MD mod-
(Continued)
hf, hp, hm
Heat-transfer coefficients of the feed, permeate, and membrane, W/m2K
H
Channel height, m
Jw
Mass flux, kg/m2h
k1, k2
Static gains
kB
Boltzmann constant
km
Membrane conductivity, W/mK
ks
Solid-phase thermal conductivity, W/mK
kg
Gas-phase thermal conductivity, W/mK
kn
Knudsen number
mhin; mhout
Inlet and outlet mass-feed rate for hot fluid, kg/h
mcin; mcout
Inlet and outlet mass-feed rate for cold fluid, kg/h
mhi
Mass-flow rate for hot side for ith cell, kg/h
mci
Mass-flow rate for cold side for ith cell, kg/h
m
Distillate mass, kg
mw
Distillate flow rate, kg/h
Mw
Molecular weight
Nu
Nusselt number
N
Number of membrane-length divisions, i.e., control elements
nt
Length of simulation time
P1, P2
Vapor pressure at feed and permeate membrane surface, Pa
Pa
Entrapped-air pressure, Pa
PD
Membrane pressure multiplied by diffusivity, Pam2/s
Pr
Prandtl number
R
Membrane pore size, m
R
Ideal gas constant,
Re
Reynold number
S
Laplace domain
T
Time
Th, Tc
Feed (hot) and permeate (cold) temperatures, K
Thb, Tcb
Feed (hot) and permeate (cold) bulk temperatures, K
Thm, Tcm
Feed and permeate membrane temperatures, K
Thout; Thin
Outlet and inlet hot feed temperatures, ˚C
Tcout; Tcin
Outlet and inlet cold stream temperatures, ˚C
TF
Transfer function
U
Overall heat-transfer coefficient, W/m2K
V
Channel volume, m3
Greek letters
Τ
Time constant, min, also membrane tortuosity
ρ
Water density, kg/m3
δ
Membrane thickness
ε
Porosity
Λ
Mean free path, m Author Contributions Author Contributions
Conceptualization: Emad Ali. Data curation: Abdullah Najib. Formal analysis: Emad Ali, Jamel Orfi. Investigation: Jamel Orfi, Abdullah Najib. Methodology: Emad Ali. Software: Emad Ali. Supervision: Jamel Orfi. Validation: Abdullah Najib. Visualization: Abdullah Najib. Writing – original draft: Emad Ali, Jamel Orfi. Writing – review & editing: Emad Ali, Jamel Orfi. Author Contributions
Conceptualization: Emad Ali. Data curation: Abdullah Najib. Formal analysis: Emad Ali, Jamel Orfi. Investigation: Jamel Orfi, Abdullah Najib. Methodology: Emad Ali. Software: Emad Ali. Supervision: Jamel Orfi. Validation: Abdullah Najib. Visualization: Abdullah Najib. Writing – original draft: Emad Ali, Jamel Orfi. Writing – review & editing: Emad Ali, Jamel Orfi. Validation: Abdullah Najib. Visualization: Abdullah Najib. Writing – original draft: Emad Ali, Jamel Orfi. Writing – original draft: Emad Ali, Jamel Orfi. Writing – review & editing: Emad Ali, Jamel Orfi. Writing – review & editing: Emad Ali, Jamel Orfi. Supporting information Supporting information
S1 Fig. Schematic of MD process: (a) lumped module, (b) ith control volume of MD mod-
ule. (TIF) 21 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0230207
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Water and Wastewater Treatment Technologies 2010. 7. Lawal D.U., Khalifa A.E. Flux prediction in direct contact membrane distillation, Int J Mech Mater Manuf,
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mance evaluation and thermal energy efficiency, Chem Eng Res Des, 2015; 100:331–340. 45. Ali E. Energy efficient configuration of membrane distillation units for brackish water desalination using
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4,2’:6’,4”- and 3,2’:6’,3”-Terpyridines: The Conflict between Well-Defined Vectorial Properties and Serendipity in the Assembly of 1D-, 2D- and 3D-Architectures
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Materials
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materials
Article
4,2’:6’,4”- and 3,2’:6’,3”-Terpyridines: The Conflict
between Well-Defined Vectorial Properties and
Serendipity in the Assembly of 1D-, 2Dand 3D-Architectures
Y. Maximilian Klein, Alessandro Prescimone, Edwin C. Constable and Catherine E. Housecroft *
Department of Chemistry, University of Basel, Spitalstrasse 51, 4056 Basel, Switzerland;
max.klein@unibas.ch (Y.M.K.); alessandro.prescimone@unibas.ch (A.P.); edwin.constable@unibas.ch (E.C.C.)
* Correspondence: catherine.housecroft@unibas.ch; Tel.: +41-61-207-1008
Received: 12 June 2017; Accepted: 28 June 2017; Published: 30 June 2017
Abstract: A comparative investigation of the coordination assemblies formed between Co(NCS)2 and
two monotopic 4,2’:6’,4”-terpyridine (4,2’:6’,4”-tpy) ligands or two related ditopic ligands is reported.
Crystals were grown by layering MeOH solutions of Co(NCS)2 over a CHCl3 or 1,2-C6 H4 Cl2 solution
of the respective ligand at room temperature. With 4’-(2-methylpyrimidin-5-yl)-4,2’:6’,4”-terpyridine
(6), the 1D-coordination polymer {[Co2 (NCS)4 (MeOH)4 (6)2 ]·2MeOH·8H2 O}n assembles with 6
coordinating only through the outer N-donors of the 4,2’:6’,4”-tpy unit; coordination by the
MeOH solvent blocks two cobalt coordination sites preventing propagation in a higher-dimensional
network. A combination of Co(NCS)2 and 1-(4,2‘:6’,4”-terpyridin-4’-yl)ferrocene (7) leads to
{[Co(NCS)2 (7)2 ]·4CHCl3 }n which contains a (4,4) net; the 2D-sheets associate through π-stacking
interactions between ferrocenyl and pyridyl units. A 3D-framework is achieved through use of
the ditopic ligand 1,4-bis(n propoxy)-2,5-bis(4,2’:6’,4”-terpyridin-4’-yl)benzene (8) which acts as a
4-connecting node in {[Co(NCS)2 (8)2 ]·2C6 H4 Cl2 }n ; the combination of metal and ligand planar
4-connecting nodes results in a {65 .8} cds net. For a comparison with the coordinating abilities of the
previously reported 1,4-bis(n octoxy)-2,5-bis(4,2’:6’,4”-terpyridin-4’-yl)benzene (3), a more flexible
analogue 9 was prepared. {[Co(NCS)2 (9)]·2CHCl3 }n contains a (4,4) net defined by both metal and
ligand planar 4-connecting nodes. The n octoxy tails of 9 protrude from each side of the (4,4) net and
thread through adjacent sheets; the arene-attached n octoxy chains associate through a combination of
van der Waals and C–H...π interactions.
Keywords: coordination polymer; coordination network; 4,2’:6’,4”-terpyridine; cobalt thiocyanate
1. Introduction
The coordination chemistry of divergent 4,2’:6’,4”-terpyridine (4,2’:6’,4”-tpy) and
3,2’:6’,3”-terpyridine ligands is now a mature field, with the former ligand in particular being
increasingly employed as a building block for the assembly of coordination polymers and
networks [1,2]. There are a few examples of 4,2’:6’,4”-tpy ligands acting in a monodentate mode [3–7]
but, typically, 4,2’:6’,4”-tpy coordinates through the two outer nitrogen atoms, with the central
nitrogen atom non-coordinated. Therefore, ligands based upon a 4,2’:6’,4”-tpy metal-binding domain
present moderately rigid, well-defined V-shaped motifs (Scheme 1a) which are ideal linkers in
coordination assemblies.
Starting with the 4,2’:6’,4”-tpy unit as a well-defined V-shaped building block, there are a
number of strategies that can be applied to direct an assembly towards 2D- or 3D-architectures
in preference to a 1D-chain. Since 4,2’:6’,4”-tpy is easily modified in the 4’-position using Kröhnke [8]
Materials 2017, 10, 728; doi:10.3390/ma10070728
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Materials 2017, 10, 728
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or Hanan [9] syntheses, one approach is the incorporation of a coordinatively non-innocent domain D
(Scheme 1b), for example D = pyridyl or carboxylate groups. The use of multitopic ligands [10–12]
is an attractive way forward but has, so far, been little exploited. Scheme 1c illustrates a generic
bis(4,2’:6’,4”-tpy) acting as a 4-connecting node. Rotation about the C–spacer bonds permits the
node to have limiting planar or approximately tetrahedral geometries, and the directionality of
the assembly can be modified by switching from 4,2’:6’,4”-tpy to 3,2’:6’,3”-tpy domains. We have
recently employed such ditopic ligands in which the spacer (Scheme 1c) is a 1,4-C6 H4 ring to assemble
parallel interpenetrating 2D→2D sheets [13–15] and 3D-metal-organic frameworks (MOFs) [15,16].
Going to a ferrocen-1,1’-yl spacer introduces rotational freedom and, in [{Zn2 (1)Cl4 }·3CHCl3 ]n where
1 = 1,1’-bis(4,2’:6’,4”-terpyridin-4’-yl)ferrocene (Scheme 2), the ligand adopts a cisoid-conformation,
leading to a double-stranded 1D-polymer chain [17].
Scheme 1. (a) Divergent V-shaped motif of 4,2’:6’,4”-tpy; (b) introduction of a donor group, D, in the
4’-position; and (c) 4-connecting node presented by ditopic bis(4,2’:6’,4”-tpy).
The discussion above focuses on bis(4,2’:6’,4”-tpy) or bis(3,2’:6’,3”-tpy) as a 4-connecting node.
However, the choice of metal is naturally important since its preferred coordination number and
geometry dictate whether it will act as a linker or node. When connected through ZnX2 units
(X = halide) as in [Zn2 Br4 (2)·H2 O]n (2 is shown in Scheme 2), ligand 2 acts as a 4-connecting node and
the zinc(II) atoms are linkers; the assembly is a MOF consisting of 2-fold interpenetrating nbo nets [15].
In contrast, when ligand 3 (Scheme 2) is combined with Co(NCS)2 , both the metal and ligand function
as 4-connecting nodes (planar and approximately tetrahedral, respectively) and assemble into a 3D
{42 .84 } lvt net [16]. Limiting the ligand to a single 4,2’:6’,4”-tpy domain as in 4 and 5 (Scheme 2) and
reacting with Co(NCS)2 leads to the formation of 3D chiral neb nets [18]. This assembly is noteworthy
in that a chiral MOF is built from achiral node and linkers. The presence of the long alkyl-tails appears
to be important in directing or stabilizing these neb nets, since related 4,2’:6’,4”-tpy ligands bearing
Materials 2017, 10, 728
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in the 4’-position t Bu, Ph, 4-MeOC6 H4 , 4-EtOC6 H4 , 4-n PrOC6 H4 , 4-HC≡CC6 H4 , 1H-imidazol-4-yl or
benzo[d][1,3]dioxol-5-yl substituents form 2D-nets with Co(NCS)2 [6,19–22]. With the exception of
the work of Mondal et al. [20], crystal growth of the coordination networks described above was by
layering under room temperature conditions.
Scheme 2. Structures of ditopic ligands 1 and 2 with 4,2’:6’,4”-tpy domains, and 3 with 3,2’:6’,3”-tpy
metal-binding units and of the monotopic ligands 4 and 5.
We now present a comparative investigation of the coordination assemblies formed between
Co(NCS)2 and two monotopic 4,2’:6’,4”-tpy ligands and two ditopic ligands. Ligand 6 (Scheme 3)
contains a 2-methylpyrimidin-5-yl substituent and we were interested to see if this would be an
additional metal-binding domain to cobalt(II). We have previously reported that the pyrimidinyl
units in 4’-(pyrimidin-5-yl)-4,2’:6’,4”-terpyridine or 6 remain uncoordinated in reactions with zinc(II)
halides [23], whereas Grafino et al. observe examples of both coordinated and non-coordinated
pyrimidinyl N-donors in reactions of 4’-(4-(pyrimidin-5-yl)phenyl)- 4,2’:6’,4”-terpyridine with
Zn(acac)2 [5]. Ligand 7 (Scheme 3) is related to ditopic ligand 1 (Scheme 2) but offers only one
4,2’:6’,4”-tpy domain. Both 1D-polymer chains and metallomacrocycles have been reported for
combinations of 7 with ZnX2 (X = Cl, Br, I, SCN or OAc) [24,25]. The assemblies are competitive for ZnI2
and both the 1D-chain [{ZnI2 (7)}·2CHCl3 ]n [25] and discrete metallosquare [Zn4 I8 (7)4 ·1.4MeOH] [24]
have been crystallographically characterized. A feature that the ferrocenyl unit in 1 or 7 brings to
crystal packing is its possibility for face-to-face π-stacking with pyridine rings [24]. Ditopic ligand
8 (Scheme 3) is a member of the 1,4-bis(alkoxy)-2,5-bis(4,2’:6’,4”-terpyridin-4’-yl)benzene family to
which 2 (Scheme 2) belongs; while we have investigated assemblies formed between such ligands
with zinc(II) halides [15], we have not described reactions with Co(NCS)2 . Finally, ligand 9 (Scheme 3)
is analogous to 3 in terms of the four N-donors. The flexibility of 9 is greater than that of 3, and we
wished to investigate how this would redirect the coordination assembly as compared to the 3D {42 .84 }
lvt net in [Co(NCS)2 (3)·4CHCl3 ]n [16].
Materials 2017, 10, 728
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Scheme 3. Structures of ligands 6–9 with atom labelling for NMR spectroscopic assignments in 8 and 9.
2. Results and Discussion
2.1. Ligand Syntheses and Characterization
Ligands 6 and 7 were prepared as previously reported [23,25]. The synthetic route to 8 is
summarized in Scheme 3 and is closely related to the synthesis of 2 [15]. Compound 8a (Scheme 3)
was prepared from the commercially available 2,5-dibromohydroquinone and then reacted with
n BuLi followed by DMF to yield the dialdehyde 8b. The 1 H NMR spectrum of 8b was consistent
with previously reported data [26]. The one-pot method of Hanan [9] (Scheme 3) was then used to
transform 8b into compound 8. The disappearance of the aldehyde signal at δ 10.52 ppm confirmed
that derivatization of both aldehyde groups had occurred (Figure 1). The 1 H and 13 C NMR spectra of
compound 8 were assigned using COSY, HMQC, HMBC and NOESY methods and were in accord
with the structure shown in Scheme 4.
Materials 2017, 10, 728
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Scheme 4. Synthetic route to compound 8. Conditions: (i) n BuLi, Et2 O, 0 ◦ C, 6 h; DMF, room
temperature, yield 56.3%; and (ii) 4-acetylpyridine, KOH, EtOH, aqueous NH3 , room temperature,
yield 21.6%.
Figure 1. Part of the 500 MHz 1 H NMR spectrum of 8 in CDCl3 showing the aromatic region of 8 and
confirming the loss of both aldehyde groups of the precursor 8b. * = residual CHCl3 .
Compound 9 was prepared by the reaction of four equivalents of 3-acetylpyridine with
2,5-bis(octoxy)benzene-1,4-dicarbaldehyde under basic conditions (Scheme 5). In the electrospray
mass spectrum of 9, the base peak at m/z 839.4 corresponded to [M+H]+ . Figure 2 shows the 1 H NMR
spectrum of 9. Both this and the 13 C NMR spectrum were assigned using 2D methods (Figure S1 shows
the HMQC spectrum) and were consistent with the structure shown in Scheme 5. In the 13 C NMR
spectrum, a resonance at δ 198.2 ppm characterized the carbonyl groups and a band at 1681 cm−1
arising from the C=O stretch was observed in the IR spectrum of 9.
Scheme 5. Synthetic route to compound 9. Conditions: (i) KOH, EtOH, room temperature, yield 31.2%.
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Figure 2. The 500 MHz 1 H NMR spectrum of 9 in CDCl3 . ** = residual CHCl3 ; * = water. See Scheme 3
for atom labelling.
2.2. 4’-(2-Methylpyrimidin-5-yl)-Functionalization: Assembly of a 1D-Chain
Layering of a methanol solution of Co(NCS)2 over a chloroform solution of 6 yielded a few
X-ray quality pink crystals under ambient conditions over a period of 2–4 weeks. Single-crystal
X-ray diffraction confirmed a formulation of {[Co2 (NCS)4 (MeOH)4 (6)2 ]·2MeOH·8H2 O}n , but there
was insufficient material for characterization of the bulk sample by powder diffraction. The compound
crystallizes in the triclinic P–1 space group, and Figure 3 shows the structure of the repeat unit in the
coordination polymer. Selected bond distances and angles are given in the figure caption. Ligand 6
binds to cobalt through atoms N1 and N3 of the 4,2’:6’,4”-tpy, leaving the central pyridine N-donor and
the two pyrimidinyl N-donors uncoordinated. The two crystallographically independent cobalt atoms
are in similar octahedral coordination spheres, and since each of Co1 and Co2 resides on an inversion
centre, the pairs of 4,2’:6’,4”-tpy, thiocyanato and MeOH ligands are necessarily trans to one another
(Figure 3). The MeOH molecule containing C1 is disordered (50:50 site occupancies), each site sharing
a common O2 atom. Since each of Co1 and Co2 binds two ligands 6 which are mutually trans, each
metal centre acts as a linear, 2-connecting node. The structure in Figure 3 propagates into a 1D-zigzag
chain and as shown in Figure 4a. Coordination by the MeOH solvent blocks two cobalt coordination
sites precluding propagation into a higher-dimensional network. The zigzag chains lie over one
another such that one pyridine ring of the 4,2’:6’,4”-tpy unit stacks over a pyrimidine ring in the next
chain. However, the stacking is not optimal. Although the pyridinecentroid ...pyrimidineplane separation
is 3.35 Å, the angle between the planes of the ring containing N1 and pyrimidine ring containing
N4iv /N5iv (symmetry code iv = − x, 1 − y, 2 − z) is 23.8◦ (Figure 4b). The presence of the methyl
substituent on the pyrimidine ring may sterically hinder a more optimal π-stacking arrangement.
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Figure 3. Structure of the repeat unit in {[Co2 (NCS)4 (MeOH)4 (6)2 ]·2MeOH·8H2 O}n with symmetry
generated atoms; H atoms and solvent molecules are omitted. Ellipsoids are plotted at 40% probability;
atom C1 was refined isotropically (see text). Symmetry codes: i = 1 − x, 2 − y, 1 − z; ii = 1 − x,
−y, 2 − z; iii = x, −2 + y, 1 + z. Selected bond parameters: Co1–N1 = 2.189(6), Co1–N6 = 2.095(7),
Co1–O1 = 2.098(6), Co2–N3 = 2.154(6), Co2–N7 = 2.069(7), Co2–O2 = 2.077(9) Å; N1–Co1–N6 = 88.8(2),
N1–Co1–O1 = 86.5(2), N3–Co2–N7 = 90.1(2), N3–Co2–O2 = 91.8(3), N7–Co2–O2 = 90.9(4)◦ .
Figure 4. Parts of two adjacent 1D chains in {[Co2 (NCS)4 (MeOH)4 (6)2 ]·2MeOH·8H2 O}n . (a) View
showing the zigzag profile of the chain and association between pyridine and pyrimidine rings in
adjacent chains; (b) The same polymer units as in (a) viewed down the crystallographic a-axis.
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Ligands based on 4,2’:6’,4”-tpy feature in a large number of 1D zigzag chains in which the metal
nodes are 2-connecting {M2 (µ-OAc)4 } paddle-wheel units [1]. A common structural motif in these
assemblies is the accommodation of the 4’-substituent of the 4,2’:6’,4”-tpy within the V-shaped cleft of a
4,2’:6’,4”-tpy domain in an adjacent chain leading to planar 2D-sheets with a herringbone packing motif.
In {[Co2 (NCS)4 (MeOH)4 (6)2 ]·2MeOH·8H2 O}n , rather than being accommodated within the V-shaped
pocket of the adjacent chain, the methyl group of the 4’-(2-methylpyrimidin-5-yl) substituent sits over
the central N2 atom of the chain beneath it (CMe ...N = 3.42(1) Å) as shown in Figure 5. This arrangement
of neighbouring chains leads to short S...H contacts [27] as shown in Figure 5. The S...H distances of
2.96 and 2.82 Å (the H atoms are in calculated positions) compare with 3.00 Å for the sum of the H
and S van der Waals radii using Bondi values [28], although this value may be an overestimate [29,30].
An analysis by Rowland and Taylor [30] of crystallographic data for intermolecular contacts in organic
compounds suggests that the Bondi van der Waals radius for H of 1.2 Å is overestimated by 0.1 Å.
Cavities in the lattice in {[Co2 (NCS)4 (MeOH)4 (6)2 ]·2MeOH·8H2 O}n are occupied by H2 O and
MeOH molecules. There are (in the asymmetric unit) two independent MeOH molecules, each
half-occupancy. The lattice water molecules comprise three independent full-occupancy molecules
and two independent half-occupancy molecules. Because of the fractional occupancies, discussion of
any hydrogen-bonded interactions involving solvent molecules is not warranted.
Figure 5. Arrangement of zigzag chains in {[Co2 (NCS)4 (MeOH)4 (6)2 ]·2MeOH·8H2 O}n showing short
S...H contacts (hashed red lines).
2.3. Ferrocenyl-Functionalization: Assembly of a 2D-Network
Layering of a methanol solution of Co(NCS)2 over a chloroform solution of 7 resulted in the growth
of orange crystals of {[Co(NCS)2 (7)2 ]·4CHCl3 }n . An X-ray quality crystal was chosen for single-crystal
structure determination and the bulk sample was characterized by powder diffraction (Figure S2).
The compound crystallizes in the monoclinic P21 /n space group. Figure 6 depicts the octahedral
coordination environment of the cobalt(II) centre and selected bond parameters are given in the figure
caption. Atom Co1 lies on an inversion centre and is bound by two thiocyanato ligands (necessarily
trans) and four ligands 7. The near square-planar arrangement of N1, N1i, N3v and N3iv (Figure 6)
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leads to the metal being a planar, 4-connecting node. The structure propagates into a (4,4) net (Figure 7)
and belongs to the family of (4,4) nets which have been reported for combinations of Co(NCS)2 and a
number of 4’-functionalized 4,2’:6’,4”-tpy ligands (functionality = t Bu, Ph, 4-MeOC6 H4 , 4-EtOC6 H4 ,
4-n PrOC6 H4 , 4-HC≡CC6 H4 , 1H-imidazol-4-yl or benzo[d][1,3]dioxol-5-yl [6,19–22]. As Figure 7b
shows, the ferrocenyl units protrude above and below the 2D-sheet, and adjacent sheets are interlocked
with each ferrocenyl unit of one sheet lying over a 4,2’:6’,4”-tpy unit in the next sheet. The 4,2’:6’,4”-tpy
unit deviates slightly from planarity with angles between the planes of the pyridine rings containing
N1/N2 and N2/N3 of 11.4 and 13.5◦ , respectively. The cyclopentadienyl ring containing C16 is
twisted 16.0◦ with respect to the pyridine ring with N2. We have previously described the role of
face-to-face π-stacking between ferrocenyl and pyridyl units in crystal packing [24]. Similar effects
are observed in {[Co(NCS)2 (7)2 ]·4CHCl3 }n and Figure 8 illustrates that adjacent (4,4) sheets associate
through stacking interactions. These involve the pyridine ring containing N1 and cyclopentadienyl
ring containing C24v (symmetry code v = −1 + x, y, z). The angle between the ring planes is 6.9◦ and
the pyridinecentroid ...C5 -ringplane distance is 3.22 Å; the distance of 4.17 Å between the ring centroids is
rather long for an optimal interaction. The pyridine ring containing N3 also lies over a ferrocenyl unit
(that with C16vi , symmetry code vi = 1 − x, −y, 1−z), but, in this case, the inter-plane angle is 19.2◦ ,
leading to an inefficient π-stacking contact.
Figure 6. Structure of the repeat unit in {[Co(NCS)2 (7)2 ]·4CHCl3 }n with symmetry generated atoms;
H atoms and solvent molecules are omitted. Ellipsoids are plotted at 50% probability. Symmetry codes:
i = −x, 1 − y, 1 − z; ii = −1 /2 + x, 3 /2 − y, −1 /2 + z; iii = 1 /2 + x, 1 /2 − y, 1 /2 + z; iv = −1 /2 + x,
1 / − y, −1 / + z; v = 1 / − x, 1 / + y, 3 / + z. Important bond parameters: Co1–N1 = 2.183(2),
2
2
2
2
2
Co1–N3iv = 2.183(2), Co1–N4 = 2.083(2), C8–C16 = 1.472(3) Å; N–Co1–N angles are in the range
86.77(8)–93.23(8)◦ .
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Figure 7. (a) TOPOS [31] representation of part of one (4,4) sheet in {[Co(NCS)2 (7)2 ]·4CHCl3 }n overlaid
with the structure; and (b) the same part of the sheet viewed down the crystallographic b-axis.
Figure 8. Parts of two adjacent sheets in {[Co(NCS)2 (7)2 ]·4CHCl3 }n showing the π-stacking between
a ferrocenyl ring in one sheet and pyridyl ring in the next sheet.
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2.4. To a 3D-Network: {[Co(NCS)2 (8)2 ]. 2C6 H4 Cl2 }n
Ligand 8 presents two 4,2’:6’,4”-tpy units connected in a “back-to-back” fashion through a spacer
which contains two n-propoxy tails. We have demonstrated that the length of such tails can have
a significant impact on the outcome of assembly processes [13–15]. Single crystals grown by layering
a MeOH solution of Co(NCS)2 over a CHCl3 solution of 8 were not of X-ray quality. However,
a change to a 1,2-dichlorobenzene solution of 8 led to crystals that were harvested after a few weeks,
and single crystal X-ray diffraction showed the formation of a coordination network of formula
{[Co(NCS)2 (8)2 ]·2C6 H4 Cl2 }n . The compound crystallizes in the monoclinic space group P21 /c and
the repeat unit in the structure is depicted in Figure 9. Atom Co1 is octahedrally sited and since it
resides on an inversion centre, the two thiocyanato ligands are necessarily trans. Atom Co1 binds to
four different ligands 8 (Figure 9) and thereby functions as a planar, 4-connecting node. Selected bond
parameters within the coordination sphere are given in the caption to Figure 9, and the Ntpy –Co–Ntpy
bond angles that define the planar node are in the range 84.44(8)–95.56(8)◦ . The asymmetric unit
contains half of a molecule of 8, and the second half is generated by inversion (Figure 9). Thus, like
the cobalt centre, the ditopic ligand also acts as a planar 4-connecting node. The 4,2’:6’,4”-tpy unit
deviates from planarity with angles between the rings containing N1/N2 and N2/N3 being 31.3 and
19.5◦ , respectively. The twist of the pyridine ring with N2 with respect to the central aryl ring is 40.5◦ ,
thereby minimizing inter-ring repulsive H...H contacts. The combination of 4-connecting metal and
ligand nodes leads to the assembly of a 3D {65 .8} cds [32,33] net, a TOPOS [31] representation of which
is shown in Figure 10. In a cds net, half of the adjacent nodes are perpendicular to one another and half
are coplanar [33]. The preference for the cds architecture is in contrast to the assembly of the {42 .84 } lvt net
which forms from Co(NCS)2 and ditopic ligand 3 (Scheme 2) [16]. Ligands 3 and 8 both act as 4-connecting
nodes, but differ in having two 3,2’:6’,3”-tpy or 4,2’:6’,4”-tpy domains, respectively. In addition to a
change in the vectorial properties of the ligands, ligand 8 possesses shorter alkoxy tails than 3 (n-propoxy
versus n-octoxy). Figure 11 shows an overlay of the structure on the TOPOS representation of the net and
this orientation reveals the accommodation of the extended propoxy tails within cavities in the network.
Remaining cavities in the cds net are occupied by 1,2-dichlorobenzene molecules (see Experimental
Section). The bulk sample was characterized by powder diffraction (Figure S3).
Figure 9. Structure of the repeat unit in {[Co(NCS)2 (8)2 ]·2C6 H4 Cl2 }n with symmetry generated atoms;
H atoms and solvent molecules are omitted. Ellipsoids are plotted at 40% probability. Symmetry codes:
i = −x, 2 − y, 1 − z; ii = 1 − x, 1 − y, 1 − z; iii = −1 + x, 1 + y, z; iv = x, 3 /2 − y, −1 /2 + z; v = −1 + x,
3 / + y, 1 / + z. Selected bond parameters: Co1–N1 = 2.173(2), Co1–N4 = 2.047(3), Co1iv –N3 = 2.193(2),
2
2
O1–C18 = 1.361(4), O1–C19 = 1.431(4) Å; N1–Co1–N4 = 90.47(10)◦ .
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Figure 10. TOPOS [31] representation of part of the cds net in {[Co(NCS)2 (8)2 ]·2C6 H4 Cl2 }n . Both Co
(maroon) and ligand (green) are planar, 4-connecting nodes.
Figure 11. Overlay of the structure of {[Co(NCS)2 (8)2 ]·2C6 H4 Cl2 }n (H atoms and solvent molecules
omitted) on a TOPOS [31] representation of part of the cds net. The ligand node (green) is defined as
the centroid of the arene ring.
2.5. Relaxing the Backbone: Going from an lvt Net to a 2D-Sheet
As described above, ditopic ligand 3 (Scheme 2) reacts with Co(NCS)2 under conditions of
crystal growth by layering to give {[Co(NCS)2 (3)]·4CHCl3 }n which possesses a {42 .84 } lvt net [16].
This assembly is rather uncommon among MOFs consisting of 4-connected nets [32] and in
{[Co(NCS)2 (3)]·4CHCl3 }n , the lvt net is produced by a combination of planar Co nodes and
approximately tetrahedral ligand nodes. We decided to investigate the effects of modifying the
ligand structure so as to retain a 4-connecting domain with similar metrics to 3 but with a more
flexible backbone. Ligand 9 (Scheme 3) conforms to these criteria. A methanol solution of Co(NCS)2
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was layered over a chloroform solution of 9 and, after 2–4 weeks, X-ray quality crystals had grown.
Single-crystal X-ray diffraction analysis confirmed the formation of {[Co(NCS)2 (9)]·2CHCl3 }n with a
Co:ligand ratio equivalent to that in {[Co(NCS)2 (3)]·4CHCl3 }n [16]; the bulk sample was characterized
by powder diffraction (Figure S4). The compound crystallizes in the triclinic space group P–1 with
atom Co1 lying on an inversion centre. The repeat unit is shown in Figure 12 and the coordination
environment of Co1 resembles that in {[Co(NCS)2 (8)2 ]·2C6 H4 Cl2 }n (Figure 9). Metrical parameters for
the coordination sphere (caption to Figure 12) are unexceptional. The closeness of each Ntpy –Co1–Ntpy
angle to 90◦ leads to a square-planar metal node. The asymmetric unit contains half of a ligand
9, and the second half is generated by inversion leading to 9 being a planar 4-connecting node
(Figure 12), the centre of which is defined by the centroid of the arene ring containing atom C16.
The planarity of the ligand 9 node in {[Co(NCS)2 (9)]·2CHCl3 }n contrasts with the tetrahedral node that
3 presents in {[Co(NCS)2 (3)]·4CHCl3 }n [16]; recall from the introduction that ditopic ligands of the
type exemplified by 2 or 3 (Scheme 2) have limiting planar or approximately tetrahedral geometries.
A comparison of the conformations of the coordinated ligands 3 and 9 in {[Co(NCS)2 (3)]·4CHCl3 }n
and {[Co(NCS)2 (9)]·2CHCl3 }n , respectively, is shown in Figure S5.
Figure 12. Structure of the repeat unit in {[Co(NCS)2 (9)]·2CHCl3 }n with symmetry generated atoms; H
atoms and solvent molecules are omitted. Ellipsoids are plotted at 40% probability. Symmetry codes:
i = −1 + x, 1 + y, 1 + z; ii = −1 + x, y, 1 + z; iii = x, −1 + y, z; iv = 2 − x, 3 − y, −z; v = x, 1 + y, z; vi = 2 − x,
2 − y, −z. Selected bond parameters: Co1–N1 = 2.196(4), Co1–N3 = 2.070(4), Co1–N2v = 2.199(4),
O1–C6 = 1.216(6), O2–C10 = 1.193(7), O3–C18 = 1.383(6), O3–C19 = 1.413(6) Å; N1–Co1–N3 = 91.13(17),
Ntpy –Co1–Ntpy range 87.68(16)–92.32(16)◦ .
Propagation of the unit in Figure 12 leads to a 2D (4,4) net.
In contrast to the
(4,4) net in {[Co(NCS)2 (7)2 ]·4CHCl3 }n , which is defined only by the metal nodes, the net in
{[Co(NCS)2 (9)]·2CHCl3 }n is defined by a combination of metal and ligand 4-connected nodes (Figure 13).
The one crystallographically independent octoxy chain in {[Co(NCS)2 (9)]·2CHCl3 }n is ordered and
has a fully extended conformation. These features are associated with the threading of the octoxy tail
through an adjacent sheet (Figure 14a) and the alignment of the tails (Figure 14b). Both van der Waals
and C–H...π interactions play a role with inter-chain C...C and C...O separations of 4.35 and 4.33 Å and
a close C–H...π contact of 3.26 Å (C...π distance = 4.04 Å) [34].
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Figure 13. Overlay of the structure of {[Co(NCS)2 (9)]·2CHCl3 }n (H atoms and solvent molecules
omitted) on a TOPOS [31] representation of part of a 2D-sheet. The ligand node (green) is defined as
the centroid of the central arene ring in 9.
Figure 14. (a) Parts of three adjacent 2D-sheets in {[Co(NCS)2 (9)]·2CHCl3 }n (H atoms and solvent
molecules omitted) showing threading of the octoxy tails through neighbouring sheets; and
(b) alignment of octoxy tails in adjacent sheets.
3. Materials and Methods
3.1. General
1H
and 13 C NMR spectra were recorded on a Bruker DRX-500 NMR spectrometer with chemical
shifts referenced to residual solvent peaks (TMS = δ 0 ppm). Electrospray ionisation (ESI) mass spectra
were measured on a Bruker esquire 3000plus spectrometer or Shimadzu LCMS-2020 instrument and
high resolution ESI mass spectra on a Bruker maXis 4G QTOF instrument. The IR spectrum of 9 was
recorded on a Perkin Elmer Spectrum Two (UATR) FT-IR.
3-Acetylpyridine, 4-acetylpyridine, 1-bromopropane, 2,5-dibromohydroquinone and
2,5-bis(octoxy)benzene-1,4-dicarbaldehyde were purchased from Sigma-Aldrich (Switzerland)
and used without further purification. Ligands 6 and 7 were prepared as previously reported [23,25].
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3.2. Synthesis of 8a
2,5-Dibromohydroquinone (2.0 g, 7.47 mmol), 1-bromopropane (1.72 mL, 2.32 g, 18.7 mmol) and
anhydrous K2 CO3 (3.1 g, 22.4 mmol) were added to dry DMF (100 mL) and the mixture was heated at
100 ◦ C for 16 h. The mixture was cooled to room temperature then poured into a beaker containing
ice water (100 mL) and the mixture stirred for 30 min. The precipitate was filtered, washed with
water (3 × 30 mL) and dried in vacuo. Compound 8a was isolated as white crystals (2.49 g, 7.07 mmol,
94.6%). 1 H NMR (500 MHz, CDCl3 ) δ/ppm 7.09 (s, 2H, HC3 ), 3.92 (t, J = 6.5 Hz, 4H, Ha ), 1.89–1.75 (m,
4H, Hb ), 1.06 (t, J = 7.4 Hz, 6H, Hc ). This matches the literature data [26].
3.3. Synthesis of 8b
Compound 8a (1.8 g, 5.11 mmol) and dry Et2 O (100 mL) were added to a dried flask and cooled
to 0 ◦ C using an ice bath. n BuLi (1.6 M in hexanes, 9.58 mL, 15.3 mmol) was added slowly to the
solution over a period of 20 min and the temperature maintained at 0 ◦ C for 6 h. Dry DMF (1.19 mL,
15.3 mmol) was added and the solution stirred for 16 h, while warming up to room temperature.
The reaction mixture was neutralized with saturated aqueous NH4 Cl and extracted with CH2 Cl2
(200 mL). The organic phase was dried over MgSO4 and concentrated in vacuo. Compound 8b was
isolated as a yellow solid (0.72 g, 2.88 mmol, 56.3%) and used without further purification. 1 H NMR
(500 MHz, CDCl3 ) δ/ppm 10.52 (s, 2H, HCHO ), 7.43 (s, 2H, HC3 ), 4.05 (t, J = 6.5 Hz, 4H, Ha ), 1.93–1.74
(m, 4H, Hb ), 1.06 (m, J = 7.4 Hz, 6H, Hc ). This matches the literature data [26].
3.4. Synthesis of 8
Compound 8b (0.3 g, 1.2 mmol) was dissolved in EtOH (100 mL), then 4-acetylpyridine (0.54 mL,
0.59 g, 4.8 mmol) and crushed KOH (0.27 g, 4.8 mmol) were added in one portion. Aqueous NH3
(32%, 3.1 mL) was added dropwise and the reaction mixture was stirred at room temperature for 16 h.
The precipitate was collected by filtration and washed with water (3 × 10 mL), EtOH (3 × 10 mL) and
Et2 O (3 × 10 mL). Compound 8 was isolated as a white solid (0.17 g, 0.26 mmol, 21.6%). Decomp.
> 330 ◦ C. 1 H NMR (500 MHz, CDCl3 ) δ/ppm 8.81 (d, J = 6.1 Hz, 8H, HA2 ), 8.12 (s, 4H, HB3 ), 8.11–8.08
(m, 8H, HA3 ), 7.16 (s, 2H, HC2 ), 4.04 (t, J = 6.4 Hz, 4H, Ha ), 1.86–1.73 (m, 4H, Hb ), 0.97 (t, J = 7.4 Hz, 6H,
Hc ). 13 C{1 H} NMR (126 MHz, CDCl3 ) δ/ppm 154.5 (CB2 ), 150.4 (CA2 ), 148.0 (CB4 ), 146.0 (CA4 ), 128.9
(CC1 ), 121.3 (CB3 ), 121.0 (CA3 ), 115.0 (CC2 ), 71.1 (Ca ), 22.4 (Cb ), 10.5 (Cc ). CC3 was not resolved. ESI-MS
m/z 657.45 [M+H]+ (calc. 657.30). High resolution ESI-MS m/z 657.2983 [M+H]+ (calc. 657.2973).
3.5. Synthesis of 9
2,5-Bis(octoxy)benzene-1,4-dicarbaldehyde (0.15 g, 0.38 mmol) was dissolved in EtOH (40 mL),
then 3-acetylpyridine (0.2 g, 1.61 mmol) and crushed KOH (0.108 g, 1.92 mmol) were added to
the solution and the reaction mixture was stirred at room temperature for ~16 h. The colourless
solution was concentrated in vacuo and left to stand in the freezer at −18 ◦ C for 3 days. Compound 9
precipitated and was isolated as an off-white powder (0.1 g, 0.12 mmol, 31.2%). M.p. = 150.5 ◦ C.
1 H NMR (500 MHz, CDCl ) δ/ppm 9.13 (dd, J = 2.3, 0.9 Hz, 4H, HA2 ), 8.74 (dd, J = 4.8, 1.7 Hz, 4H,
3
HA6 ), 8.19 (ddd, J = 8.0, 2.3, 1.7 Hz, 4H, HA4 ), 7.38 (ddd, J = 7.9, 4.8, 0.9 Hz, 4H, HA5 ), 6.69 (s, 2H, HB3 ),
4.17 (p, J = 6.8 Hz, 2H, Hx ), 3.85 (t, J = 6.6 Hz, 4H, Ha ), 3.5–3.34 (m, 8H, Hy ), 1.77–1.69 (m, 4H, Hb ),
1.42 (m, 4H, Hc ), 1.36–1.19 (m, 16H, Hd/e/f/g ), 0.91–0.75 (m, 6H, Hh ). 13 C{1 H} NMR (126 MHz, CDCl3 )
δ/ppm 198.2 (CC=0 ), 153.5 (CA6 ), 150.4 (CB2 ), 149.8 (CA2 ), 135.5 (CA4 ), 132.4 (CA3 ), 129.7 (CB1 ), 123.7
(CA5 ), 113.5 (CB3 ), 68.7 (Ca ), 43.2 (Cy ), 33.5 (Cx ), 31.9 (Cf ), 29.7 (Cb ), 29.5 (Cd/e ), 29.4 (Cd/e ), 26.5 (Cc ),
22.8 (Cg ), 14.3 (Ch ). ESI-MS m/z 839.4 [M+H]+ (calc. 839.5). IR (solid, ν/cm−1 ) 2953 (w), 2927 (m),
2852 (m), 1681 (s), 1585 (s), 1572 (m), 1511 (m), 1469 (m), 1417 (s), 1379 (m), 1357 (m), 1340 (m), 1285
(m), 1243 (m), 1222 (m), 1200 (s), 1154 (m), 1065 (m), 1040 (w), 1026 (m), 978 (m), 879 (m), 840 (w), 799
(m), 702 (s), 673 (w), 630 (m), 620 (m), 509 (w), 405 (w). Found C 69.41, H 7.11, N 6.43; required for
C52 H62 N4 O6 ·3H2 O C 69.93, H 7.67, N 6.27.
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3.6. {[Co2 (NCS)4 (MeOH)4 (6)2 ]·2MeOH·8H2 O}n
A solution of Co(NCS)2 (1.75 mg, 0.01 mmol) in MeOH (8 mL) was layered over a solution of 6
(9.76 mg, 0.03 mmol) in CHCl3 (5 mL). A few pink crystals of {[Co2 (NCS)4 (MeOH)4 (6)2 ]·2MeOH·8H2 O}n
were obtained after 2–4 weeks.
3.7. {[Co(NCS)2 (7)2 ]·4CHCl3 }n
A solution of Co(NCS)2 (1.75 mg, 0.010 mmol) in MeOH (8 mL) was layered over a solution
of 7 (4.17 mg, 0.010 mmol) in CHCl3 (5 mL). Orange crystals of [Co(NCS)2 (7)2 ·4CHCl3 ]n (1.7 mg,
0.0011 mmol, 22% based on 7) were obtained after 2–4 weeks. The bulk sample was characterized by
powder diffraction (see Figure S2).
3.8. {[Co(NCS)2 (8)2 ]·2C6 H4 Cl2 }n
A solution of Co(NCS)2 (1.75 mg, 0.010 mmol) in MeOH (8 mL) was layered over a solution of
8 (6.57 mg, 0.010 mmol) in 1,2-dichlorobenzene (5 mL). Pink crystals of {[Co(NCS)2 (8)2 ]·2C6 H4 Cl2 }n
(3.9 mg, 0.0035 mmol, 70% based on 8) were obtained after 2–4 weeks. The bulk sample was
characterized by powder diffraction (see Figure S3).
3.9. {[Co(NCS)2 (9)]·2CHCl3 }n
A solution of Co(NCS)2 (0.875 mg, 0.005 mmol) in MeOH (8 mL) was layered over a solution of 9
(12.6 mg, 0.015 mmol) in CHCl3 (5 mL). Pink crystals of [Co(NCS)2 (9)·2CHCl3 ]n (0.6 mg, 0.00048 mmol,
9.6%) were obtained after 2–4 weeks. The bulk sample was characterized by powder diffraction
(see Figure S4).
3.10. Crystallography
Single crystal data were collected on a Bruker APEX-II diffractometer; data reduction, solution
and refinement used APEX2, SuperFlip and CRYSTALS, respectively [35–37]. Structure analysis
used Mercury v. 3.7 [38,39]. In {[Co(NCS)2 (8)2 ]·2C6 H4 Cl2 }n , one 1,2-dichlorobenzene molecule was
disordered and was refined isotropically, being treated as a rigid body. SQUEEZE [40] was used to
treat the remainder of the solvent region in {[Co(NCS)2 (8)2 ]·2C6 H4 Cl2 }n and electron density removed
equated to one extra molecule of 1,2-dichlorobenzene per formula unit. Powder diffraction data were
collected on a Stoe Stadi P powder diffractometer.
{[Co2 (NCS)4 (6)2 (MeOH)4 ]·2MeOH·8H2 O}n : C50 H70 Co2 N14 O14 S4 , M = 1337.32, pink block,
triclinic, space group P–1, a = 10.7199(6),b = 11.3886(7), c = 14.7671(11) Å, α = 86.374(5), β = 85.892(5),
γ = 88.031(4)◦ , U = 1793.80(13) Å3 , Z = 1, Dc = 1.238 Mg m−3 , µ(Cu-Kα) = 5.235 mm−1 , T = 123 K.
Total 24308 reflections, 6218 unique, Rint = 0.051. Refinement of 3947 reflections (364 parameters) with
I > 2σ (I) converged at final R1 = 0.1347 (R1 all data = 0.1693), wR2 = 0.3635 (wR2 all data =0.3934),
gof = 1.0474. CCDC 1550589.
{[Co(NCS)2 (7)2 ]·4CHCl3 }n : C56 H42 Cl12 CoFe2 N8 S2 , M = 1487.19, orange block, monoclinic, space
group P21 /n, a = 10.5796(7),b = 17.4860(12), c = 16.9126(11) Å, β = 99.047(3)◦ , U = 3089.8(4) Å3 ,
Z = 2, Dc = 1.598 Mg m−3 , µ(Cu-Kα) = 11.565 mm−1 , T = 123 K. Total 21236 reflections, 5706 unique,
Rint = 0.030. Refinement of 5555 reflections (367 parameters) with I > 2σ (I) converged at final
R1 = 0.0473 (R1 all data = 0.0483), wR2 = 0.0964 (wR2 all data =0.0966), gof = 0.9222. CCDC 1550588.
{[Co(NCS)2 (8)2 ]·2C6 H4 Cl2 }n : C56 H44 Cl4 CoN8 O2 S2 , M = 1125.89, pink block, monoclinic, space
group P21 /c, a = 10.2136(9),b = 19.3452(17), c = 16.2214(15) Å, β = 107.027(3)◦ , U = 3064.6(5) Å3 , Z = 2,
Dc = 1.22 Mg m−3 , µ(Cu-Kα) = 4.787 mm−1 , T = 123 K. Total 20265 reflections, 5638 unique, Rint = 0.029.
Refinement of 4472 reflections (298 parameters) with I > 2σ (I) converged at final R1 = 0.1258 (R1 all
data = 0.1287), wR2 = 0.1436 (wR2 all data =0.1437), gof = 0.9942. CCDC 1550587.
{[Co(NCS)2 (9)]·2CHCl3 }n : C56 H64 Cl6 CoN6 O6 S2 , M = 1252.94, pink block, triclinic, space group
P–1, a = 9.5196(10),b = 11.1662(13), c = 14.4682(16) Å, α = 80.088(6), β = 78.780(5), γ = 86.642(6)◦ ,
Materials 2017, 10, 728
17 of 19
U = 1485.5(3) Å3 , Z = 1, Dc = 1.400 Mg m−3 , µ(Cu-Kα) = 5.838 mm−1 , T = 123 K. Total 18364 reflections,
5368 unique, Rint = 0.049. Refinement of 4997 reflections (349 parameters) with I >2σ (I) converged at
final R1 = 0.1210 (R1 all data = 0.1256), wR2 = 0.2862 (wR2 all data =0.2891), gof = 0.9924. CCDC 1550586.
4. Conclusions
In conclusion, we have described coordination polymer and network assemblies that result from
crystal growth under ambient conditions when Co(NCS)2 is combined with monotopic ligands 6
and 7, and ditopic ligands 8 and 9. Although ligand 6 contains both pyrimidine and 4,2’:6’,4”-tpy
metal-binding domains, it binds cobalt only through the outer N-donors of the 4,2’:6’,4”-tpy
unit. In 7, the ferrocenyl functionality is coordinatively innocent and, like 6, ligand 7 acts as
a V-shaped linker. {[Co(NCS)2 (7)2 ]·4CHCl3 }n contains a (4,4) net defined by 4-connecting cobalt
nodes. In contrast, coordinated MeOH (a solvent used in all the experiments reported here)
in {[Co2 (NCS)4 (MeOH)4 (6)2 ]·2MeOH·8H2 O}n blocks two of the octahedral sites and the resulting
assembly is a 1D-coordination polymer. This result highlights the role of serendipity in the assembly
process. On the other hand, inter-sheet π-stacking interactions between ferrocenyl and pyridyl units in
{[Co(NCS)2 (7)2 ]·4CHCl3 }n may assist in directing this assembly.
By moving to the ditopic ligand 8 which may adopt limiting 4-connecting planar or approximately
tetrahedral nodal geometries, it is possible to realize a 3D-framework. {[Co(NCS)2 (8)2 ] 2C6 H4 Cl2 }n
consists of a {65 .8} cds net which is defined by a combination of metal and ligand planar 4-connecting
nodes. This contrasts with the 3D {42 .84 } lvt net present in [Co(NCS)2 (3)·4CHCl3 ]n where the metal and
ligand act as 4-connecting planar and approximately tetrahedral nodes, respectively [16]. On going
from 3 to 9, a more flexible backbone is introduced to the ligand, and reaction of 9 with Co(NCS)2
leads to {[Co(NCS)2 (9)]·2CHCl3 }n which contains a (4,4) net defined by both metal and ligand planar
4-connecting nodes. The n octoxy tails of 9 protrude from each side of the (4,4) net and thread through
adjacent sheets; the arene-attached n octoxy chains associate through a combination of van der Waals
and C–H...π interactions.
Taking Co(NCS)2 as a common building block with a preference for octahedral coordination,
this investigation has once again [24,41] illustrated the difficulties of predicting and understanding
coordination assembly algorithms, even when the ligand has well-defined directional properties, in
particular in assembly environments containing potentially coordinating solvent molecules.
Supplementary Materials: The following are available online at www.mdpi.com/1996-1944/10/7/728/s1.
Supplementary information contains Figure S1: HMQC spectrum of ligand 9. Figures S2–S4: Comparisons
of powder diffraction patterns for bulk samples and predicted patterns from single crystal diffraction;
Figure S5: Comparison of the conformations of coordinated ligands 3 and 9 in {[Co(NCS)2 (3)]·4CHCl3 }n and
{[Co(NCS)2 (9)]·2CHCl3 }n .
Acknowledgments: We thank the Swiss National Science Foundation (Grant number 200020_162631) and the
University of Basel for support. The Swiss National Science Foundation through the NCCR Molecular Systems
Engineering is acknowledged for partial funding of the powder diffractometer.
Author Contributions: Y. Maximilian Klein (synthesis, powder diffraction, contribution to writing of manuscript);
Alessandro Prescimone (crystallographer); Edwin C. Constable (project concepts); Catherine E. Housecroft (project
concepts, writing of manuscript).
Conflicts of Interest: The authors declare no conflict of interest.
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© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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1 Commission Regulation (EU) No 283/2013 of 1 March 2013 setting out
the data requirements for active substances, in accordance with Regulation
(EC) No 1107/2009 of the European Parliament and of the Council con-
cerning the placing of plant protection products on the market, and Com-
mission Regulation (EU) No 284/2013 of 1 March 2013 setting out the data
requirements for active substances, in accordance with Regulation (EC) No
1107/2009 of the European Parliament and of the Council concerning the
placing of plant protection products on the market. © 2016 The Author(s). 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. Background for the observed amphibian decline, PPPs undergo an
authorisation process and are used deliberately, so that
regulatory intervention for the protection of non-target
organisms is possible. Plant protection products (PPPs) are biologically active
substances, which means that their application in the
field can have side effects on non-target organisms. In
2013, the European Union1 has explicitly called for
amphibian toxicity data to be considered when authoris-
ing the use of PPPs. To date however, neither the EU nor
Switzerland has produced any concrete suggestions or
guidelines for the regulatory risk assessment of PPPs to
amphibians.h Abstract Background: The majority of Swiss amphibians are threatened. There is a range of factors which have been dis-
cussed as possible causes for their decline, including plant protection products (PPPs). Results: The influence of PPPs on amphibian populations has not yet been studied to any great extent, neither for
active ingredients nor for the wetting agents, breakdown products or tank mixtures. A further topic of discussion was
how to better protect amphibians by reducing their exposure to PPPs in agricultural fields. Conclusion: Experts at a workshop concluded that further research is needed. Keywords: Amphibians, Pesticides, Ecological risk assessment, Toxicity, Exposure, Workshop Aldrich et al. Environ Sci Eur (2016) 28:17
DOI 10.1186/s12302-016-0085-6 Aldrich et al. Environ Sci Eur (2016) 28:17
DOI 10.1186/s12302-016-0085-6 Open Access Amphibians and plant‑protection
products: what research and action is needed? Annette Aldrich1*†, Marion Junghans2†, Caroline Aeberli3, Carsten A. Brühl4, Franz Streiss
and Benedikt R. Schmidt6,7 *Correspondence: annette.aldrich@agroscope.admin.ch
†Annette Aldrich and Marion Junghans contributed equally to this work
1 Agroscope, Schloss, 8820 Wädenswil, Switzerland
Full list of author information is available at the end of the article Current status of knowledge As an introduction to the subject matter, lectures were
given on six topics, which are outlined in brief below: • •
Natural history and endangerment of amphibians
(Benedikt Schmidt, karch) Amphibians are the most
endangered class of vertebrates [5]. There are numer-
ous reasons for their decline. The most important
ones are the loss of habitat (quantity and quality),
emerging diseases and the overexploitation of popu-
lations [5, 6]. In addition, various threats may inter-
act: e.g. PPP with predators (e.g. [7, 8]) or with dis-
eases (e.g. [9]). In the case of habitat loss, the absence
of temporary ponds is a particular problem in Swit-
zerland [10]. p
g
p p
Effect of pesticides on the terrestrial life phase
(Carsten Brühl, University of Koblenz-Landau Several
amphibian species like the fire-bellied toad (Bombina
bombina) or the common spadefoot (Pelobates fus-
cus) inhabit the agricultural landscape during their
summer activity period, resulting in possible terres-
trial exposure [2]. Owing to the biology of the spe-
cies, it is to be expected that dermal exposure is the
major route of exposure [17, 18]. The skin of amphib-
ians has a unique structure and function that is not
comparable to that of mammals. Since it is consider-
ably more permeable, amphibians are more vulner-
able to PPPs than other terrestrial vertebrates. The
susceptibility of terrestrial amphibians has not yet
been described to any great extent, although some
studies have shown that PPPs at environmentally rel-
evant concentrations can be toxic for terrestrial life
stages of amphibians [19]. Postmetamorphic juvenile
European common frogs were directly sprayed with
different PPPs in a laboratory study. The mortality of
the animals in the case of all seven PPP formulations PPP could also contribute to the decline, because
amphibians use different habitats due to their com-
plex life cycle and annual cycle. This means that they
may come into contact with PPPs in food, water, land
and air. Natterjack toads, for example, have been
observed on arable land, e.g. in cereal fields, where
during the day they sit on the ground or bury them-
selves [11]. PPPs can also be detected in amphib-
ian breeding sites, although little data are currently
available on this topic. The analysis of all Swiss Red
Lists shows that aquatic species face a greater risk of
extinction compared to terrestrial species [12]. Aldrich et al. Environ Sci Eur (2016) 28:17 Aldrich et al. Environ Sci Eur (2016) 28:17 Page 2 of 8 Although variability in the sensitivity of different species
to PPPs has not been significantly researched to date,
there seem to be certain phylogenetic patterns [13]. gathered proactively. Given that the practitioners were
from Switzerland, the situation in Swiss farmland was
chosen as an example to facilitate discussions on con-
crete field situations. The effect on the population is not necessarily pre-
dictable by observing the effects on the individual. Thus, it is often not possible to scale up from mor-
tality of individuals to population-level effects [14]. Population models however, suggest that the survival
of the postmetamorphic juveniles is of crucial impor-
tance for population dynamics [15]. Populations fluc-
tuate greatly in size from year to year, so long-term
observations are essential in order to permit an accu-
rate statement on the impact of PPPs on amphibians
[16]. Furthermore, the factors influencing population
dynamics (e.g. population density or the vulnerabil-
ity of various life stages) are currently not sufficiently
understood to explain changes in populations. i
The participants included experts and stakehold-
ers from all involved sectors, namely academia, pub-
lic authorities, agriculture, industry and environmental
associations (Fig. 1). To our knowledge, this is the first
workshop on this topic and within such a framework to
be held in Europe. Expert workshop for knowledge exchange In order to increase our understanding of the potential
impacts of PPPs on amphibians (individuals and popu-
lations) and to discuss the protection of this imperiled
(and protected) group of organisms, an expert workshop
was held on 17 June 2015 in Dübendorf, Switzerland, by
Agroscope, the Swiss Amphibian and Reptile Conserva-
tion Program (karch) and the Swiss Centre for Applied
Ecotoxicology Eawag-EPFL (Ecotox Centre). The aim of
this workshop was to establish a network of experts on
this topic and to promote knowledge exchange between
scientists, regulators, practitioners and stakeholders. The current state of knowledge was analysed by experts
and unresolved issues as well as ideas for projects were The International Union for Conservation of Nature
(IUCN) considers worldwide “habitat loss and degrada-
tion as the greatest threat by far to amphibians at present. The number of species impacted this way is almost four
times greater than the next most common threat, pollu-
tion” [1]. PPPs fall within the category of pollution and
could be important contributors, given that in Europe,
the presence of some amphibian species and pesticides in
fields overlap regularly [2], and there are studies detect-
ing PPP residues in amphibians from agriculturally influ-
enced areas [3, 4]. Unlike other contributory factors *Correspondence: annette.aldrich@agroscope.admin.ch
†Annette Aldrich and Marion Junghans contributed equally to this work
1 Agroscope, Schloss, 8820 Wädenswil, Switzerland
Full list of author information is available at the end of the article Current status of knowledge Public Authorities
Academia
Industry
Agriculture
Non Governmental Organisations
Herpetologists
Environmental Lawyers
Fig. 1 Composition of the participants in terms of their affiliation Public Authorities Public Authorities
Academia
Industry
Agriculture
Non Governmental Organisations
Herpetologists
Environmental Lawyers Aldrich et al. Environ Sci Eur (2016) 28:17 Page 3 of 8 Page 3 of 8 investigated (4 fungicides, 2 herbicides and 1 insec-
ticide) was high, reaching 100 % at field application
rates and 40 % at 10 % field application rate, despite
the fact that products and application quantities
authorised in Germany and Switzerland have been
tested [19]. The authors have noted that in addition
to the active substance, co-formulants can also deci-
sively influence the toxicity of PPPs to amphibians. any additional stress in order to avoid population col-
lapse. Because of the unanswered questions on risk
assessment for amphibians in the authorisation pro-
cess, one should think about how specific measures
can be used to reduce exposure to PPPs, thereby
increasing the level of protection. The migration cor-
ridors in the amphibian spawning areas of national
importance are mentioned here as an example of
places in which the use of PPPs is to be monitored,
particularly in the spring. In principle, the regula-
tory authority can mandate risk mitigation measures
(obligations) through which a potential risk can be
reduced. yl
y
p
Ecotoxicological risk assessment for the authorisation
of PPP (Annette Aldrich, Agroscope) In Switzerland,
the Ordinance on Plant Protection Products (PSMV)
forms the legal basis for authorisation, and the same
data requirements and assessment criteria apply as
in other European countries, with the effects of the
individual PPP on birds, mammals, arthropods, non-
target plants, soil macro- and microorganisms, fish,
aquatic invertebrates and aquatic plants being inves-
tigated. Surrogate species of the various groups are
tested as representatives of all organisms. However,
it is the aim of the PSMV that PPP have no unaccep-
table effects on the environment as a whole, and on
non-target organisms in particular. Until now, direct
tests with amphibians have not been required. It was
assumed that risks to tadpoles could be assessed by
considering toxicity data from surrogate aquatic
organisms such as, e.g. fish, and that effects on ter-
restrial amphibians could be assessed by considering
toxicity data from surrogate terrestrial vertebrates
such as birds or mammals. Current status of knowledge To enable assessment of
the declared aim of the PSMV, the protection goal
must be defined, and relevant information regarding
exposure and toxicity must be available. Ultimately,
risk is assessed by comparing toxicity and predicted
exposure. Both parameters are based on results from
standardised studies and models, so that extrapo-
lating to the actual condition in the environment is
fraught with uncertainty. The more the situation to
be assessed differs from the studied situation, and
the smaller the number of studies, the greater the
uncertainty. This means that although it seems that
the sensitivity of tadpoles to PPPs is comparable to
that of fish [20, 21], an accurate statement cannot
be made on the risk of PPPs for aquatic amphib-
ians, since no specific information is yet available on
exposure, nor is the variability in sensitivity known. At present, we do not yet know how the sensitivity
of various species, populations and life stages var-
ies, and which species could serve as representative
organisms. It is likewise still unclear how amphibians
take up PPPs, and how exposure can be calculated
with models. Finally, it should be considered that
amphibians represent a strongly imperiled group of
organisms for which it seems mandatory to minimise Coincidence of pesticide application and presence of
amphibians (Carsten Brühl) Amphibians can come
into direct contact with PPPs on agricultural land in
two ways: firstly, by inhabiting cultivated fields, and,
secondly, by crossing the areas in question on the
way to spawning or after spawning and metamor-
phosis. Because of their annual cycle (migrating to
their breeding sites and back, foraging in their sum-
mer habitat), amphibians can be found on agricul-
tural fields over the entire six-month summer activ-
ity period and are thus potentially directly exposed to
PPPs, although interception by plants may seasonally
reduce exposure. The percentage of individuals in a
population active on arable land during PPP appli-
cation varied between 0.8 and 74.6 %, depending on
species and year of study [2]. Particularly high per-
centages were affected in the case of PPP use in win-
ter cereals and winter oilseed rape. In many cases,
species were present during several applications of
insecticides, herbicides or fungicides. The likelihood
of exposure is lower for species migrating to their
breeding sites early in the year than for those migrat-
ing to the ponds later in the year [2]. Current status of knowledge EFSA activities concerning the preparation of a risk
assessment of PPP for amphibians (Franz Streissl,
EFSA) The European Food Safety Authority (EFSA)
is developing guidance documents for environmen-
tal risk assessment of PPP in Europe. The evaluation
of the effects on amphibians is an important topic
which will be part of a future Guidance Document. A scientific opinion summarising the state of the sci-
ence on the risk assessment for amphibians and rep-
tiles will provide the scientific basis. EFSA organises
rounds of public consultation in order to have the
feedback of the different stakeholders. As a prepara-
tion, a report was drafted on the sensitivity, occur-
rence, habitat use and exposure of amphibian species
in agricultural environments [18]. The authors con-
cluded that absorption through the skin is likely to be
the main route of exposure. Toxicity data for terres- Aldrich et al. Environ Sci Eur (2016) 28:17 Page 4 of 8 harmful or a nuisance in the foreseeable future…” is
sufficient. Owing to its legal nature, however, the
precautionary principle is to be classified as strongly
programmatic, which means that there will always be
a debate about the ‘what’ and ‘how’. This even applies
when it has already undergone a process of concreti-
sation at, e.g. ordinance level. Thus, in article 1 para. 4 of the PSMV, reference is made to the precaution-
ary principle as the basis for the provisions of the
PSMV, and the legislation for the placing on the mar-
ket of PPPs (art. 14 PSMV) represents a fleshing-out
of the precautionary principle. In these cases, the
precautionary principle is already considered, and
the taking of further-reaching measures whilst invok-
ing the precautionary principle is difficult in legal
terms [23]. This does not, however, mean that the
precautionary principle cannot be applied in a spe-
cific case or cannot be used in a further-reaching
manner within the scope of revisions of the concrete
definitions. Thus, articles 148a and 165a of the Law
on Agriculture stipulate in what instances precau-
tionary measures can be taken, what form these may
take and how long they are to apply. In principle, the
plausibility of an unacceptable side effect must exist,
and the likelihood of its occurrence must be rated as
substantial or its consequences must be far reaching. The precautionary principle applies essentially in the
same way in the EU. 3 Tschannen, USG commentary, Art. 1 marginal no. 33; Vereinigung für
Umweltrecht/Keller Helen (Publ.), Kommentar zum Umweltschutzgesetz,
1st and 2nd editions, Zurich 1998 ff. 2 see decisions BGE 124 II 219 and BGE 131 II 431 of the Swiss Federal
Court. Current status of knowledge trial species or life stages are scarce, and therefore, it
is difficult to estimate their sensitivity to PPPs. Based
on available data, the sensitivity of tadpoles seems
to be comparable to fish [20, 21]. Aquatic life stages
occurring in permanent waters are therefore covered
by the existing first tier risk assessment for fish [22]. EFSA initiated a study to collect data on population
effects and toxicity data for terrestrial life stages, and
compared these with toxicity endpoints observed
in studies with standard test organisms. The aim is
to use toxicity endpoints of standard test organisms
as a surrogate for the risk assessment of amphib-
ians to avoid additional toxicity tests with amphib-
ians. A further aim is to be able to extrapolate effects
observed in the laboratory to population-level effects
in the field. i
The precautionary principle (Caroline Aeberli) The
precautionary principle is a way of dealing with sci-
entific uncertainty, based on available scientific
knowledge, the degree of uncertainty and societal
values (e.g. legal or political assessments). In Switzer-
land, the precautionary principle is anchored in the
Federal Constitution (BV) and the Environmental
Protection Act (USG). The aim of the USG is to pro-
tect people, animals and plants, as well as their biotic
communities and habitats, from harmful effects or
nuisances. Pursuant to art. 1 paragraph 2 of the USG,
“In keeping with the precautionary principle, […],
impacts which could become harmful or create a nui-
sance are to be limited at an early stage”. The precau-
tionary principle also applies in further decrees, inter
alia in the Water Protection Act (GSchG) and the
Chemicals Act (ChemG), or at ordinance level, for
example, in the Plant Protection Product Ordinance
(PSMV). The precautionary principle is chiefly
underpinned by the concept of avoiding or limiting
incalculable risks. It creates a safety margin (in Swiss
legal terms) taking the uncertainties vis-à-vis the
longer-term effects of environmental pollution into
account.2 The precautionary principle can be applied
in the form of measures even when there is not yet
any concrete danger, and it is intended to be effective
where scientific uncertainty still exists. This principle
is meant to provide preventive protection against
risks as well as environmental protection focused on
the long term (“prevention instead of cure”). Accord-
ing to Tschannen,3 “a plausible probability, based on
empirical values, that the impacts could become Risk potential was a problem, and hence a need for action. A further
33 % rated the topic ‘effects of plant-protection products
on amphibians’ as interesting. Only 8 % of the partici-
pants saw no problem, and hence no need for action. Exposure and effects are the factors that determine the
risk posed by PPPs to amphibians. As regards to the
effects, it was largely uncontested that PPPs can have a
toxic effect on amphibians. What was disputed, however,
was to what extent amphibians are exposed in the field
and how well the existing experimental studies reflect
this exposure. Whilst some participants noted a temporal
and spatial coincidence of amphibians and PPPs, others
observed that there was still a lack of studies taking par-
ticular account of the behaviour of amphibians (e.g. bury-
ing behaviour). It was pointed out that multiple factors
besides PPP such as habitat loss, diseases and predators
could contribute to the amphibian decline. The relative
contribution of PPPs for amphibian population declines
is currently unclear and it was not the aim of the work-
shop to discuss all factors for amphibian decline. How-
ever, field observations suggest that there are important
factors besides the loss of habitat [6]. According to the
statements of participants from the amphibian protec-
tion sector, in some areas even apparently suitable ponds
are not inhabited. In the opinion of these participants,
the possible influence of PPPs should be more closely
investigated in these cases. The unique life cycle of
amphibians, with both an aquatic and terrestrial phase,
as well as the measured PPP residues in waters [24] and
the permeable amphibian skin put them at potential risk
from PPPs. PPPs can also represent an additional stress
for amphibians already at risk, and any additional stress
can be problematic. These are all observations leading to
the conclusion that PPPs pose a risk, especially in agri-
cultural landscapes, although the extent of this risk com-
pared to that posed by other factors is unclear. On the
other hand, it was mentioned by some participants that
they know of successful conservation projects in which
no particular emphasis was placed on the reduction of
exposure to PPP. It was pointed out that studies have
often been carried out with active ingredients that are no
longer approved and that studies with current formulated
products would be helpful. Discussion After the lectures, participants were divided into two
groups in order to discuss the following questions: • •
How high do the experts estimate the risk potential
of PPPs for amphibians to be in the field? i
• •
What research questions should be addressed to sup-
port the development of a risk assessment scheme
for amphibians? • •
What measures can be implemented on a voluntary
basis or on the basis of the precautionary principle to
reduce pressure on amphibian populations in agri-
cultural areas? The participants had time before the group discussions
to formulate their own ideas on the issues, so that eve-
ryone had the opportunity to contribute. Their ideas and
questions formed the starting point for the group discus-
sions. The aim was to collect the full range of opinions,
not to reach a consensus. After each question, par-
ticipants could rate, and hence prioritise, the individual
ideas. Below, the breadth of the suggestions is summa-
rised, with the spontaneously favoured ideas by the par-
ticipants shown in the box. Aldrich et al. Environ Sci Eur (2016) 28:17 Page 5 of 8 Risk potential Another issue brought up was
that toxicity studies using direct overspray at field appli-
cation rates might not represent a realistic exposure sce-
nario, because interception by the crop canopy will likely
reduce the exposure of the amphibians. What, in your opinion, is indicative of the presence or absence of a signifi-
cant threat to amphibians from PPPs? Presence of threat:
Spatial and temporal coincidence of PPP application with the pres-
ence of amphibians
Exposure in water and on land owing to their special life cycle
Laboratory studies in which effects are observed at full and reduced
application rate
Population declines in nature
Absence of threat:
Uncertainty in terms of actual exposure
Relative impact of other factors
Relevance to population trend is unclear
Exposure reduced by crop canopy What, in your opinion, is indicative of the presence or absence of a signifi-
cant threat to amphibians from PPPs? Presence of threat:
Spatial and temporal coincidence of PPP application with the pres-
ence of amphibians
Exposure in water and on land owing to their special life cycle
Laboratory studies in which effects are observed at full and reduced
application rate
Population declines in nature
Absence of threat:
Uncertainty in terms of actual exposure
Relative impact of other factors
Relevance to population trend is unclear
Exposure reduced by crop canopy Research issues In agreement with EFSA, it was deemed necessary to
define the protection goal for amphibians in greater
detail, and in particular to evaluate the relevance of
effects on populations. Therefore, it can be helpful to
develop population models that allow us to assess long-
term effects at population level on the basis of effects on
individuals. To this end, it was suggested that observa-
tions in the field are increased. Which species and devel-
opmental stages are exposed in which crop to which PPP
at what point in time, and to what extent must be deter-
mined. Coincidence studies, in which the application
of PPP is correlated with the presence and behaviour of
the species, are helpful for this, but direct, indirect and
large-scale exposure in the field should also be observed
in order to develop exposure models. There are still many
unanswered questions regarding the toxicity of PPPs for
amphibians, including co-formulants, mixtures, multiple
stressors and interactive effects. Is it possible to predict
the effects according to the mode of action of the PPP? Effects on terrestrial life stages have still not been studied
enough. The question of which species is most suitable
for testing is also still unresolved. Many studies are car-
ried out with the African clawed frog, but its representa-
tiveness for native species has been questioned by the
participants. The variability between species and popula-
tions should be studied so that uncertainty and applica-
bility may be estimated. For an efficient risk assessment
for amphibians, it was proposed to define entry criteria
to identify quickly problematic active substances and co-
formulants on the basis of certain substance properties. Standard inhalation toxicity studies with birds and mam-
mals might be helpful to identify substances which could After the discussion, workshop participants (for back-
ground, see Fig. 1) were invited to give their spontane-
ous assessment of the topic. Here, they were given the
choice between three responses: (i) “There is a problem”. (ii) “It is unclear whether or not there is a problem, but it
is an interesting topic”. (iii) “There is not a problem”. The
query was made on a flipchart, and each participant had
one sticker to place by their selected response. According
to the results, 58 % of the participants thought that there Aldrich et al. Research issues Environ Sci Eur (2016) 28:17 Page 6 of 8 Page 6 of 8 the industry to reduce or withdraw problematic applica-
tions or PPPs from the market, or to develop recommen-
dations on how and when the use of the PPP in question
could be reduced or avoided in a professional or private
context. The ‘advisory and awareness-raising activi-
ties’ topic took up the latter subject, determining that
farmers should be informed of the dangers of PPP use
for amphibians particularly where amphibians migrate
from land habitats to ponds. It was stressed that advice
is also necessary in the context of PPP use in domestic
gardens. Advice and increasing awareness could also
lead to a reduction in and optimisation of the use of PPP. Approaches such as alternative plant protection, absten-
tion from the use of certain PPP, or the use of PPP strictly
in accordance with Integrated Production (IP) rules
would be conceivable here. Where mechanical alterna-
tives to PPP use are recommended, the possible influence
of these alternatives on amphibians should also be con-
sidered. One mitigation measure could be based on the
prediction of amphibian migration so that PPP are not
used when amphibians are present in large numbers on
farmland. A variety of measures under the heading ‘habi-
tat promotion’ were discussed—for example, untreated
corridors could be set up on farmland where amphibians
are present or to which they migrate. Direct payments
to farmers could be a possibility to promote the imple-
mentation of amphibian friendly management measures
including amphibian-specific ecological compensation
areas or biodiversity-promoting areas, or even in IP agri-
culture. Particular attention should also be paid to the
amphibian breeding sites of national importance, as well
as the migration corridors (e.g. a ban on application, even
if only temporary). The question of costs and benefit was
also of importance in the subject area of ‘measures’. be acutely toxic to amphibians via dermal exposure, since
the percutaneous absorption rates of substances through
the amphibian skin is deemed to be very high. Ultimately,
risk mitigation measures should be available so that the
risk both inside and outside the agricultural field can be
reduced. Part of this consists e.g. in research on cultiva-
tion methods without the use of PPP. The effect of buffer
strips to reduce the input into ponds should be investi-
gated, as should the design of habitats, e.g. Risk mitigation measures Advice and awareness raising for private users in their home gardensi In the discussion on measures which could be taken, the
topics of authorisation, reduction/optimisation of PPP
use, advisory and awareness-raising activities and habi-
tat improvement were at the forefront. For authorisation,
one important topic was the need to adapt risk assess-
ment schemes in order to identify PPPs with unaccepta-
ble affects and prevent their authorisation. Furthermore,
it was hoped that comparative assessments would allow
differentiation between problematic and less problem-
atic active substances or formulations and rank them
according to their risk potential. The aim would be for Compensation measures (e.g. amphibian-specific ecological compensa-
tion areas) Promotion of amphibians via direct payments and/or existing label
schemes, e.g. IP-Suisse Reduction of PPP use through alternative plant protection strategies
where possible and sensible Research issues Avoidance of PPP application during migration in affected areas with
the help of prediction models How relevant are other factors for the decline in the amphibian popula-
tion, compared to PPPs? Recommended avoidance of specific products in vulnerable areas or
critical time periods Advice and awareness raising for farmers Research issues existing biodi-
versity-promoting areas (a type of agricultural set-aside),
so that they too benefit the amphibians. How other fac-
tors, e.g. tillage or mechanical weed control, affect the
amphibians, and the extent to which these factors con-
tribute to the decline of the amphibians compared to
the impact of PPPs was also discussed. A weighing of
the various factors should be conducted. Interactions
between factors make it difficult to deal with them sepa-
rately. Here, developments in research on multiple stress-
ors should be pursued. A further overarching issue was
addressed: What approach or approaches to protect
amphibians are the most efficient in terms of costs (for
the farmer) and effect (on the amphibians)? What research issues should be addressed? How can realistic exposure of amphibians to PPPs be assessed? How great is the risk to which species, at what developmental stage, in
which crop and from which PPP? Which effects on individuals and at what life stages are most relevant
for population trends? Which population models can be used to estimate the effects observed
in the laboratory with individuals, at the population level as a whole? What effect do co-formulants have on toxicity? How high is the variability in sensitivity between the various amphibian
species and populations? Can entrance criteria be developed in order to identify especially
problematic substances for ecotoxicological risk assessment in the
authorisation of PPP? What risk-management options are there both inside and outside of
the field? How relevant are other factors for the decline in the amphibian popula-
tion, compared to PPPs? What research issues should be addressed? How can realistic exposure of amphibians to PPPs be assessed? How great is the risk to which species, at what developmental stage, in
which crop and from which PPP? Which effects on individuals and at what life stages are most relevant
for population trends? Which population models can be used to estimate the effects observed
in the laboratory with individuals, at the population level as a whole? What effect do co-formulants have on toxicity? How high is the variability in sensitivity between the various amphibian
species and populations? Can entrance criteria be developed in order to identify especially
problematic substances for ecotoxicological risk assessment in the
authorisation of PPP? What risk-management options are there both inside and outside of
the field? How relevant are other factors for the decline in the amphibian popula-
tion, compared to PPPs? Received: 31 January 2016 Accepted: 1 May 2016 Received: 31 January 2016 Accepted: 1 May 2016 Acknowledgements 17. Brühl CA, Pieper S, Weber B (2011) Amphibians at risk?—Susceptibility
of terrestrial amphibian life stages to pesticides. Environ Toxicol Chem
30:2465–2472 We are grateful to Katja Knauer (BLW), Francis Cordillot (BAFU), Marcel Liner
(ProNatura) and Anna Bozzi (scienceindustries) for their valuable comments
to the draft. Furthermore, we would like to thank all participants for their con-
structive and active contribution at the workshop. Additionally, the support
of Agroscope, the Swiss Amphibian and Reptile Conservation Program (karch)
and the Swiss Centre for Applied Ecotoxicology Eawag-EPFL (Ecotox Centre)
was greatly appreciated, as well as the Agroscope translation service. A previous version of this paper was published in German [25]. 18. Fryday S, Thompson H (2012) Toxicity of pesticides to aquatic and terres-
trial life stages of amphibians and occurrence, habitat use and exposure
of amphibian species in agricultural environments. Supporting Publica-
tions 2012:EN-343, p 348. http://www.efsa.europa.eu/publications 19. Brühl CA, Schmidt T, Pieper S, Alscher A (2013) Terrestrial pesticide expo-
sure of amphibians: an underestimated cause of global decline? Sci Rep
3:1135. doi:10.1038/srep01135 20. Aldrich AP (2009) Empfindlichkeit von Amphibien gegenüber Pflanzens-
chutzmitteln. AGRARForschung 16:466–471 Author details
1 13. Hammond JI, Jones DK, Stephens PR, Relyea RA (2012) Phylogeny meets
ecotoxicology: evolutionary patterns of sensitivity to a common insecti-
cide. Evol Appl 5:593–606 1 Agroscope, Schloss, 8820 Wädenswil, Switzerland. 2 Swiss Centre for Applied
Ecotoxicology Eawag-EPFL (Ecotox Centre), Überlandstrasse 133, 8600 Düben-
dorf, Switzerland. 3 EWP AG, 8307 Effretikon, Switzerland. 4 Institute for Envi-
ronmental Sciences, University Koblenz-Landau, Fortstraße 7, 76829 Landau,
Germany. 5 European Food Safety Authority (EFSA), Pesticide Unit, Via Carlo
Magno 1A, 43100 Parma, Italy. 6 KARCH, Passage Maximilien‑de‑Meuron 6,
2000 Neuchâtel, Switzerland. 7 Department for Evolutionary Biology and Envi-
ronmental Studies, University of Zurich, Winterthurerstrasse 190, 8057 Zurich,
Switzerland. 14. Forbes VE, Calow P (2002) Population growth rate as a basis for ecological
risk assessment of toxic chemicals. Phil Trans R Soc Lond 357:1299–1306 15. Schmidt BR (2011) Die Bedeutung der Jungtiere für die Populationsdyna-
mik von Amphibien. Zeitschrift für Feldherpetologie 18:129–136 16. Böll S, Schmidt BR, Veith M, Wagner N, Rödder D, Weimann C, Kirschey T,
Lötters S (2013) Anuran amphibians as indicators of changes in aquatic
and terrestrial ecosystems following GM crop cultivation: a monitoring
guideline. BioRisk 8:39–51 Authors’ contributions
AA MJ
d BS h 11. Schweizer E (2014) Raumnutzung der Kreuzkröte (Bufo calamita) im
Ackerbaugebiet. Bachelor’s thesis. Zürcher Hochschule für Angewandte
Wissenschaften, Wädenswil 11. Schweizer E (2014) Raumnutzung der Kreuzkröte (Bufo calamita) im
Ackerbaugebiet. Bachelor’s thesis. Zürcher Hochschule für Angewandte
Wissenschaften, Wädenswil AA, MJ and BS have organised the workshop and drafted the manuscript. Additionally, as presenters of the respective topics at the workshop, CA, CB
and FS have significantly contributed to the manuscript. The authors read and
approved the final manuscript. 12. Cordillot F, Klaus G (2011) Gefährdete Arten in der Schweiz. Synthese Rote
Listen, Stand 2010. Bundesamt für Umwelt, Bern. Umwelt-Zustand Nr. 1120:111 S Conclusions and outlookh The workshop showed that there is a need for additional
knowledge on the subject of amphibians and PPP, and that
this topic should be pursued further. Much information Aldrich et al. Environ Sci Eur (2016) 28:17 Page 7 of 8 was exchanged and many ideas were gathered and con-
tacts established. The broad-based cooperation between
stakeholders from agriculture, industry, environmental
associations, public authorities and herpetologists has
proven useful, and must be developed further. Workshop
participants were made aware of the topic of amphibians
and PPP, and this awareness must now be carried on into
the various areas represented at the workshop. Seemingly
crucial is the question of whether further research is nec-
essary in the first instance, or whether preventive meas-
ures should be taken in order to improve the situation
for the amphibians. Speaking for the latter is the fact that
amphibian populations are at risk; for the former, a higher
level of knowledge will allow measures to be applied in a
more strategic and effective fashion, as the relative con-
tribution of the different PPP to the risk is not yet quanti-
fied. The protection of amphibians should be considered
particularly when elaborating new concepts, e.g. as part of
the National Action Plan for risk reduction and sustain-
able use of PPP, as well as in existing concepts concerning
agricultural policy, such as the Direct Payment Ordinance
(specific incentives to remunerate farmers for services of
public and common interest). At the end of the workshop,
it was proposed that a pilot project be launched to gather
more information on the exposure of amphibians to PPP
in the field. Such a project requires good collaboration,
the foundation of which was laid during the workshop. was exchanged and many ideas were gathered and con-
tacts established. The broad-based cooperation between
stakeholders from agriculture, industry, environmental
associations, public authorities and herpetologists has
proven useful, and must be developed further. Workshop
participants were made aware of the topic of amphibians
and PPP, and this awareness must now be carried on into
the various areas represented at the workshop. Seemingly
crucial is the question of whether further research is nec-
essary in the first instance, or whether preventive meas-
ures should be taken in order to improve the situation
for the amphibians. 1.
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amphibians and pesticide applications on arable fields during spring
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pesticides in Pacific chorus frogs (Pseudacris regilla) from California’s Sierra
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Pierce CL (2015) Pesticides concentrations in frog tissue and wetland
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Pierce CL (2015) Pesticides concentrations in frog tissue and wetland
habitats in a landscape dominated by agriculture. Sci Total Environ
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Waller RW (2004) Status and trends of amphibian declines and extinc-
tions worldwide. Science 306:1783–1786 6. Collins JP, Storfer A (2003) Global amphibian declines: sorting the hypoth-
eses. Divers Distrib 9:89–98 7. Relyea RA (2003) Predator cues and pesticides: a double dose of danger
for amphibians. Ecol Appl 13:1515–1521 8. Relyea RA, Hoverman JT (2008) Interactive effects of predators and a
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10. Schmidt BR, Zumbach S, Tobler U, Lippuner M (2015) Amphibien
brauchen temporäre Gewässer. Zeitschrift für Feldherpetologie
22:137–150 Conclusions and outlookh Speaking for the latter is the fact that
amphibian populations are at risk; for the former, a higher
level of knowledge will allow measures to be applied in a
more strategic and effective fashion, as the relative con-
tribution of the different PPP to the risk is not yet quanti-
fied. The protection of amphibians should be considered
particularly when elaborating new concepts, e.g. as part of
the National Action Plan for risk reduction and sustain-
able use of PPP, as well as in existing concepts concerning
agricultural policy, such as the Direct Payment Ordinance
(specific incentives to remunerate farmers for services of
public and common interest). At the end of the workshop,
it was proposed that a pilot project be launched to gather
more information on the exposure of amphibians to PPP
in the field. Such a project requires good collaboration,
the foundation of which was laid during the workshop. be an EFSA output. The positions and opinions presented are those of the
authors alone and are not intended to represent the views of the EFSA. 24. Moschet C, Wittmer I, Simovic J, Junghans M, Piazzoli A, Singer H,
Stamm C, Leu C, Hollender J (2014) How a complete pesticide screening
changes the assessment of surface water quality. Environ Sci Technol
48:5423–5432 25. Aldrich A, Junghans M, Aeberli C, Brühl CA, Streissl F, Schmidt BR (2016)
Amphibien und Pflanzenschutzmittel – Forschungs - und Handlungsb-
edarf. Aqua&Gas 4:14–20 Aldrich et al. Environ Sci Eur (2016) 28:17 24. Moschet C, Wittmer I, Simovic J, Junghans M, Piazzoli A, Singer H,
Stamm C, Leu C, Hollender J (2014) How a complete pesticide screening
changes the assessment of surface water quality. Environ Sci Technol
48:5423–5432
25. Aldrich A, Junghans M, Aeberli C, Brühl CA, Streissl F, Schmidt BR (2016)
Amphibien und Pflanzenschutzmittel – Forschungs - und Handlungsb-
edarf. Aqua&Gas 4:14–20 22. EFSA (2013) Guidance on tiered risk assessment for plant protection
products for aquatic organisms in edge-of-field surface waters. EFSA J
11(7):3209. doi:10.2903/j.efsa2013.3290 22. EFSA (2013) Guidance on tiered risk assessment for plant protection
products for aquatic organisms in edge-of-field surface waters. EFSA J
11(7):3209. doi:10.2903/j.efsa2013.3290
23. BAG (overall control), BUWAL (BAFU), BLW, BVET, seco, EDA (2003), Das
Vorsorgeprinzip aus schweizerischer und internationaler Sicht, Synthese-
papier der interdepartementalen Arbeitsgruppe „Vorsorgeprinzip“August
2003, Bern 2003 Competing interests The authors declare no competing interests. The workshop, the drafting of
the manuscript and the article processing charge of this publication were
financially supported by Agroscope and Ecotox Centre. This publication was
drafted under the sole responsibility of the authors, and is not considered to 21. Weltje L, Simpson P, Gross M, Crane M, Wheeler J (2013) Comparative
acute and chronic sensitivity of fish and amphibians: a critical review of
data. Environ Toxicol Chem 32:984–994 Aldrich et al. Environ Sci Eur (2016) 28:17 Page 8 of 8 Page 8 of 8 Page 8 of 8 22. EFSA (2013) Guidance on tiered risk assessment for plant protection
products for aquatic organisms in edge-of-field surface waters. EFSA J
11(7):3209. doi:10.2903/j.efsa2013.3290 j
23. BAG (overall control), BUWAL (BAFU), BLW, BVET, seco, EDA (2003), Das
Vorsorgeprinzip aus schweizerischer und internationaler Sicht, Synthese-
papier der interdepartementalen Arbeitsgruppe „Vorsorgeprinzip“August
2003, Bern 2003
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Switzerland: Diversity in the Classroom, Uniformity in the Faculty
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M. Beck (*)
St. Gallen University of Teacher Education, St. Gallen, Switzerland
e-mail: michael.beck@phsg.ch
C. Mantel · S. Bischoff
University of Teacher Education, St. Gallen, Switzerland Diversity and Uniformity – The Swiss Case With regard to the concept of minorities teaching in a majority society, this article
deals with the perception of otherness and othering in the context of transnational
migration in Switzerland. Despite relatively strict immigration laws, Switzerland is
an immigration country with a high percentage of inhabitants with an immigrant
background which are very heterogenous in terms of ethnic or national origin,
socioeconomic history or reasons for migration (Federal Statistical Office (FSO)
Switzerland, 2017). At the same time, Switzerland’s educational system is considered to be relatively
highly structured, with an early division of students into different achievement lev
els, which usually takes place at the age of 12. In the current literature, this transi
tion is often cited as being responsible for a strong relationship between educational
success and social as well as immigrant background. When it comes to naming
migration-related diversity, words are (obviously) important. Surveys and opera
tionalizations of the so-called “immigrant background” in the context of (official)
statistics as well as (educational) scientific studies have three possible consequences:
First, different data collection types and operationalizations often have very differ
ent implications for theory-based explanations of the studied ‘faits sociaux’, in
Emile Durkheim’s words, which in our view are not always made sufficiently
explicit. Second, the nature of data collection and operationalization naturally
affects the nature and strength of the effects and results as well as their interpreta
tions. And third, the terms, definitions, and operationalizations used, in turn, natu
rally influence the ‘faits sociaux’, for example, by not only not reflecting but
possibly also preserving stereotypes, minority status and thus power relations. In contrast to the heterogeneity and abundance of different immigrant back
grounds and histories in Switzerland, there has been little research on teachers with
an immigrant background. This article shows the current state of research, and at the
same time, points out the gaps in the state of research, based on different theoretical
backgrounds. Throughout this article, we look at the construction of difference in the choice of
studying to become a teacher. We assume that the choice of apprenticeship or study
is a result of weighing the advantages and disadvantages one expects from the asso
ciated career options. These advantages and disadvantages can be, for example,
more economic/material factors such as salary or job security. Chapter 4
Switzerland: Diversity in the Classroom,
Uniformity in the Faculty Michael Beck
, Carola Mantel, and Sonja Bischoff Abstract Switzerland is a country with a long immigration history. Today, 27% of
all pupils in compulsory education are foreign nationals (Federal Statistical Office
(FSO) Switzerland, Obligatorische Schule: Lernende nach Grossregion,
Schulkanton, Bildungstyp und Staatsangehörigkeit (je-d-15.02.01.05). Retrieved
from
https://www.bfs.admin.ch/bfs/de/home/statistiken/bildung-wissenschaft/
personen-ausbildung/obligatorische-schule.assetdetail.11787900.html, 2020). On
the other hand, teachers of immigrant background constitute a small minority even
though the demand for teachers is high, with secure jobs that pay well. Moreover,
there is a debate on the question whether teachers with an immigrant background
are especially qualified for teaching culturally diverse classes. Given that persons
with an immigrant background tend to aspire to higher educational and occupa
tional goals than non-immigrant individuals (Van De Werfhorst & Van Tubergen,
Ethnicities 7(3):416–444, 2007), the question arises why individuals with an immi
grant background do not choose to become teachers more often. In light of this question, we provide an overview of studies from Switzerland and
examine transitions of individuals into and out of Universities of Teacher Education
in Switzerland as well as studies concerning active teachers. We will discuss current research evidence, that suggests that social background
as well as motivations for choosing the fields of study (which usually play a signifi
cant role in explaining differing educational decisions) do not provide an explana
tion for the low enrolment rate into teacher education among students with immigrant
background. Evidence indicates that dichotomizing into immigrant and non-
immigrant might conceal differences in attitudes which particularly those from stig
matized cultural backgrounds face. These findings are highlighted by empirical
insights on practicing teachers who experience a lack of recognition and who
develop a range of strategies in response to these experiences. M. Beck (*)
St. Gallen University of Teacher Education, St. Gallen, Switzerland
e-mail: michael.beck@phsg.ch C. Mantel · S. Bischoff
University of Teacher Education, St. Gallen, Switzerland 45 © The Author(s) 2023
M. Gutman et al. (eds.), To Be a Minority Teacher in a Foreign Culture,
https://doi.org/10.1007/978-3-031-25584-7_4 45 M. Beck et al. M. Beck et al. 46 Diversity and Uniformity – The Swiss Case They can also be
more psychological/non-material factors such as self-worth and a sense of belong
ing, but also anticipated interactions (for example, the risk of experiencing discrimi
nation on the basis of origin in daily interactions) with superiors, colleagues and
“customers” (in the case of teachers: students and parents). This article is structured as follows: First, we will show examples of the prob
lems involved in constructing the immigrant background on an operational and
theoretical level. We then provide theoretical considerations with regard to explain
ing differing educational pathways, leading to differential outcomes concerning the
choice of studying to become a teacher. Following this, the Swiss educational 4 Switzerland: Diversity in the Classroom, Uniformity in the Faculty 47 4 system is briefly explained, with a focus on the training of teachers for compulsory
education. Then, based on figures from official education statistics, we present distributions
of the proportions of persons with an immigrant background in relation to the total
population as well as various indicators of educational success. Furthermore, we
show the results of studies to choose a course of study to become a teacher and the
experiences of teachers with a migrant background when on the job or looking for
a job. The article concludes with implications of the results for research and educa
tional policy.
1
2014
2016
2015
2020
2019
2017
2000
2017 2020
2006 2008
1 Due to limited space and the focus of this paper, we only use data from the focus group discus
sions with students. 2 This is an assumption on our part, to our knowledge there are currently no systematic studies
on this.
3 And with it, differences in socioeconomic background and in basic norms and values. Who? Otherness and Othering To analyze differences in outcomes for different groups of a population, one must
first define the different groups. In doing so, there is a danger of reproducing social
categories and the inequalities they entail. Therefore, it should be pointed out that
operational definitions are artificial and must always be interpreted with regard to
the object of investigation. Starting from a (as far as possible) value-free approach,
the distinctions presented here should therefore be understood as theory-based. Their construction should neither be understood as a justification of unequal 48 M. Beck et al. M. Beck et al. treatment nor as a negation of other relevant distinctions. In terms of content, the
following descriptions largely follow the considerations in Beck and Edelmann
(2016), which in turn are strongly oriented on Gresch and Kristen (2011) and
Kemper (2010).
2013
2014
2020
2
3
Using statistical categorizations (or variables) that already exist in data comes
with different problems: First, when analyzing such data, researchers rely on the
variables collected to construct exhaustive categories. Second, these constructions Switzerland: Diversity in the Classroom, Uniformity in the Faculty 49 4 should also be able to represent categories that are valid and have theoretical con
tent. One danger of such approaches, which is not emphasized enough by Beck and
Edelmann (2016), is the following: An approach that is predominantly oriented
towards statistical categories may overlook the fact that these categories are not
“objectively given”. Rather, the relevance of these categories in relation to outcomes
(such as experiences of discrimination in the job search) may be rooted deeply in
the social processes that both helped to create such categorizations, as well as work
ing as a mechanism responsible for the phenomena of interest. Especially studies with qualitative research approaches proceed in a different
way by primarily recording or interpreting self-attributions or perceived attributions
by others with reference to an immigrant background. The relevance of the catego
rization is either left to the actors themselves or revealed with the help of analytical
(mostly hermeneutical) methods in combination with attributions from others. This
has the advantage for different lines of difference to be drawn when analyzing dif
ferent situations. Furthermore, the subjective view of the actors can be better docu
mented. Who? Otherness and Othering Of course, this approach also poses challenges that have to be addressed by
the researcher with the help of adequate methodology: Potentially important pro
cesses that take place outside the perception of the actors can almost not be cap
tured. Furthermore, there is a danger that certain situations and their consequences
are over- or under-interpreted with regard to the underlying lines of difference. Relevant attributions of an immigrant background can also be discovered within the
framework of a specific analysis by analyzing patterns. Ideally, this can also uncover
patterns of action and interpretations hidden from the persons involved in the social
processes under investigation. The use of existing categories should therefore be theory-guided so that they do
not only represent otherness, but also take the processes of categorizing in the sense
of othering into account. Thus, existing differences should not be interpreted as
objectively and permanently existing differences but should be understood as situ
ational as well as addressed and interpreted as a consequence of social processes
and resulting attributions. 4 As the Thomas theorem states: “If men define situations as real, they are real in their conse
quences” (Thomas & Thomas, 1928, p. 572). From Diverse Schools to Universities: Choice or Restriction? 52 2013 2013 probably not be explained by the career choice motives considered in the study
(Beck & Edelmann, 2016, p. 185). Second, the extent of the underrepresentation
depends strongly on what counts as an immigrant background. It is particularly
interesting to note that the definition of an immigrant background as made by the
official statistics of Switzerland, takes into account the nationality in addition to the
countries of birth of the respondents and their parents (Federal Statistical Office
(FSO) Switzerland, 2017). This has consequences, for example, when people have
one parent born in Switzerland and one born abroad; in this case, nationality at birth
determines whether a person is categorized as having an immigrant back
ground or not. probably not be explained by the career choice motives considered in the study
(Beck & Edelmann, 2016, p. 185). Second, the extent of the underrepresentation
depends strongly on what counts as an immigrant background. It is particularly
interesting to note that the definition of an immigrant background as made by the
official statistics of Switzerland, takes into account the nationality in addition to the
countries of birth of the respondents and their parents (Federal Statistical Office
(FSO) Switzerland, 2017). This has consequences, for example, when people have
one parent born in Switzerland and one born abroad; in this case, nationality at birth
determines whether a person is categorized as having an immigrant back
ground or not. From Diverse Schools to Universities: Choice or Restriction? 2019
M. Beck et al. 50 M. Beck et al. 2020
2019
2019
1974
2006
2007
2007
1995
2020
2019
2019
1974
2006
2007
2007
1995
Switzerland: Diversity in the Classroom, Uniformity in the Faculty 51 4 4 Suter (2016) found evidence of high parental educational aspirations among edu
cationally successful students with a migrant background in Switzerland in the con
text of a guideline-based interview study. Beck (2015) however, finds no evidence
for increased aspirations among parents of primary school students with an immi
grant background from “typical” migration countries (former Yugoslavian states,
Turkey and Portugal). Glauser (2015) finds no clear influence of migration status on
the educational motivation of school leavers in Switzerland, while Tjaden and
Scharenberg (2017), using a different data set, find more favorable transitions after
lower secondary school for students with an immigrant background (controlling
for social origin and school performance) in Switzerland, which they attribute
predominantly to immigrant optimism. Similarly, Griga and Hadjar (2014) finds
that second-generation immigrant women in Switzerland are more likely to enter
university than natives, controlling for social background and school performance. However, it is not clear whether this is due to higher educational aspirations or
expected discrimination in the search for apprenticeships. Overall, one might consider the profession of a teacher in Switzerland as very
secure nowadays due to a shortage of teachers (Dachverband Lehrerinnen und
Lehrer Schweiz (LCH), 2020). It is also well paid compared to the level of demands
of the training (Wolter et al., 2003) and since pay is based on established salary
categories, there is actually little possibility of wage discrimination. So based on
these considerations and assumptions, there are actually many reasons why people
with a migrant background could choose to study to become a teacher more often,
as long as they meet the entry requirements. But this consideration is also based on
the restriction that no discrimination on the basis of origin is to be expected in the
search for a job or in the job itself. 2005
2009 2010
2016
4.1 Two findings stand out (Beck & Edelmann, 2016): First, students with an immi
grant background are significantly underrepresented at Swiss universities of teacher
education compared to universities and universities of applied sciences, and this can M. Beck et al. At University: One of Us? For prospective teachers, the path from educational decision to the profession nor
mally leads through the University of Teacher Education. Among other things, fel
low students and the interactions with the lecturers can have an influence on whether
the training can be successfully completed. Literature on dropping out of higher
education with a focus on students with an immigrant background is relatively 4 Switzerland: Diversity in the Classroom, Uniformity in the Faculty 53 4 scarce until now, especially when it comes to teacher education. One of the prob
lems is the adequate operationalization of dropout and the associated problems in
the study of such phenomena. However, there are indications that a low socioeco
nomic status as well as general conditions of study have an influence on the risk of
dropping out from university in general (see, for example Wolter et al., 2014). One
conceivable factor here for students with an immigrant background would be, how
integrated they feel at university and the sense of belonging conveyed to them by
fellow students and lecturers. Furthermore, research on the sense of belonging at
universities in Switzerland is rare to non-existent (see Federal Statistical Office
(FSO) Switzerland, 2018 for results related to overall wellbeing). Recent studies
from the United States of America (Gopalan & Brady, 2020) show a significant dif
ference depending on the racial-ethnic origin of students in the sense of belonging,
even if the reported effect sizes are rather small. We use selected examples to show
the extent to which integration and a sense of belonging at university might play a
role for prospective teachers. Results from the survey of 14 students with an immigrant background as part of
the project DIVAL (cf. Paragraph 1) indicate that the question of representativeness
and belonging is relevant to some of the students with an immigrant background and
that they would welcome an increased proportion of students with an immigrant
background at university. This is illustrated with the following selected statements
from the interviews (cf. Edelmann et al., 2015, p. 217): When I started here, I was, I believe, the only dark-skinned student. And now at under
graduate level there are, I think, two or three more dark-skinned students that have recently
started. And I think that’s good. 5 The original study describes four ideal types. However, the fourth type does not have sufficient
data saturation and is therefore not presented here (for details, see Mantel, 2017, 2020). At University: One of Us? I have asked myself this question before I started teacher education: If I become a
teacher as a person with an immigrant background, how many other such students are cur
rently at this university? Will I be the only one? And if you already know, aha, yes, there are
other students with an immigrant background at this university and it is seen as something
positive, then you simply don’t need to have this discussion with yourself and worries at the
beginning. Other students emphasized that they hardly perceive an immigrant background as a
relevant distinguishing feature at St. Gallen University of Teacher Education. One
reason mentioned is that all students have already completed a successful educa
tional path in Switzerland and acquired the Certificate of Access to Higher Education
(Edelmann et al., 2015, p. 217). These results show several aspects: On the one hand, a perceived relevance of the
sense of belonging, which, however, is rather defined by belonging to the minority
(in this case there seems that “migration” and “skin color/race” are mixed together). The risk of not feeling a sense of belonging can be interpreted as a cost aspect,
which can lead to an overall negative evaluation of the teacher education program,
even among those who are inclined to study. On the other hand, a meritocratic atti
tude is reported, where only the entrance qualification counts. M. Beck et al. 54 M. Beck et al. Into and on the Job: What to Expect? The decision as to whether students choose to enter the teaching profession may
also depend on what they anticipate encountering as teachers. This anticipation may
be based on what they experience during their practical courses as well as what they
observe about and hear from practicing teachers. There are indications from the DIVAL and DIVAL_transition projects that pro
spective teachers with a so-called immigrant background are indeed concerned
about their future role, as they tend to think much more about certain aspects than
their fellow students. For example, considerations are made regarding the extent to
which languages shared with students or parents may or may not be used in a school
context or to what extent a common national or ethnic origin with the pupils and
their parents may lead to more closeness and when it is appropriate or necessary to
make delimitations. So, the question arises, what kind of stories may reach the students during their
pre-service teacher education – or, in other words: What are the experiences of
teachers for whom a so-called immigrant background has become relevant? Edelmann (2006, 2008) has studied with a content analysis approach (Mayring,
2010), how teachers in the city of Zurich who consider themselves as someone
‘with immigrant background’ (and therefore using self-identification as an opera
tionalization) deal with cultural diversity among their students. Surprisingly, she
has found these teachers to feel fully recognized. They considered their ‘back
ground’ as a resource for their teaching by – for instance – acting as role models for
their students or by being particularly empathetic to those with similar migration
experiences. Edelmann (2008) suggests to interpret these results against the back
ground that the teachers in the city of Zurich at the time of research had a particu
larly high working satisfaction, as the working conditions were attractive and there
was an overall teacher shortage so that they were usually able to choose the particu
lar school neighborhood they most preferred (ibid., p. 200ff). g
y
p
p
2017 2020
2013
1921–1922
1969
1982
1995
1983
5 4 Switzerland: Diversity in the Classroom, Uniformity in the Faculty 55 The first type has an overall orientation of striving towards appreciation for all. Into and on the Job: What to Expect? This type has biographically experienced only lightly effective processes of bound
ary making which he or she has been able to influence by constantly modifying
boundaries in claiming both sides to be equally valuable in some way. As a teacher,
this type avoids schools in the rural context and consciously prefers schools in
urban-immigrant neighborhoods for a negative as well as a positive reason: The
negative reason is the anticipation to be less exposed to stigmatization or denigra
tion as would presumably be the case in the rural context, while the positive reason
is to feel familiar with the milieu of the urban-immigrant environment. Nevertheless,
this type experiences precarious lack of belonging and recognition as a teacher, is
particularly cautious not to offend anyone and tends to think of those ‘with immi
grant background’ – including his or her own students – as those who will always
be limited in their career opportunities. The second type orientates towards a struggle against social exclusion and has
experienced social boundaries that have been almost impossible to overcome,
including physical and/or psychological violence such as being strongly stigma
tized based on one’s national or ethnic family history, which in turn leads to a con
stant feeling of vulnerability. As a teacher, this type avoids the migration context
altogether and seeks to find an environment in which migration is on the agenda as
little as possible for fear of again being addressed as someone of foreign origin. Due
to the feeling of vulnerability, this type seeks to keep and defend the relatively high
social position as a teacher, while at the same time teaching the students not to
exclude or denigrate anyone. The third type is structured along self-determined belonging through upward
mobility. In this case, there is a strong desire for change because of unsatisfying
family conditions and limiting socioeconomic circumstances. Social upward mobil
ity becomes a main biographical orientation. Having reached this social advance
ment by becoming a teacher, the social position feels strong and secure. Consequently,
this type feels free to choose a school neighborhood independently of concerns of
being recognized as a teacher, while sometimes urban-immigrant neighborhoods
are chosen for reasons of familiarity. Conclusions and Implication In this article, we have tried to summarize the current state of research on teachers
with a migration history in Switzerland. The focus was on the construction of the
immigrant background as an object of investigation in scientific works, on the
choice of a study program to become a teacher as well as on the experiences of
prospective and practicing teachers with an immigrant background. Based on the
results presented here, we would like to briefly discuss what we already know and,
building on this, focus on the question of what we do not know. Although we first discussed the question of operationalizing immigrant back
ground, this discussion is by no means completed. Especially in a strongly hetero
geneous immigration society like Switzerland, it is important that this heterogeneity
is also anticipated in research. For this, studies with a focus on specific immigrant
backgrounds would be promising in the first instance, since dichotomizing opera
tionalizations (with/without an immigrant background) are mainly demarcations in
relation to the autochthonous population, but they blur the boundaries within the
immigrant population. At the same time, the examples of skin color/race and reli
gion described here show once more that national origin in this context is not suf
ficient to examine relevant boundary demarcations and questions of representation
and belonging. This raises the problem of which categories can be used in research
and how. Categorizations like skin color or race are easily misused for biologizing
explanations and therefore subject to data protection for good reasons.i We have seen that differences in operationalization, which seem trivial at first
glance, already result in large quantitative differences in the categorization into
“minority” and “majority”. If, for example, nationality is taken into account in the
construction of the “2nd generation”, we could see that the proportion of students
with an immigrant background is clearly underestimated according to the official
statistical definition of Switzerland. This leads to the question: Does “opening up”
the term “immigrant background” in the true sense of the word (here, for example,
if at least one parent was born abroad, regardless of current nationality), which leads
to a markedly higher proportion of students with an immigrant background, enable
the normalization of diversity in the context of migration? Does the “assimilation”
by definition of persons with an actual immigrant background as described above
lead to a stronger delimitation of the then so-called “immigrants”? Into and on the Job: What to Expect? These teachers typically distance themselves
from the ‘new immigrants’ who – in their eyes – have not yet ‘earned’ their belong
ing while showing strongly optimistic performance expectations towards all of their
students combined with the idea that assimilation is needed in order to achieve full
integration and educational success. In all three types, the boundaries that are most strongly experienced and lead to
strongly contoured type structures are those that refer to socioeconomic background
as well as migration, typically having a parental labor migration history. In sum, the overall picture of experienced teachers for whom a so-called immi
grant background has become significant, shows various kinds of tensions between
different resources in dealing with student diversity and limitations based on expe
rienced boundary making processes. Those feeling vulnerable in their recognition
as teachers tend to avoid rural environments and choose urban-immigrant neighbor
hoods instead, while nevertheless experiencing precarious conditions of belonging
(Ha & Bischoff, 2020; Mantel, 2020). M. Beck et al. M. Beck et al. 56 Conclusions and Implication A promising approach could be to use extended concepts of (self-identified)
ancestry to measure specific group memberships in future (quantitative) studies. While measurements of origin via cultural and ethnic background as well as self-
identified ancestry are already standard in surveys in countries such as Australia or
the USA, such operationalizations have only recently been applied in the European
context (cf. European Standard Classification of Cultural and Ethnic Groups
(ESCEG) by Heath et al., 2016). It has been shown that these are also suitable for
identifying distinctions based on sub-national and regional categories (Heath et al.,
2016, p. 45) and are suitable for uncovering connections with, for example,
experiences of discrimination that cannot be identified exclusively with 4 Switzerland: Diversity in the Classroom, Uniformity in the Faculty 57 4 operationalizations based on countries of birth and nationalities (Schneider &
Heath, 2020, p. 549). A central question remains to what extent one overlooks relevant lines of differ
ence with too rigid categorizations based on simple observable facts (such as coun
try of birth). At the same time, however, focusing on the complexity of heterogeneity,
especially in the Swiss context, can obscure the bigger picture. This an make it
difficult to distinguish structural realities and mechanisms from the assessment of
individual preferences and choices. Another aspect of this is to return to the fit
between operationalization and theoretical explanation: perhaps the question should
not be who chooses to study to become a teacher, but who chooses not to. Further
studies thereby address the question of the extent to which “objective” or self-
assessed factors are relevant in determining who becomes a teacher, or whether
there is also the question of who, why, and what categorizations of “otherness” are
made and whether this may already be discouraging school leavers from becoming
teachers. Certainly, studies that start much earlier in the career choice process could
provide more clarity here. In the context of recognition and belonging, this is imme
diately followed by the question of who drops out of teacher training or leaves the
teaching profession prematurely and why. This could provide valuable clues as to
which aspects of categorization can help explain the low representation of minority
teachers in Switzerland. ourdieu, P. (1982). Die feinen Unterschiede. Kritik der gesellschaftlichen Urteilskraft. Suhrkamp. References Barth, F. (1969). Introduction. In F. Barth (Ed.), Ethnic groups and boundaries: The social organi
zation of culture difference (pp. 9–38). Allen & Unwin. Beck, M. (2015). Bildungserfolg von Migranten: der Beitrag von Rational-Choice-Theorien bei de
Erklärung von migrationsbedingten Bildungsungleichheiten in Bern und Zürich. Haupt Verlag Beck, M., & Edelmann, D. (2016). Migrationshintergrund und Gender: Eine Überprüfung der
statistischen Konstruktion von Differenz am Beispiel der Lehrerinnen- und Lehrerbildung in
der Schweiz. In I. Kriesi, B. Liebig, I. Horwarth, & B. Riegraf (Eds.), Gender und Migration
an Universitäten, Fachhochschulen und in der höheren Berufsbildung (Forum Frauen- und
Geschlechterforschung, Band 46). (pp. 168–192). Westfälisches Dampfboot. Beck, M., Bischoff, S., & Edelmann, D. (2014). Migrationsbedingte und soziale Diversität
von Studierenden der Pädagogischen Hochschule St. Gallen. Pädagogische Hochschule St. Gallen (PHSG). Bischoff, S., & Edelmann, D. (2017). «Ich habe zwar den Schweizerpass, bin aber türkisch, und
dann kommt halt diese Religionsfrage…» – Einblicke in das qualitative Forschungsprojekt
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Weber, M. (1921–1922). Wirtschaft und Gesellschaft. J.C.B. Mohr (Paul Siebeck). Wimmer, A. (2013). Ethnic boundary making. Oxford University Press. Witzel, A. (2000). Das problemzentrierte Interview. Forum Qualitative Sozialforschung, 1(1), 8. (
)
p
Q
f
g
( )
Wolter, S. C., Denzler, S., & Weber, B. a. (2003). Betrachtungen zum Arbeitsmarkt der Lehrer
in der Schweiz. Vierteljahrshefte zur Wirtschaftsforschung, 72(2), 305–319. https://doi. org/10.3790/vjh.72.2.305 Wolter, S. C., Diem, A., & Messer, D. (2014). Drop-outs from Swiss universities: An empirical
analysis of data on all students between 1975 and 2008. European Journal of Education, 49(4),
471–483. https://doi.org/10.1111/ejed.12096 M. Beck et al. M. Beck et al. 60 Michael Beck is a research fellow and lecturer at St. Gallen University of Teacher Education,
Switzerland. He studied social sciences with a focus on sociology and research methodology in
Manheim, Germany, and received his Ph.D. in Educational Sociology at the University of Bern,
Switzerland. His fields of interest are social mobility, educational inequality, research in teacher
education and assessment of student competencies. Carola Mantel is head of the Institute for International Cooperation in Education at the University
of Teacher Education Zug, Switzerland. She studied Social Anthropology, International Law and
Religious Studies at the University of Zurich. From the same University, she received her PhD on
her research about minority teachers. Her research interests include social justice in and through
education, recognition, social boundary making, migration-related diversity, professional develop
ment of teachers, learning through study abroad and South-North cooperation. Sonja Bischoff is a researcher fellow and lecturer at the St. Gallen University of Teacher
Education, Switzerland. She is a qualified primary school teacher and studied Social Anthropology
and Sociology at the University of Zurich. Sonja Bischoff received her Ph.D. in Social Anthropology
at the University of Bern, Switzerland. Her fields of interests are educational inequality, migration-
related diversity and qualitative research methodology. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give appropriate
credit to the original author(s) and the source, provide a link to the Creative Commons license and
indicate if changes were made. The images or other third party material in this chapter are included in the chapter’s Creative
Commons license, unless indicated otherwise in a credit line to the material. References If material is not
included in the chapter’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.
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P091: Timely administering prophylactic antibiotics
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Antimicrobial resistance and infection control
| 2,013
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cc-by
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P091: Timely administering prophylactic antibiotics
Y Yau1*, ICNT1, I Wong2
From 2nd International Conference on Prevention and Infection Control (ICPIC 2013)
Geneva, Switzerland. 25-28 June 2013 Y Yau1*, ICNT1, I Wong2 From 2nd International Conference on Prevention and Infection Control (ICPIC 2013)
Geneva, Switzerland. 25-28 June 2013 - Infection rate was 0.13% over these 4 years. Methods Published: 20 June 2013 Published: 20 June 2013 During the period 2009 to 2012, all anesthetic records of
orthopedic operation performed at the Duchess of Kent
Children’s Hospital at Sandy Bay were reviewed. Data on
time, administrator of prophylactic antibiotic, logistic of
antibiotics being issued, methods of safety guide for
administration and infection control rate were collected. doi:10.1186/2047-2994-2-S1-P91
Cite this article as: Yau et al.: P091: Timely administering prophylactic
antibiotics. Antimicrobial Resistance and Infection Control 2013 2(Suppl 1):P91. doi:10.1186/2047-2994-2-S1-P91
Cite this article as: Yau et al.: P091: Timely administering prophylactic
antibiotics. Antimicrobial Resistance and Infection Control 2013 2(Suppl 1):P91. Author details
1 1Nursing ICNT, Hong Kong, Hong Kong. 2Nursing CND, DKCH, Hong Kong,
Hong Kong. 1Nursing ICNT, Hong Kong, Hong Kong
Full list of author information is available at the end of the article Introduction It is well documented that prophylactic antibiotic could
reduce surgical site infection significantly providing that
it is given timely with right drug and right dose. How-
ever, administering prophylactic antibiotic in a timely
manner is not easy. Several factors such as low awareness
of importance of timing on prophylactic antibiotic given,
the workflow, ease of administration and perception of
individual responsibility toward the administration could
all contribute to the failure of given timely antibiotic to
reduce surgical infection. Objectives None declared. To investigate how these problems were tackled in an
elective surgical hospital in order to achieve timely
administering surgical prophylactic antibiotic. © 2013 Yau 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. Yau et al. Antimicrobial Resistance and Infection Control 2013, 2(Suppl 1):P91
http://www.aricjournal.com/content/2/S1/P91 Yau et al. Antimicrobial Resistance and Infection Control 2013, 2(Suppl 1):P91
http://www.aricjournal.com/content/2/S1/P91 Yau et al. Antimicrobial Resistance and Infection Control 2013, 2(Suppl 1):P91
http://www.aricjournal.com/content/2/S1/P91 Open Access Open Access Conclusion Clearly defined roles and responsibility in the process of
prophylactic antibiotic administration were essential. Safety check list helped to enforce the guidelines. Monitor-
ing the outcome alerted stakeholders to take necessary
action. Successful transfer evidence-based guidelines into
daily practice required multiple interventions and support
from top management to frontline staff were vital. Results - Total 6061 cases were reviewed. - 99.9% of the prophylactic antibiotics were given
within 15-45 minutes interval before operation. - 99.9% of the prophylactic antibiotics were given
within 15-45 minutes interval before operation. - 100% cases went through the safety check list by
nurse. - 100% cases went through the safety check list by
nurse. - Anesthesiologist administered all the antibiotics
while surgeon prescribed all prophylactic antibiotics. - All prophylactic antibiotics were given in operating
theater except Vancomycin. - However, 99% antibiotics went through their regular
route to OT. 1Nursing ICNT, Hong Kong, Hong Kong
Full list of author information is available at the end of the article © 2013 Yau et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
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The impact of the COVID-19 pandemic on mental health and well-being of people living with a long-term physical health condition: a qualitative study
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BMC public health
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Fisher et al. BMC Public Health (2021) 21:1801
https://doi.org/10.1186/s12889-021-11751-3 Fisher et al. BMC Public Health (2021) 21:1801
https://doi.org/10.1186/s12889-021-11751-3 Open Access The impact of the COVID-19 pandemic on
mental health and well-being of people
living with a long-term physical health
condition: a qualitative study A. Fisher*†
, A. Roberts†, A. R. McKinlay, D. Fancourt and A. Burton Abstract Background: The COVID-19 pandemic and associated restrictions caused major global disruption. Individuals with
long-term physical health conditions (LTCs) are at higher risk of severe illness and often subject to the strictest
pandemic guidance, so may be disproportionally affected. The aim of this study was to qualitatively explore how
living with a LTC during the COVID-19 pandemic affected people’s mental health and wellbeing. Methods: Participants were people living with LTCs who participated in telephone/video call interviews based on a
semi-structured topic guide. Key themes and subthemes were determined using deductive and inductive thematic
analysis. Results: The sample included 32 participants with LTCs (most commonly cancer, respiratory conditions or
cardiovascular diseases), mean age 57 (SD 13) years, 66% female and 72% white British. There were four overarching
themes specific to living with a LTC. These were 1) high levels of fear and anxiety related to perceived
consequences of catching COVID-19, 2) impact of shielding/isolation on mental health and wellbeing, 3) experience
of healthcare during the pandemic and 4) anxiety created by uncertainty about the future. Fourteen subthemes
were identified, including concerns about accessing essential supplies and the importance of social support. Individuals who lived alone and were advised to shield could be profoundly negatively affected. Conclusions: This study found that there were a number of aspects of living with a LTC during the pandemic that
had a significant impact on mental health and well-being. There should be focus on how best to provide practical
and social support to people with LTCs during a pandemic, particularly if they have to shield or isolate. Keywords: Chronic disease, Pandemics, Mental health, Interview, Social isolation * Correspondence: abigail.fisher@ucl.ac.uk
Fisher A and Roberts A are joint first author. †A. Fisher and A. Roberts contributed equally to this work. Department of Behavioural Science and Health, University College London,
1-19 Torrington Place, London WC1E 7HB, UK © The Author(s). 2021 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. * Correspondence: abigail.fisher@ucl.ac.uk
Fisher A and Roberts A are joint first author.
†A. Fisher and A. Roberts contributed equally to this work.
Department of Behavioural Science and Health, University College London,
1-19 Torrington Place, London WC1E 7HB, UK Background g
The COVID-19 pandemic caused global disruption, with
nearly every country introducing social distancing mea-
sures. Individuals with long-term physical health condi-
tions (LTCs) like diabetes, hypertension, and respiratory
conditions are particularly susceptible to severe illness if
infected by the virus that causes COVID-19 (Sars-Cov-2)
[1–3] and therefore were often subject to the strictest re-
strictions. Indeed, if considered extremely clinically vul-
nerable, people living with LTCs in the UK were advised
to ‘shield’ at the beginning of the pandemic (not leaving
their home/garden and avoiding social contact) [4]. In
addition, diversion of healthcare resources, more limited
access to routine care and disruption to medical supplies
during pandemics and other health emergencies can dis-
proportionately affect people living with LTCs [5–7]. High levels of stress and negative changes to diet or
physical activity (likely intensified by the enhanced pan-
demic restrictions among those who have to shield/iso-
late) can also exacerbate some LTCs [7]. These factors
can lead to widening of health inequalities between
those with and without LTCs during and after a pan-
demic. Understanding the impact of the challenges faced
by people living with LTCs during the pandemic is
therefore crucial in determining who has been negatively
affected, what support they may need during and beyond
the pandemic, and how they could be supported should
future pandemics occur. Quantitative surveys suggest that the impact of lock-
down and shielding had negative effects on mental
health and well-being for some, but not all, people with
LTCs. An Office for National Statistics (ONS) report es-
timated that that 35% (of 2.2 million people) living in
the UK experienced poorer mental health as a result of
shielding [8]. The COVID Social Study, a longitudinal
survey of 70,000 respondents, found that those with
LTCs were more adherent to UK Government lockdown
guidelines than those without, but reported higher levels
of stress, depression and anxiety throughout [9]. Other
longitudinal surveys also suggest that the pandemic re-
strictions had a negative impact on mental health and
well-being in individuals with LTCs, although some
showed no difference compared to the general popula-
tion [10–12]. However, long-term physical and mental
health problems often co-occur (e.g. [13]), so the extent
to which these findings were a result of the pandemic
and associated restrictions is unclear. © The Author(s). 2021 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. Fisher et al. BMC Public Health (2021) 21:1801 Page 2 of 12 Page 2 of 12 Fisher et al. BMC Public Health (2021) 21:1801 infectious disease emergencies, like the spread of Ebola
[15]. However, at the time of writing, there was limited
qualitative data from individuals with LTCs collected
during the COVID-19 pandemic. In an early-phase sur-
vey of 7039 individuals with long-term respiratory con-
ditions, qualitative analysis of free text suggested that
perceived vulnerability to infection, limited access to
healthcare and necessities, uncertainty about the future
and inadequacy of the UK Government response were
having extremely negative impacts on mental health
[16]. Free text responses are valuable, but limited by the
inability to probe. 41 participants living with LTCs in
India were interviewed about their experiences of the
pandemic and most discussed stress and anxiety related
to adverse economic consequences, as well as difficulties
accessing healthcare [17]. A qualitative interview study
of 30 young adults with Type 1 diabetes in India found
that participants had low awareness of their elevated risk
of severe illness from COVID-19 and low awareness of
COVID-19 symptoms or preventative measures [18]. Further, in a qualitative study of the impact of COVID-
related delays in 31 adults awaiting kidney transplant-
ation in Australia, participants described disappoint-
ment/devastation,
concerns
about
vulnerability,
additional burdens (like financial) and high levels of
stress created by uncertainty [19]. Background Indeed, limited in-
depth research has been undertaken to understand how
individuals with LTCs experience a pandemic and what
unique challenges they may face. However, these stud-
ies focussed on specific health conditions and/or coun-
tries where economic and healthcare impacts could be
different to the UK. Therefore the aim of this study was
to qualitatively explore how living with a LTC during
the COVID-19 pandemic in the UK affected people’s
mental health and wellbeing. Data analysis y
Interviews were audio recorded and transcribed by a
professional transcription service with a UCL data shar-
ing agreement. Transcripts were de-identified by remov-
ing names and locations and checked for accuracy
before being transferred to NVivo 12 for analysis. The-
matic analysis was conducted following the steps out-
lined by Braun and Clarke [24]. AR undertook line-by-
line coding of 23 transcripts during ongoing data collec-
tion using an initial deductive coding framework based
on the interview schedule. AM and AR double-coded
three
transcripts
and
compared
codes
to
ensure
consistency of approach. AR continued coding tran-
scripts using a modified coding framework based on
content described by participants as coding progressed
(inductive approach) until no new themes were gener-
ated. Results were presented to a group of qualitative
COVID-19 Social Study researchers, who provided feed-
back and comments on the analysis over several weeks. AM applied the coding framework to the final 9 tran-
scripts then AF, AM and AB reviewed, defined and
named the themes, selected the quotes and produced
the report. Themes and sub-themes are presented with
illustrative quotes, with participant sex, age range (in
years) and brief description of LTC. reported in full in Table 2, but most commonly reported
were cancer (n = 12/38%), respiratory conditions (n = 10/
31%) and cardiovascular diseases (CVD) (n = 9/28%). Six participants (19%) reported a co-occurring mental
health condition. Interviews were between 31 and 150
(mean 65) minutes long. g
Themes and subthemes are summarised in Table 3. Themes relating to living with a LTC were 1) high levels
of fear and anxiety related to perceived outcomes of
catching COVID-19, 2) major impact of shielding/isola-
tion on mental health and wellbeing, 3) experience of
healthcare during the pandemic and 4) anxiety created
by uncertainty about the future. Other more general
themes about the impact of the pandemic, that were
common across the COVID-19 Social Study groups, are Methods Interviewers had no prior relationship with
the
participants
they
interviewed. The
interviews
followed a topic guide which was developed for this
study using existing theories on behaviour change [21],
social networks and health [22] and stress, health and
coping [23]. Questions were designed to explore how
the COVID-19 pandemic has affected mental health and
wellbeing, social lives, and social behaviours. The topic
guide is presented as Supplementary Material. presented in line with the Consolidated Criteria for
Reporting Qualitative Research (COREQ) checklist [20]. Participants completed a pre-interview demographic
questionnaire reporting age, gender, ethnicity, education,
marital status and details of LTCs. The question about
gender was an open response, but participants reported
identifying as male or female so it is herein referred to
as ‘sex’. LTCs were coded into 12 categories for descrip-
tive purposes. Interviews were conducted by four female
Health
Psychology/Social
Science
postdoctoral
re-
searchers experienced in qualitative interviewing (AR,
AM, RC, SE). Interviewers had no prior relationship with
the
participants
they
interviewed. The
interviews
followed a topic guide which was developed for this
study using existing theories on behaviour change [21],
social networks and health [22] and stress, health and
coping [23]. Questions were designed to explore how
the COVID-19 pandemic has affected mental health and
wellbeing, social lives, and social behaviours. The topic
guide is presented as Supplementary Material. Methods This study was part of a wider investigation of the psy-
chological and social impacts on different groups of
people living in the UK during the COVID-19 pandemic:
the COVID-19 Social Study www.covidsocialstudy.org. One-to one semi-structured qualitative interviews were
conducted via video or telephone call to explore the im-
pact of living with a long-term physical health condition
during the pandemic. For inclusion, participants had to
be adults (> 18 years), be fluent in spoken English and
have been diagnosed with any condition they perceived
to be a long-term physical health condition. Participants
were recruited using targeted advertisements via social
media, the COVID-19 Social Study website and newslet-
ter (3919 subscribers) and purposively sampled to try
and capture different age groups, genders, ethnic back-
grounds and types of LTCs. Ethical approval was pro-
vided by UCL Ethics Committee (Project ID 14895/005)
and all participants provided informed consent to par-
ticipate prior to interview. Methods and results are Qualitative research can elucidate such experiences
and help untangle seemingly discrepant findings [14]. Qualitative methodologies have also proved invaluable in
understanding the mental health impacts during other Fisher et al. BMC Public Health (2021) 21:1801 Page 3 of 12 Table 1 Participant characteristics
Demographics
Age (years)
Range
32–75
Mean (SD)
57 (13)
Sex n (%)
Female
21 (66)
Male
11 (34)
Ethnicity, n (%)
White British
23 (72)
White - Other
3 (9)
Indian
2 (6)
Mixed ethnic groups
2 (6)
Black - Other
2 (6)
Marital status, n (%)
Married/Civil Partnership
16 (50)
Divorced/separated
7 (22)
Single (never married)
8 (25)
Widowed
1 (3)
Education, n (%)
GSCE (or equivalent)
3 (9)
A-levels (or equivalent)
5 (16)
Undergraduate degree
12 (38)
Postgraduate degree
12 (38)
Employment, n (%)
Employed
9 (28)
Unemployed
19 (60)
Furloughed
3 (9)
Studying
1 (3)
Living situation, n (%)
Live alone
9 (28)
Live with others
23 (72) presented in line with the Consolidated Criteria for
Reporting Qualitative Research (COREQ) checklist [20]. Participants completed a pre-interview demographic
questionnaire reporting age, gender, ethnicity, education,
marital status and details of LTCs. The question about
gender was an open response, but participants reported
identifying as male or female so it is herein referred to
as ‘sex’. LTCs were coded into 12 categories for descrip-
tive purposes. Interviews were conducted by four female
Health
Psychology/Social
Science
postdoctoral
re-
searchers experienced in qualitative interviewing (AR,
AM, RC, SE). Fear of hospitalisation, ventilation and death Other participants described the specific risks that
COVID-19 infection could lead to, including long-term
health problems, risk of hospital admission, ventilation,
and dying, and acknowledged the negative impact of this
on mental health: the subject of seperate papers, for example Burton et al.,
2021 [25]. High levels of fear and anxiety related to perceived
consequences of COVID-19 infection
Heightened awareness of risk due to LTC
Participants described fear and anxiety caused by aware-
ness of the potential implications of catching COVID-
19. Some described a heightened sense of risk, coupled High levels of fear and anxiety related to perceived
consequences of COVID-19 infection
Heightened awareness of risk due to LTC “I don’t want to catch it, [I have] already experienced
being on a ventilator before for 17 days and I never want
that experience again…” (female, 60–69, cancer, CVD). Participants described fear and anxiety caused by aware-
ness of the potential implications of catching COVID-
19. Some described a heightened sense of risk, coupled “I’m terrified of getting this virus, because I know that
if I get it, it probably is the end of me. My lungs are not
good … I don’t want to die in hospital and I don’t want
to have that intubation and sedation” (female, 70–79, re-
spiratory condition). Table 3 Impact of COVID-19 pandemic on people with LTCs
Summary of themes (and subthemes) “I’m really scared of getting [COVID-19] … I think I
probably would not survive if I got it, so I’m trying to
keep away from it … when I heard about [COVID-19], I
just automatically went, oh my God, I’m going to die. It
wasn’t great” (female, 30–39, CVD and respiratory
condition). Heightened awareness of risk due to LTC Heightened awareness of risk due to LTC
Fear of hospitalisation, ventilation and death Impact of shielding/isolation on mental health and
wellbeing The COVID-19 restrictions imposed on the participants’
lives led to a number of negative consequences for men-
tal health. Many were in the extremely clinically vulner-
able group and therefore advised to shield, or had made
the decision to isolate. This led to specific concerns, as
well as discussion of factors that alleviated these worries. Results 32 people with LTCs took part and their characteristics
are presented in Table 1. Individuals were aged 32–75
years and the majority identified as female (n = 21/66%)
and white British (n = 23/72%), but a wide range of
demographic
groups
were
represented. LTCs
are Fisher et al. BMC Public Health (2021) 21:1801 Page 4 of 12 Page 4 of 12 Table 2 Long-term health conditions (LTCs)
Health conditions, n (%)a
Respiratory (e.g. asthma, COPD)
10 (31)
Cardiovascular (e.g. high blood pressure, heart disease)
9 (28)
Musculoskeletal (e.g. osteoporosis, osteoarthritis)
8 (25)
Cancer (in remission)
7 (22)
Current cancer diagnosis (incl. Advanced/incurable cancer)
5 (16)
Diabetes
5 (16)
Autoimmune (e.g. rheumatoid arthritis)
5 (16)
Neurological (e.g. Parkinson’s Disease, Multiple Sclerosis)
4 (13)
Renal (e.g. kidney condition, interstitial cystitis)
2 (6)
Inflammatory Bowel Disease (e.g. Crohn’s)
2 (6)
Organ removal/transplant (e.g. spleen removed, liver transplant)
2 (6)
Comorbid mental health condition (e.g. anxiety, depression,
schizophrenia)
6 (19)
a percentages do not equal 100% due to comorbidity among participants Table 2 Long-term health conditions (LTCs)
a with
an
understanding
of
the
need
to
protect
themselves: “There’s been some degree of added stress, I suppose,
because it became relatively clear, relatively early, that
[COVID-19’s] something that really, given my circum-
stances, I should be careful not to catch.” (male, 30–39,
cancer). However, heightened awareness of risk did not always
lead to additional worry, with some participants discuss-
ing elements of chance or fate in the outcome: “My medical condition, I’ve had it all my life so if that
places me in a higher risk category, you’ve just got to do
your absolute best to not catch it and then after that it’s
sort of in the lap of the Gods really isn’t it? So it didn’t
really upset me or stress me or worry me” (female, 40–49,
respiratory condition). Concerns about access to essential supplies Participants reported issues around receiving the ne-
cessary shielding certification that would allow access to
(e.g.) priority status online supermarkets: “I didn’t get a letter [from the Government/National
Health Service (NHS)] for ages because they weren’t able
to identify me and the therapy from that essential health
data. So, yes it was a bloody nightmare to be honest” (fe-
male, 40–49, cancer). “[my son] was living at home … he left during the lock-
down because I had the letter from the NHS saying I was
vulnerable. At the beginning, his girlfriend came and
then she went. And he wanted her to come back and I
had to say, no … so he left … It was awful … I felt abso-
lutely devastated … The other [son] had gone a few
months before he’d gone. I was left on my own, my part-
ner had left me [last year] after 22 years … I blamed my-
self. I didn’t know. I was very down, very depressed”
(female, 50–59, cancer, respiratory condition). Those who had Government certification/other shield-
ing arrangements (like social and neighbourhood net-
works) in place acknowledged the positive impact of this
on their mental health: “I registered very early with the supermarket vulnerable
list, I think, that the government was sharing with them. And I think because I got my shielded letter, very early
on I got an email from [supermarket] saying, you’re on
the vulnerable list and we’ll prioritise slots. So we’ve been
very lucky that we actually managed to get slots when we
need” (female, 30–39, autoimmune condition). Some participants also reported feeling aggravated by
friends and family who they believed were not following
the social distancing rules relevant to them and this put-
ting strain on relationships: Others spoke about how they stockpiled supplies to
reduce anxiety: “I actually made sure that I had an extra stock [of
medication]. I put an extra repeat prescription in before
the lockdown and I’ve just put another one in..” (female,
40–49, cancer). “we’ve got friends who are in their 70s, and they’re so
gung-ho about the whole thing. We’ve had arguments
with them to get them to stay in, because they’re all
really social … we had a hell of a job persuading them
not to do that because of what’s happened, because it’s
all just dissolved into a shambles” (male, 60–69, neuro-
logical condition). Concerns about access to essential supplies Participants discussed the ability to access essential sup-
plies (e.g. food, medical supplies) as having strong influ-
ences on mental health. Those who found it difficult to
access food and medical supplies were fearful and
anxious: Page 5 of 12 Fisher et al. BMC Public Health (2021) 21:1801 Fisher et al. BMC Public Health (2021) 21:1801 “they ended up cancelling my vulnerable status on their
[online shopping] system, cancelling my delivery slot. … .. And now the next delivery slot is three weeks away. So,
what am I meant to do? How the hell am I going to cope
now? I haven’t got three weeks’ worth of food” (male, 30–
39, cancer, respiratory condition, low immunity). “Most of the time [the family are] in the house …
Everybody’s on the same page … Maybe if I hadn’t had
gone through everything I went through last year and
when kids see you poorly and what have you, and it’s
cancer, so it’s the C word, isn’t it? It’s made everybody
think on the same wavelength … it’s had its moments …
But as a family, we’ve done well with it to be honest” (fe-
male, 50–59, cancer). “… the pharmacy’s not providing any service of delivery
to vulnerable people, and the GP’s are arguing with me,
to say, why are you ordering medication a week before
it’s due date? Or whatever, but I can’t order one item at
a time, who’s going to go and pick it up, for a start? And
when it runs out, it’s going to run out, and I’m not going
to have anything to replace it…” (female, 50–59, multiple
conditions). However, some described challenges that arose from
having to justify their need to shield. This led to worries
about offending others, arguments, and had an ex-
tremely detrimental impact on mental health: “Without sounding harsh, my sister just didn’t get this
idea of me needing to isolate. So, she kept trying to come
up and see us. We were relying on her to bring us food
and things like that. But she just kept hanging around.. … I think that was probably one of the tougher ones be-
cause I think from her point of view, she just seemed to
think that I just wanted her to not be around, whereas
this is the advice I’d been given” (male, 30–39, cancer, re-
spiratory condition). Loss of independence and reliance on others Although social networks were important, participants
discussed the need to rely on others and loss of inde-
pendence as difficult: Concerns about access to essential supplies “I suppose I panicked a bit and I got a delivery thinking
I’d better be prepared. I do have a certain amount of food
in anyway because living alone, I’ve got to be prepared
for a bad cold or something … I thought oh, a couple of
months, I’d better order all the tinned stuff …” (female,
70–79, neurological and musculoskeletal conditions). Experience of healthcare during the pandemic a sense, I felt overly reliant on her … .. So yes, that was
difficult” (male, 40–49, blood condition). Experience of healthcare during the pandemic Experiences of the impact on healthcare and treatments
were commonly discussed, with both positive and nega-
tive aspects reported. ff
The difficulty in relying on others resulted in one par-
ticipant going out to shop despite guidance against it: “I don’t like relying on people. I hated to have to ring
people up. At the very beginning, lots of people were get-
ting in touch … Then it tailed off a little bit and I don’t
like ringing people and asking for help … I just felt really
guilty for it. I just thought, I won’t bother anybody, I’ll go
and do it myself” (female, 50–59, cancer, respiratory
condition). Move to remote interaction with health services mostly
positive Many participants described how their healthcare con-
sultations had changed to telephone/video appointments
during the pandemic, and this was often regarded as a
more convenient alternative: “there’s been a big push to change some things, like
phone clinics and video clinics … it’s been brilliant. And
my care is split over three different hospitals, so rather
than spend 45 minutes each way on the Tube to get into
my hospital, wait in a busy clinic and then get back …
It’s so much easier to just be able to phone...” (female,
30–39, bowel condition). Feeling safe versus feeling isolated Some participants reported that, despite missing friends
and family, the enhanced feeling of safety and security
through lockdown was reassuring and protective for
mental health and a worthwhile trade-off: “we’re in lockdown, it’s not great, but … I think the
sense of relief kind of outweighed any frustrations really
… … I’m just happy to proceed like this until we see
what’s
going
to
happen
…”
(female,
30–39,
bowel
condition). “I have a routine discussion with the oncology unit at
the hospital, which has been by telephone ever since I
moved down here. I’ve had one face-to-face meeting with
the oncologist and he said, well you have to have tele-
phone routine checks, and I said that’s fine with me, I
don’t have to drive over, park the car, hang around for
an hour or two. So this actually works better …” (male,
70–79, cancer). However, others found not seeing friends and family
one of the most challenging aspects to deal with: “I’m desperately worried about [my mother-in-law]. She’s got serious Alzheimer’s. She’s alone, she’s not seen
any of her family for months and she’s just sitting in a
room. She’s starving to death because she’s not eating
any more. She’s going to die at some point probably in
the next six weeks at this rate. And it’s possible that we’re
not going to be able to see her. It’s horrendous” (male,
40–49, autoimmune condition, CVD). Although one participant felt it wasn’t as satisfactory
and another talked about missing the reassurance nor-
mally gained from a physical assessment and having
concerns that it was not as medically rigorous: “my clinic appointments have been over the phone. The
bit that I’ve missed out on is somebody physically check-
ing my lymph nodes …” (female, 40–49, cancer). Some participants who were shielding and living alone
suffered particularly with social isolation. Two disclosed
that the extent of the social isolation (in combination
with other factors related to the pandemic including loss
of work through furlough/redundancy and worries about
management and treatment of their LTC) led them to
contemplate/attempt suicide: Immediate social network strongly influenced experience of
shielding “I was especially jealous of my girlfriend, who didn’t
have to shield. She made sure that before she starts work
in the morning, she goes for a walk for at least 45 min to
an hour … She also had to do the grocery shopping. And
there was once or twice during lockdown that I needed
my prescriptions to be refilled. She had to do that. So, in In addition to the impact of social networks in support-
ing shielding, participants described polarised experi-
ences of whether they felt supported by friends and
family members to shield. Those who felt supported
described the positive impact: Fisher et al. BMC Public Health (2021) 21:1801 Page 6 of 12 a sense, I felt overly reliant on her … .. So yes, that was
difficult” (male, 40–49, blood condition). The difficulty in relying on others resulted in one par-
ticipant going out to shop despite guidance against it:
“I don’t like relying on people. I hated to have to ring
people up. At the very beginning, lots of people were get-
ting in touch … Then it tailed off a little bit and I don’t
like ringing people and asking for help … I just felt really
guilty for it. I just thought, I won’t bother anybody, I’ll go
and do it myself” (female, 50–59, cancer, respiratory
condition). a sense, I felt overly reliant on her … .. So yes, that was
difficult” (male, 40–49, blood condition). Mixed views of attending healthcare settings in person Participants described varying emotions regarding at-
tending healthcare services during lockdown. Some felt
that the precautions taken by healthcare professionals
(wearing
personal
protective
equipment,
promoting
hand-washing, triaging appointments) alleviated anxiety,
and they felt safe and reassured: “because you’re stuck at home and if you don’t talk on-
line to somebody or over the phone to somebody then
you’re on your own and you’re just going to go downhill. There’s that fear that I could get back to being suicidal
… I’ve had periods where I have felt very low and strug-
gling and very alone, and I think living on your own is
just shit. People that are part of a couple or a family and
whatever have got other people around” (female, 50–59,
neurological condition). “I felt okay about [going for a blood test], I know the
GP surgery quite well and they’re all very friendly and
competent as far as I can see … if there had been about
20 people in the waiting room, I wouldn’t have been too
happy about that, but they’re actually managing the flow
of people extremely well....” (male, 70–79, cancer). Others described feeling anxious initially, however for
those attending regularly, this anxiety diminished: Others discussed loss of physical contact as particu-
larly difficult: “The first time I went, I don’t know because the nurses
have not been tested. And I thought I’m shielding at
home and yet I’m coming here and you’re giving me
treatment intravenously and you’ve not been tested and “there’s the desire to hug and shake hands, just human
contact sometimes … I miss a little bit of human contact
obviously” (male, 50–59, CVD). Page 7 of 12 Fisher et al. BMC Public Health (2021) 21:1801 Page 7 of 12 that didn’t sit right with me … But now I’m fine, I go
every three weeks... It’s fine” (female, 50–59, cancer). that didn’t sit right with me … But now I’m fine, I go
every three weeks... It’s fine” (female, 50–59, cancer). that didn’t sit right with me … But now I’m fine, I go
every three weeks... It’s fine” (female, 50–59, cancer). important at the moment … but I can understand why”
(male, 60–69, neurological condition). Mixed views of attending healthcare settings in person Some felt extremely anxious about catching COVID-
19 during hospitalisation for their LTC, particularly if
accident and emergency (A&E) admission was required,
whilst others felt the risk of this was low: “I was supposed to have a surgical review with a view
to having surgery this year, but obviously that’s all
stopped. The review was cancelled. Surgery will not be
any time soon. It can wait, but it’s also something that’s
disappointing. But I do understand that there will be a
massive backlog now and there are many more urgent
things that need sorting” (female, 40–49, respiratory
condition). “In my head, if I went to A&E and went into the hot
side with Coronavirus, that’s just a death sentence”
(male, 30–39, multiple conditions). “I didn’t feel any anxiety about going into hospital be-
cause most hospitals are designated as red zones and
green zones. Certainly, all the nurses etc. wore masks,
gloves and all the rest of it. But I had excellent care and
I wasn’t at any point worried that I would actually catch
[COVID-19] in hospital” (female, 60–69, respiratory
condition). Threat of disruption to cancer treatments created stress and
anxiety Participants undergoing cancer treatment spoke about
the stress and anxiety caused by threat of disruption to
treatment. One participant was undergoing chemother-
apy with curative intent: Some participants described evaluating the perceived
seriousness of health problems they were experiencing
versus the potential risk of catching COVID-19 in decid-
ing whether to access healthcare services: “There was a bit of stress at some point because … [the
oncologist] was hinting, basically, that potentially they
would have to cut my treatment short to a degree … I
guess the consequence of that is that you increase the risk
of things coming back and all that, so it wasn’t ideal. But
15 days later, when I went back for my next cycle, that
was out of the window and things have improved in [the
hospital]” (male, 30–39, cancer). “the night I had the chest pain, it mentally went
through well, I’ll give it another 10 minutes and see. If it
hasn’t gone, I will go to hospital. So I wasn’t just going to
sit there and think I’m going to put up with chest pain
just because I might get COVID-19 if I go to hospital. I
was sensible enough to realise that really, the more press-
ing thing’s getting what seemed to be a heart attack
sorted than worrying about a theoretical risk of getting
[COVID-19]” (female, 60–69, respiratory condition). Another was receiving treatment for incurable cancer
that was intended to limit cancer progression and pro-
long her life, and was relieved that her treatment was
not affected: f
p
y
However, others reported real reluctance to engage
with healthcare services. One described not attending an
appointment at the hospital because of their concerns
about the risk of contracting COVID-19, and another
delayed help seeking for suspected cancer recurrence:
“I had possible signs of a resurgence of the cancer. I
had a lump come up on my neck. And we were already
in lockdown at that point … And so I put it off, of course,
as we usually do, and seeing if it would go away. But two
weeks later it was still there … And it was clear I had to
do something because I was tearing myself apart with
panic...” (female, 70–79, cancer in remission). However, others reported real reluctance to engage
with healthcare services. Anxiety created by uncertainty about the future Whilst a number of participants recognised that the ex-
perience of living with a LTC may have somewhat pre-
pared them to cope with uncertainty, they still described
a number of issues specific to the pandemic that were
challenging for mental health: “[uncertainty’s] something that I wouldn’t say I’m an
expert in dealing with, but I’m very experienced in deal-
ing with it. But it doesn’t make it any easier. But it seems
to be something I’ve had to deal with, at least on the
back burner, all my adult life” (male, 50–59, diabetes). Postponement of non-essential treatments Some participants had non-essential treatments or ap-
pointments postponed. Some were in agreement with
this decision, while others experienced disappointment
(despite understanding the necessity of it): Threat of disruption to cancer treatments created stress and
anxiety One described not attending an
appointment at the hospital because of their concerns
about the risk of contracting COVID-19, and another
delayed help seeking for suspected cancer recurrence: “my treatment’s carried on, they were talking about
cancelling it, but they didn’t … I was glad, because you
never know. It’s under control at the minute, but I know
things can change, so I was glad mine carried on.”
(female, 50–59, cancer). “I had possible signs of a resurgence of the cancer. I
had a lump come up on my neck. And we were already
in lockdown at that point … And so I put it off, of course,
as we usually do, and seeing if it would go away. But two
weeks later it was still there … And it was clear I had to
do something because I was tearing myself apart with
panic...” (female, 70–79, cancer in remission). Impact of pandemic on treatment access with progression “I should have seen my neurologist in January … but
that got cancelled. I contacted [the hospital] and got an
answer phone, then I was told that the neurologist would
be getting in touch and she never was …. A bit disap-
pointed and I do feel like I think what I’ve got is a pretty
serious condition, but it’s obviously regarded as not that Not having an end in sight Despite some reporting they would electively remain in
isolation after restrictions were relaxed, several partici-
pants mentioned that the uncertainty around how long
the pandemic would last was particularly difficult: “healthcare is my main priority, that really worries me,
that I’m not going to get the same level of treatment as I
was getting before, because there won’t be sufficient
money around, and a lot of services will be cut” (female,
50–59, cancer, autoimmune and respiratory conditions). “how long’s the virus going to be floating around for? How long have we got to take these precautions?...I can’t
anticipate whether we’re talking weeks, months or a
couple of years and the long-term effects are going to be
floating around. The anxiety I’m sure will lessen but I
think it’s going to be there for a good while” (female, 50–
59, neurological condition). This worry seemed less pronounced in those with can-
cer, who emphasised that their main worries about the
future were about the cancer, and the pandemic had not
changed that: Some described a sense of loss or grief in not knowing
when (or if) social lives would return to normal: “[cancer’s] always a worry, really. I think I’m very
much of the opinion that it will come back at some point,
and that’s kind of what the numbers show. I’m not par-
ticularly emotionally worried about that … that’s not
changed
because
of
this
situation”
(female,
40–49,
cancer). “It’s fine for the moment, but, obviously if I think I’m
never going to see the Royal Ballet again, I can get quite
tearful. And it’s things like dancing, we do dance quite a
lot … obviously we can dance together, but it’s not the
same as going dancing, so yes. It’s a grieving for how
quickly those things come back” (female, 50–59, cancer). One participant, who was currently receiving cancer
treatment, described how they tried not to look beyond
completion of this: “it’s almost like a sense of mourning for the life that
we’ve lost. Because it’s hard to see how it can ever go
back to being quite what it was before … that grieves me
a bit” (female, 70–79, cancer in remission). Not having an end in sight “if I’ve got any worries about the future, in the ranking,
cancer comes a clear first and the pandemic is some way
behind that … I’ll need to process what it means to the
future of me, in the future, once the treatment is gone”
(male, 30–39, cancer). Many participants also expressed that they felt that
normal life would not resume until a vaccine had been
found and placed their hopes for a return to normality
on a successful vaccine: “…. This has crippled the world and will continue to
do so until a vaccine is found. That’s quite a daunting
prospect
on
normality”
(female,
40–49,
respiratory
condition). “…. This has crippled the world and will continue to
do so until a vaccine is found. That’s quite a daunting
prospect
on
normality”
(female,
40–49,
respiratory
condition). p
of LTC For some participants, the pandemic exacerbated their
worries about future treatment, progression of their
LTC, and ability to access healthcare in the future if
their health deteriorated: Page 8 of 12 Fisher et al. BMC Public Health (2021) 21:1801 Fisher et al. BMC Public Health (2021) 21:1801 where I sit here. I’m not going to be part of the second
wave” (male, 30–39, cancer). “there is the underlying anxiety, as well, about how will
I be treated in the future?...The healthcare worry is obvi-
ously a lot more intense, because if I need to go back into
treatment, I will have to be isolated to a much greater
degree, and will be much more dangerous … it’s always
been there, but [now] it’s an increased anxiety” (female,
50–59, cancer). Fear of restrictions being relaxed and plans to continue
isolating Many participants discussed anxiety around relaxation
of guidelines and plans to continue shielding or isolating
even if/when restrictions were lifted: “to me, the only thing that will stop this is a vaccine …
that’s the real hope” (male, 50–59, CVD). “Even now shops are opening and stuff. I’m too anxious
to go out and about …. I’ll just keep monitoring what’s
going on and make my own decision as to when I feel it’s
safe for me to get back into the big world” (female, 50–
59, neurological condition). “I think the uncertainty of it and just not being sure
how it’s going to end. And when they also say things like
oh, we may never get a vaccine, for f**k sake, and then
what? Do I never go out again? I think that’s it. Probably
the worst bit” (female, 30–39, CVD and respiratory
condition). “And I personally feel that it will take a while before I
would feel comfortable going out. So, regardless of what
the government says, I will be preferring my own guide-
lines” (female, 60–69, cancer in remission, CVD). Discussion This study found living with a LTC during the COVID-
19 pandemic had a major impact on multiple different
aspects of mental health and wellbeing. Whilst some ex-
periences of people living with LTCs mirrored those of
people without [26], this study identified a range of
themes with subthemes that were particular to living
with a LTC during a pandemic. Themes included anxiety
created by perceived consequences of catching COVID-
19, the impact of shielding/isolation, impact on health-
care and anxiety created by uncertainty about the future. Each of these findings has clear implications for the con-
tinued management of the COVID-19 pandemic but also
preparation for future epidemics, with these implications
discussed below. g
Social support to shield or isolate was also a factor in
influencing how detrimental the experience of shielding/
isolation was to mental health in our sample. In over 1
million people in the UK who felt able to shield, 74% re-
ported that regular contact from friends and family was
a main factor in enabling them to adhere [8]. For partici-
pants in our study, immediate social networks were the
most important social connections and this raises aware-
ness of how strongly immediate social support influences
the experience of people with LTCs. Acceptance, sup-
port and understanding for the needs of the person with
the LTC to shield/isolate supported coping, but partici-
pants also discussed challenges including their perceived
loss of independence and reliance on others. Loss of in-
dependence was related to worsening mental health in a
large longitudinal cohort prior to the pandemic [27] and
considering the negative impact of loss of independence
on mental and well-being is part of National Institute
for Health and Care Excellence (NICE) guidance for
working with older adults [28]. Considering how to bal-
ance the impact of loss of independence, whilst ensuring
people with LTCs get the support they need when
shielding/isolating is an important area for future research. Participants commonly reported that anxiety about
the perceived consequences of catching COVID-19
were having very negative impacts. This is in line
with a free text analysis of 7039 participants with re-
spiratory disease in the UK, a qualitative analysis of
patients awaiting kidney transplant in Australia, a
sample of people living with LTCs in India [16–18]
and a large quantitative survey [9]. Discussion Individuals with
certain LTCs are at elevated risk of worse outcomes
if they catch COVID-19, and awareness of this ap-
pears high. This highlights the importance of provid-
ing helpful information on strategies to reduce risk,
along with balanced and accurate messaging about
level of risk for people with LTCs during epidemics,
to help alleviate anxiety. In a rapid review of the psy-
chological impact of quarantine in previous infectious
disease outbreaks, lack of adequate information about
level of risk was a key psychological stressor (as was
fear of infection), so the authors recommend giving
as much reliable information as possible as a mitigat-
ing strategy [26]. g
g
p
Participants who were required to shield and lived
alone discussed how challenging it could be for their
mental health. It was extremely concerning that two
people reported having felt suicidal or attempting suicide
due to shielding/isolating without social support, in
combination with other concerns like loss of work and
worry about management of their LTC during the pan-
demic. This is broadly in line with a quantitative survey
of 6607 US adults that found that COVID-19 related
stress, social isolation and financial strain were related to
suicidal ideation and self-harm, although this study did
not focus on those with LTCs [29]. Future research
should explore these associations in people living with
LTCs, particularly in those who are extremely clinically
vulnerable and advised to shield. Healthcare profes-
sionals who have contact with people living with LTCs
during pandemics should feel able/encouraged to en-
quire about their mental health and wellbeing and feel
confident that there are appropriate referral pathways in
place where required. Information issued to inform
people they need to shield could also signpost to avail-
able support, for example to mental health charities that
support people in crisis. Building on this, it is also important that support is
available to ensure that any guidance introduced to re-
duce risk (like social distancing/isolating) are feasible
and tolerable. For example, participants reported not al-
ways being able to access essential supplies whilst shield-
ing, with concerns over this access playing a key role in
determining how negatively lockdown guidance (particu-
larly shielding/isolation) was experienced. This echoes
findings from previous studies, including the study of
participants with respiratory disease and a rapid review
of the psychological impact of quarantine [16, 26]. Acceptance most protective for mental health Despite the challenges of uncertainty, many participants
described that they had found trying to accept the situ-
ation as the most protective coping mechanism for their
mental health: However, participants undergoing active cancer treat-
ment felt less concerned about relaxation of the lock-
down, since their treatment meant they would have to
isolate anyway: “I guess it’s just a massive thing that’s outside anyone’s
control, so you just have to adapt. You have to be very
flexible and adapt to it” (male, 50–59, diabetes). “I’m not concerned so much, because … I’m going to be
isolating for longer than everybody else, so when the
country starts to reopen, I’m going to be here. And if
there’s a second wave, I’m going to witness that from “what’s the point of railing against it? All you’re doing
is making yourself upset. The situation is the situation … Page 9 of 12 Fisher et al. BMC Public Health (2021) 21:1801 Page 9 of 12 you’ve just got to accept that it will be what it will be
and you make the best of it” (female, 70–79, neurological
and musculoskeletal conditions). need to access essential supplies was the most common
reason for people breaking shielding [8]. Our partici-
pants reported practical factors, like timely receipt of
Government shielding status, as having a major impact
on whether they were eligible for priority schemes to re-
ceive deliveries of supplies, providing some clear targets
for support for the continuing COVID-19 pandemic and
future health emergencies. Strengths and limitations Many participants discussed how they
felt that a vaccine was their only chance of return to
normal life. The interviews in this study were conducted
before a COVID-19 vaccine was available, and it would
be pertinent to explore the impact on people who can
be protected by available vaccines, versus those who To the best of our knowledge, this was the first study to
explore the impact of the COVID-19 pandemic and as-
sociated restrictions in the UK on the mental health and
well-being of people with LTCs. In this paper we fo-
cussed on the themes that were specific to living with an
LTC. There were other more general themes discussed
so this study alone may not reflect the full experience of
living through the pandemic. However, since this work
was conducted in parallel with studies in (e.g.) older
adults [33], people with mental health conditions [34],
parents [35] and young people [36] and many others, the
more general overlapping themes will be presented in
future papers. p p
Our findings support large-scale quantitative surveys,
but build on these by providing valuable depth and con-
text, as well as factors that could potentially mitigate
some of the negative impacts. The strength of qualitative
research is that it can provide rich insights into people’s
lived experience, which is not attainable through quanti-
tative methods. Interviews were conducted via telephone
or video call, so could have missed some of the non-
verbal cues and ability to build rapport and trust be-
tween interviewer and participant provided by in-person
interviews [37]. However, pandemic restrictions meant
that face-to-face interviewing was not feasible, and the
length and content of interviews suggest that partici-
pants still felt able to have an authentic discussion. In
addition, it is also feasible that some people would be
willing to share more over the phone. The interviews
were conducted during a period when shielding was ad-
vised, and views might change as restrictions and guide-
lines change. However, we did capture anxieties about
the future and consideration of how participants would
feel when/if rules were relaxed. In addition, work con-
ducted on previous pandemics helps shape the under-
standing of, and response to, future pandemics, so
building this evidence base is important. Overall, the
current study adds to the literature suggesting that
people with LTCs can be very negatively impacted by
pandemics. Strengths and limitations Our findings support the recommendations
to mitigate the negative impact of pandemics on people
with LTCs outlined by Hartman-Boyce and Mahtani
(2020) [7] which outline the importance of collaboration
(e.g. establishing community partnerships) communica-
tion (providing support, information and emergency
points of contact for people with LTCs) and pandemic
continuity planning for providers to ensure access to
essential supplies. Finally, a consistent theme in qualitative studies con-
ducted in people with LTCs during the pandemic (in-
cluding ours) was uncertainty about the future having a
negative impact on mental health [16, 18]. Whilst people
without LTCs may share these concerns, the reasons
identified in our study were specifically related to having
LTCs (e.g. fear of their conditions progressing and treat-
ments not being available due to COVID-related pres-
sure on health services). Participants reported that the
most effective way they had found to mitigate the nega-
tive impact of the pandemic-related uncertainty on their
mental health was to develop acceptance. Acceptance
Commitment Therapy incorporates this central idea of
acceptance and has shown early promise in people with
LTCs [32] so could be worth exploring for pandemic-
related distress. Many participants discussed how they
felt that a vaccine was their only chance of return to
normal life. The interviews in this study were conducted
before a COVID-19 vaccine was available, and it would
be pertinent to explore the impact on people who can
be protected by available vaccines, versus those who
cannot. Discussion These
challenges led to some participants going out against ad-
vice to access essential supplies. Quantitative data from
the ONS Shielding Behavioural Survey suggest that the Page 10 of 12 Page 10 of 12 Fisher et al. BMC Public Health (2021) 21:1801 Fisher et al. BMC Public Health (2021) 21:1801 In line with the aforementioned free text study of indi-
viduals with respiratory conditions [16], participants in
the current study very commonly discussed impact on
healthcare as one of the most prominent factors affect-
ing
their
mental
health
and
wellbeing
during
the
COVID-19 pandemic. In our study there were encour-
aging positives; for most, the move to remote consulta-
tions
was
viewed
as
a
convenient
and
acceptable
alternative. Some of the factors that were causing fear
and anxiety appeared based on perceptions (i.e. fear of
potential disruption to cancer treatments, or fear of be-
ing admitted to COVID-19 zones in hospital) and did
not materialise in practice in our sample. Indeed those
who were initially fearful and then had to attend regular
appointments found their fears diminished. However,
there were occasions of participants reporting weighing
up presentation of potentially serious symptoms with
fear of attending healthcare. This is concerning as it
could have implications for the development of diseases
and burden on the healthcare service if such diseases are
diagnosed later. Our findings reflect quantitative data
showing decline in healthcare attendance during the
pandemic. For example, data from the UK NHS suggest
that A&E attendance was 29% lower during pandemic
restrictions than in the same month the previous year
[30]. The US Centre for Disease Control estimated that
41% of people with one underlying health condition, and
55% with two or more, were delaying or avoiding med-
ical care due the COVID-19 pandemic (compared to
30% without) [31]. Future research should explore how
people with LTCs can be reassured that it is safer to
attend healthcare settings than delay presenting symptoms. ll
h
l
d Strengths and limitations y
viduals with respiratory conditions [16], participants in
the current study very commonly discussed impact on
healthcare as one of the most prominent factors affect-
ing
their
mental
health
and
wellbeing
during
the
COVID-19 pandemic. In our study there were encour-
aging positives; for most, the move to remote consulta-
tions
was
viewed
as
a
convenient
and
acceptable
alternative. Some of the factors that were causing fear
and anxiety appeared based on perceptions (i.e. fear of
potential disruption to cancer treatments, or fear of be-
ing admitted to COVID-19 zones in hospital) and did
not materialise in practice in our sample. Indeed those
who were initially fearful and then had to attend regular
appointments found their fears diminished. However,
there were occasions of participants reporting weighing
up presentation of potentially serious symptoms with
fear of attending healthcare. This is concerning as it
could have implications for the development of diseases
and burden on the healthcare service if such diseases are
diagnosed later. Our findings reflect quantitative data
showing decline in healthcare attendance during the
pandemic. For example, data from the UK NHS suggest
that A&E attendance was 29% lower during pandemic
restrictions than in the same month the previous year
[30]. The US Centre for Disease Control estimated that
41% of people with one underlying health condition, and
55% with two or more, were delaying or avoiding med-
ical care due the COVID-19 pandemic (compared to
30% without) [31]. Future research should explore how
people with LTCs can be reassured that it is safer to
attend healthcare settings than delay presenting symptoms. Finally, a consistent theme in qualitative studies con-
ducted in people with LTCs during the pandemic (in-
cluding ours) was uncertainty about the future having a
negative impact on mental health [16, 18]. Whilst people
without LTCs may share these concerns, the reasons
identified in our study were specifically related to having
LTCs (e.g. fear of their conditions progressing and treat-
ments not being available due to COVID-related pres-
sure on health services). Participants reported that the
most effective way they had found to mitigate the nega-
tive impact of the pandemic-related uncertainty on their
mental health was to develop acceptance. Acceptance
Commitment Therapy incorporates this central idea of
acceptance and has shown early promise in people with
LTCs [32] so could be worth exploring for pandemic-
related distress. Funding This Covid-19 Social Study was funded by the Nuffield Foundation [WEL/FR-
000022583], but the views expressed are those of the authors and not neces-
sarily the Foundation. The study was also supported by the MARCH Mental
Health Network funded by the Cross-Disciplinary Mental Health Network Plus
initiative supported by UK Research and Innovation [ES/S002588/1], and by
the Wellcome Trust [221400/Z/20/Z]. DF was funded by the Wellcome Trust
[205407/Z/16/Z]. The funders had no final role in the study design; in the
collection, analysis and interpretation of data; in the writing of the report; or
in the decision to submit the paper for publication. All researchers listed as
authors are independent from the funders and all final decisions about the
research were taken by the investigators and were unrestricted. 10. Bargon CA, Batenburg MCT, van Stam L, van der Molen DR, van Dam IE, van
der Leij F, Bass IO, Ernset MF, Maarse W, Vermulst N et al: The impact of the
COVID-19 pandemic on quality of life, physical and psychosocial wellbeing
in breast cancer patients and survivors – a prospective, multicenter cohort
study MedRix preprint server 2020. 11. Pierce M, Hope H, Ford T, Hatch S, Hotopf M, John A, et al. Mental health
before and during the COVID-19 pandemic: a longitudinal probability
sample survey of the UK population. Lancet Psychiatry. 2020;7(10):883–92. https://doi.org/10.1016/S2215-0366(20)30308-4. 11. Pierce M, Hope H, Ford T, Hatch S, Hotopf M, John A, et al. Mental health
before and during the COVID-19 pandemic: a longitudinal probability
sample survey of the UK population. Lancet Psychiatry. 2020;7(10):883–92. https://doi.org/10.1016/S2215-0366(20)30308-4. 12. Alonzi S, La Torre A, Silverstein MW. The psychological impact of preexisting
mental and physical health conditions during the COVID-19 pandemic. Psychol Trauma. 2020;12(S1):S236–8. https://doi.org/10.1037/tra0000840. Availability of data and materials The datasets generated and/or analysed during the current study are not
publicly available because they consist of transcripts from audio-recorded in-
terviews where participants may discuss sensitive topics and describe mul-
tiple health conditions and treatments. 13. Yohannes AM, Willgoss TG, Baldwin RC, Connolly MJ. Depression and
anxiety in chronic heart failure and chronic obstructive pulmonary disease:
prevalence, relevance, clinical implications and management principles. Int J
Geriatr Psychiatry. 2010;25(12):1209–21. https://doi.org/10.1002/gps.2463. Acknowledgements The researchers are grateful for the support of a number of organisations
with their recruitment efforts including: the UKRI Mental Health Networks,
Alzheimers’s Society, University of Hertfordshire, NCRI Consumer forum, Third
Aid Project, Yorkshire Cancer Community, Asian Women Cancer Group,
Asthma UK. We would like to thank the Covid-19 Social Study team for on-
going discussion about analyses and Dr. Rana Conway and Sara Esser for
conducting some of the interviews. 6. Chudasama YV, Gillies CL, Zaccardi F, Coles B, Davies MJ, Seidu S, et al. Impact of COVID-19 on routine care for chronic diseases: a global survey of
views from healthcare professionals. Diabetes Metabol Syndr. 2020;14(5):
965–7. https://doi.org/10.1016/j.dsx.2020.06.042. 6. Chudasama YV, Gillies CL, Zaccardi F, Coles B, Davies MJ, Seidu S, et al. Impact of COVID-19 on routine care for chronic diseases: a global survey of
views from healthcare professionals. Diabetes Metabol Syndr. 2020;14(5):
965–7. https://doi.org/10.1016/j.dsx.2020.06.042. 7. Hartmann-Boyce J and Mahtani KR (2020) Supporting people with long-
term conditions (LTCS) during national emergengies. Centre for Evidence
Based Medicine Review. www.cebm.net/covid-19/supporting-people-with-
long-term-conditions-ltcs-during-national-emergencies. Accessed 07/07/21. Competing interests Competing interests
The authors declare that they have no competing interests The authors declare that they have no competing interests Received: 17 February 2021 Accepted: 8 September 2021 Received: 17 February 2021 Accepted: 8 September 2021 Abbreviations Abbreviations
ONS: Office for National Statistics; LTCs: long-term conditions;
COREQ: Consolidated Criteria for Reporting Qualitative Research;
CVD: cardiovascular disease; NHS: National Health Service; NICE: National
Institute for Health and Care Excellence 3. Sanyaolu A, Okorie C, Marinkovic A, Patidar R, Younis K, Desai P, et al. Comorbidity and its impact on patients with COVID-19. SN Compr Clin Med. 2020;2(8):1–8. https://doi.org/10.1007/s42399-020-00363-4. 4. PHE (2020) COVID-19: guidance on shielding and protecting people defined
on medical grounds as extremely vulnerable. Report from the Department
of Health & Social Care and Public Health England URL: www.gov.uk/
government/publications/guidance-on-shielding-and-protecting-extremely-
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Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Publisher’s Note A&E Attendances and Emergency Admissions March
2020 Statistical Commentary. https://www.england.nhs.uk/statistics/wp-
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Opportunities and limitations related to the application of plant-derived lipid molecular proxies in soil science
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Opportunities and limitations related to the application of
plant-derived lipid molecular proxies in soil science plant-derived lipid molecular proxies in soil science
Boris Jansen1 and Guido L. B. Wiesenberg2
1Institute for Biodiversity and Ecosystem Dynamics, P.O. Box 94240, University of Amsterdam, 1090 GE
Amsterdam, the Netherlands
2Department of Geography, University of Zürich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
Correspondence to: Boris Jansen (b.jansen@uva.nl)
Received: 14 March 2017 – Discussion started: 20 April 2017
Revised: 14 August 2017 – Accepted: 24 September 2017 – Published: 16 November 2017 Boris Jansen1 and Guido L. B. Wiesenberg2
1Institute for Biodiversity and Ecosystem Dynamics, P.O. Box 94240, University of Amsterdam, 1090 GE
Amsterdam, the Netherlands
2Department of Geography, University of Zürich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
Correspondence to: Boris Jansen (b.jansen@uva.nl) Boris Jansen1 and Guido L. B. Wiesenberg2
1Institute for Biodiversity and Ecosystem Dynamics, P.O. Box 94240, University of Amsterdam, 1090 GE
Amsterdam, the Netherlands
2Department of Geography, University of Zürich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
Correspondence to: Boris Jansen (b.jansen@uva.nl) Abstract. The application of lipids in soils as molecular proxies, also often referred to as biomarkers, has
dramatically increased in the last decades. Applications range from inferring changes in past vegetation compo-
sition, climate, and/or human presence to unraveling the input and turnover of soil organic matter (SOM). The
molecules used are extractable and non-extractable lipids, including ester-bound lipids. In addition, the carbon
or hydrogen isotopic composition of such molecules is used. While holding great promise, the application of soil
lipids as molecular proxies comes with several constraining factors, the most important of which are (i) variabil-
ity in the molecular composition of plant-derived organic matter both internally and between individual plants,
(ii) variability in (the relative contribution of) input pathways into the soil, and (iii) the transformation and/or
(selective) degradation of (some of) the molecules once present in the soil. Unfortunately, the information about
such constraining factors and their impact on the applicability of molecular proxies is fragmented and scattered. The purpose of this study is to provide a critical review of the current state of knowledge with respect to the
applicability of molecular proxies in soil science, specifically focusing on the factors constraining such applica-
bility. Variability in genetic, ontogenetic, and environmental factors influences plant n-alkane patterns in such
a way that no unique compounds or specific molecular proxies pointing to, for example, plant community dif-
ferences or environmental influences, exist. SOIL, 3, 211–234, 2017
https://doi.org/10.5194/soil-3-211-2017
© Author(s) 2017. This work is distributed under
the Creative Commons Attribution 3.0 License. SOIL, 3, 211–234, 2017
https://doi.org/10.5194/soil-3-211-2017
© Author(s) 2017. This work is distributed under
the Creative Commons Attribution 3.0 License. SOIL Published by Copernicus Publications on behalf of the European Geosciences Union. 1
Introduction - changes in vegetation composition inferred from ex-
tractable and/or ester-bound lipids of plant origin and/or
their carbon isotopic composition (e.g., Huang et al.,
1996; Zech et al., 2009a; Le Milbeau et al., 2013); - changes in climate, i.e., mean annual temperature and/or
precipitation inferred from bacterial membrane lipids
and/or the hydrogen isotopic composition of plant-
derived lipids (e.g., Weijers et al., 2006; Krull et al.,
2006; Rao et al., 2009); - changes in paleo-elevation inferred from bacterial mem-
brane lipids and/or the hydrogen isotopic composition
of plant-derived lipids (e.g., Sachse et al., 2006; Bai et
al., 2011; Ernst et al., 2013); - changes in human impact or settlement inferred from
compound-specific N-isotope analysis or transforma-
tion products of plant-derived lipids, e.g., through burn-
ing or manure-derived lipids (e.g., Bull et al., 1999;
Eckmeier and Wiesenberg, 2009; Zocatelli et al., 2012); - and the contribution of fossil-fuel-derived carbon to
soil assessed according to lipid molecular composition
and compound-specific isotopes (e.g., Lichtfouse et al.,
1995, 1997; Rethemeyer et al., 2004). In the broadest sense, molecular proxies allow for the de-
termination of the presence, absence, or certain characteris-
tics of a (set of) molecule(s) that are indicative of a process
in or the state or composition of a system of interest. For
instance, in the clinical sciences molecular proxies among
other applications are used as indicators of the presence of
a disease or response to treatment (Brennan et al., 2013;
Van Bon et al., 2014). In toxicology they are used to as-
sess the effect of toxicant exposure on biota (Clemente et
al., 2014), and in the forensic sciences they can link suspects
to a crime scene (Concheri et al., 2011). In limnology they
are used to examine past lacustrine environmental conditions
(Castañeda and Schouten, 2011), and in organic geochem-
istry they can be used to follow oil formation and transloca-
tion in source and reservoir rocks (Curiale, 2002). Input, transformation and/or decomposition of soil organic
matter inferred from or traced through extractable and/or
ester-bound lipids of plant origin and/or bacterial membrane
lipids and/or their carbon isotopic composition. (e.g., Nierop
et al., 2001; Amelung et al., 2008; Hamer et al., 2012). In Table 1 an overview is given of the classes of molecules
frequently used as molecular proxies in soil archives in rela-
tion to their application and total and recent (last 10 years)
publications including the respective keywords. 1
Introduction For more than a century, various compounds deriving from
the substance class of lipids have been investigated in plant
and soil science. Some of the earliest publications in plant
science date back to the first half of the 19th century (Liebig
et al., 1837; Wöhler and Liebig, 1839) and in soil science to
the early 20th century as reviewed by Stevenson (1966). One
of the main interests to study lipids was the large heterogene-
ity of compounds included in this substance class. Some of
the individual compounds have been described as “biomark-
ers” or “biogenic markers”, i.e., compounds that “may be di-
agnostic of specific organisms, classes of organism, or gen-
eral biota that contribute organic matter to the atmosphere,
aqueous or sedimentary environment” (Peters et al., 2005). In addition, in environmental sciences, anthropogenic and
petroleum markers were also highlighted that have the ability
to be preserved with “no or only minor change” (Tissot and
Welte, 1984; Peters et al., 2005). Because the term biomarker
has been used in a narrow sense for the differentiation of bio-
logical tissues of different origin in environmental matrices,
recently the term “molecular proxy” has come into vogue. This term allows for the inclusion of biomarkers as indi-
vidual compounds characterizing specific biogenic sources,
but also individual compounds acting as a specific proxy
(e.g., for anthropogenic impact or thermal alteration). Fur-
thermore, it accommodates the use of groups of compounds
used in the before-mentioned way. Finally, it implies the use
of molecular ratios of compounds like the carbon preference
index (CPI) or the average chain length (ACL) that could
also be indicative for biogenic sources, alteration, or over-
print of organic matter. Therefore, in the present work we
use the term molecular proxy rather than biomarker. SOIL, 3, 211–234, 2017 Opportunities and limitations related to the application of
plant-derived lipid molecular proxies in soil science Other components, such as n-alcohols, n-fatty acids, and cutin- and
suberin-derived monomers, have received far less attention in this respect. Furthermore, there is a high diversity
of input pathways offering both opportunities and limitations for the use of molecular proxies at the same time. New modeling approaches might offer a possibility to unravel such mixed input signals. Finally, the transforma-
tion and turnover of SOM offer opportunities when tracing such processes is the purpose of applying a molecular
proxy while imposing limitations when they obliterate the molecular proxy signals linked to other phenomena. For n-alkanes several modeling approaches have recently been developed to compensate for (selective) degrada-
tion. Still, such techniques are in their infancy and information about their applicability to classes of components
other than n-alkanes is lacking. All constraining factors considered can have a significant influence on the appli-
cability of molecular proxies in soil science. The degree of influence strongly depends on the type of molecular
proxy and the environmental context in which it is applied. However, the potential impact of the constraining
factors should always explicitly be addressed whenever molecular proxies are applied in a soil scientific context. More importantly, there is still a serious lack of available information, in particular for compound classes other
than the n-alkanes. Therefore, we urgently call for the consideration of more holistic approaches determining
various factors during sampling and using as many compound classes as possible. Published by Copernicus Publications on behalf of the European Geosciences Union. B. Jansen and G. L. B. Wiesenberg: Plant-derived lipid molecular proxies: opportunities and limitations 212 soils (Frostegard and Bååth, 1996) to the application of pre-
served retene / caldalene ratios to infer paleoecological veg-
etation shifts (Hautevelle et al., 2006). Also, the archives of
the molecular proxies in soil sciences that are used are di-
verse and, in addition to soils themselves, include lacustrine
and terrestrial sediments, peat deposits, and paleosols (Zhang
et al., 2006; Bai et al., 2009; Andersson et al., 2011; Berke
et al., 2012). However, in spite of this large variety a limited
number of scientific topics can be discerned that encompass
the great majority of molecular proxy application in the soil
sciences. These are outlined as follows: 1
Introduction When using molecular proxies to answer research ques-
tions in any of the areas identified, in particular when soils
are used as an archive, several constraining factors have to
be taken into account that vary with the type of application
and research question to be answered. The most important
ones are the following: Also in soil science, molecular proxies have been used for
decades, and their application has exponentially increased in
the last decade as indicated by the number of related articles
published in Web of Science indexed journals (Table 1). The
types of molecular proxies used are as diverse as the field
of soil science itself. They range from the use of phospho-
lipid fatty acids to estimate bacterial and fungal biomass in i. variability in the source of plant-derived organic matter,
i.e., the abundance and composition of the molecular i. variability in the source of plant-derived organic matter,
i.e., the abundance and composition of the molecular www.soil-journal.net/3/211/2017/ SOIL, 3, 211–234, 2017 B. Jansen and G. L. B. Wiesenberg: Plant-derived lipid molecular proxies: opportunities and limitations B. Jansen and G. L. B. Wiesenberg: Plant-derived lipid molecular proxies: opportunities and limitations 213 1. Compounds frequently used as molecular proxies in soils. Compound (the ones
Most commonly
Examples of
Number of articles
considered in this review
used as a proxy for
recent
published until 2017
indicated in bold)
publicationsa
(publications 2007–
2016)b
Molecules of plant origin
n-Alkanes, n-alcohols
(Groups of) plant
Zhang et al. (2006), Zeng et
n-Alkane: 1588 (1025)
(n-alkanol), n-fatty
species
al. (2011),
n-Alcohol: 1972 (1123);
acids (n-alkanoic acid)
Jansen et al. n-Alkanol: 18 (11)
(2013), Gocke
n-Fatty acids: 43 (27);
et al. (2013)
n-Alkanoic acid: 67 (41)
n-Methyl ketones
Degradation/
Bai et al. n-Methyl ketone 104 (50)
transformation
(2006), Jansen
of soil organic
and Nierop
matter
(2009), B. K. Lei
et al. (2010)
Plant sterols and
(Groups of) plant
Volkman
Plant sterol: 1682 (590)
pentacyclic
species
(2005), Jansen
Pentacyclic triterpenoid:
triterpenoids
et al. (2007),
25 (10)
Lavrieux et
al. (2011)
Lignin monomers
Coniferous species
Nierop et al. Lignin monomer: 115 (74)
versus broadleaf
(2006), Heim
species versus grasses
and Schmidt
and organic matter
(2007),
transformation
Thevenot et
al. (2010),
Simpson and
Simpson
(2012), Moingt
et al. (2016)
Cutin and suberin
Root versus
(Mendez-
Cutin monomer: 25 (17)
monomers
aboveground
Millan et al. Suberin monomer: 32 (18)
biomass input
(2011), Hamer
et al. ii. variability in (the relative contribution of) input path-
ways into the soil, in particular microbial versus vege-
tation input and root versus aboveground biomass input
(e.g., Jackson et al., 1996; Schefuß et al., 2003; Mam-
belli et al., 2011); 1
Introduction (2012)
Molecules of animal or bacterial origin
Manure compounds
Human impact,
D’Anjou et
Coprostanol: 35 (17)
such as coprostanol,
animal husbandry
al. (2012),
Stigmastanol: 12 (7)
5β-stigmastanol,
Birk et al. Sitosterol: 70 (47)
sitosterol, and their
(2012), Prost et
epimers
al. (2017)
Glycerol dialkyl glycerol
Mean ambient air
Luo et al. GDGT: 148 (144)
tetraethers (GDGTs)
temperature, paleo-
(2011),
elevation and soil
Weijers et al. pH
(2011), Peterse
et al. (2012),
Ernst et al. (2013), De
Jonge et al. (2014) Table 1. Compounds frequently used as molecular proxies in soils. SOIL, 3, 211–234, 2017 www.soil-journal.net/3/211/2017/ B. Jansen and G. L. B. Wiesenberg: Plant-derived lipid molecular proxies: opportunities and limitations 214 Table 1. Continued. Compound (the ones
Most commonly
Examples of
Number of articles
considered in this review
used as a proxy for
recent
published until 2017
indicated in bold)
publicationsa
(publications 2007–
2016)b
Molecules of animal or bacterial origin
Phospholipid fatty acids
Microbial biomass
Kramer and
Phospholipid fatty acid:
(PLFAs)
Gleixner
2157 (1628)
(2006), Kindler
PLFA: 1525 (1140)
et al. (2009),
Ngosong et
al. (2012),
Malik et al. (2013)
Compound-specific stable isotope signal of one or more of the abovec
δ13C
C3 versus C4 plants
Feng et al. 13C: 13 (11)
and tracing carbon
(2010),
transformations,
Mendez-
e.g., by free-air CO2
Millan et al. enrichment
(2012)
(FACE)
δ15N
(Past) land
Bol et al. 15N: 2 (2)
management
(2005)
Griepentrog
et al. (2014)
δ2H (deuterium)
Precipitation and
Peterse et
2H: 6 (4)
paleo-elevation
al. (2009), Bai
Deuterium: 9 (7)
et al. (2011),
Luo et al. (2011), Sachse
et al. (2012),
Hermann et
al. (2017)
114C (radiocarbon)
Age and
Marschner et
14C: 3 (1)
contamination
al. (2008),
Radiocarbon: 35 (30)
determination
Mendez-
Millan et al. (2014)
a Published from 2005 until 2017. b According to ISI Web of Science, checked for “soil” and “target compound” in the topics
of articles on 27 February 2017 included in all available databases. c “Compound-specific” and the respective isotope
(i.e., 13C, 15N, 2H, and 14C) were used as separate keywords in addition to “soil”. Table 1. Continued. a Published from 2005 until 2017. b According to ISI Web of Science, checked for “soil” and “target compound” in the topics
of articles on 27 February 2017 included in all available databases. c “Compound-specific” and the respective isotope
(i.e., 13C, 15N, 2H, and 14C) were used as separate keywords in addition to “soil”. iii. 1
Introduction and the transformation and/or (selective) degradation of
(some of) the compounds once present in the soil when
it is not the aim of the study to use the molecular proxies
to study such transformations (e.g., De Leeuw and Baas,
1986; Nguyen Tu et al., 2004; Andreetta et al., 2013). iii. and the transformation and/or (selective) degradation of
(some of) the compounds once present in the soil when
it is not the aim of the study to use the molecular proxies
to study such transformations (e.g., De Leeuw and Baas,
1986; Nguyen Tu et al., 2004; Andreetta et al., 2013). iii. and the transformation and/or (selective) degradation of
(some of) the compounds once present in the soil when
it is not the aim of the study to use the molecular proxies
to study such transformations (e.g., De Leeuw and Baas,
1986; Nguyen Tu et al., 2004; Andreetta et al., 2013). proxies in different plant species, plant specimens, and
plant parts as a result of genetic or life stage variations
and/or external factors such as climate, seasonality, or
exposure to the sun (e.g., Nødskov Giese, 1975; Lock-
heart et al., 1998; Shepherd and Griffiths, 2006); However, the information about such constraining factors
and their impact on the applicability of molecular proxies
is fragmented and scattered over different publications in-
side and outside the discipline of soil sciences. For instance,
much of the available information about variation in leaf wax SOIL, 3, 211–234, 2017 www.soil-journal.net/3/211/2017/ B. Jansen and G. L. B. Wiesenberg: Plant-derived lipid molecular proxies: opportunities and limitations 215 lipid composition is presented in the plant physiological lit-
erature in studies that were not conducted with the applica-
tion of such lipids as a molecular proxy for past vegetation
composition from soil archives in mind (e.g., Tulloch, 1973;
Avato et al., 1984; Kim et al., 2007). The fragmentation of
the information makes it difficult for researchers to assess
the potential influence of constraining factors on the appli-
cation of molecular proxies. It also hinders the identification
of hiatuses in the available knowledge about the constraining
factors and the designation of potential strategies to compen-
sate or correct for such constraints. 1
Introduction constitute the proxy, e.g., n-alkanes of different chain lengths
in the leaf waxes of a certain species and (ii) variation in
the absolute abundance of the molecules used as a proxy ei-
ther between different specimens or between different parts
of a single specimen. Depending on the research question,
intraspecies variability in molecular proxies may be desir-
able or not. For instance, when preserved leaf wax lipid pat-
terns are used to reconstruct past vegetation composition, the
implicit assumption is that the intraspecies variability in the
source vegetation is small compared to the interspecies vari-
ability. There are two main causes of intraspecies variability
in molecular proxies: internal variation related to genetics
and/or ontogeny and external variation related to the grow-
ing environment. Both are related in the sense that differ-
ences in the response to environmental factors are also of-
ten genetically determined (Shepherd and Griffiths, 2006). Here we discuss both causes separately with a third para-
graph devoted to studies in which combined effects were ex-
amined. For a detailed description of the biomolecular mech-
anisms of wax genesis and all potential sources of change,
the reader is referred to the review provided by Shepherd and
Griffiths (2006). Therefore, the purpose of the present study is to provide
a critical review of the current state of knowledge with re-
spect to the applicability of molecular proxies in soil sci-
ence, specifically focusing on the factors constraining such
applicability. Based on this we will identify areas for future
research both with respect to the application of molecular
proxies in soil science and the constraints thereof. The vastness of the field of molecular proxies forced us
to restrict the scope of the present study. With respect to the
molecules to consider, a first restriction was to focus on those
related to the earlier-mentioned main areas of application
for molecular proxies in soil science. A second restriction
was to focus on the main classes of components as used by
several researchers. Finally, in spite of their common appli-
cation, we explicitly excluded lignin and phospholipid fatty
acids (PLFAs) as lignin was the subject of another recent re-
view (Thevenot et al., 2010) and PLFAs are considered in
such a large set of studies (cf. Table 1) that they would require
a separate review. 1
Introduction Finally, GDGTs were excluded because
their application is predominantly in aquatic sediments rather
than soils and they have been recently reviewed (Schouten et
al., 2013). This leaves the component classes labeled in bold
in Table 1 to be considered in the present study. Our study
is relevant to the application of compound-specific isotope
analysis inasmuch as such analysis is directly affected by the
variability and transformation of the underlying molecules. However, we did not explicitly consider the sources and ef-
fects of variation in the stable δ13C and δ2H isotope signature
of specific molecules themselves; this is a research area of its
own and also the subject of other recent reviews by Sachse et
al. (2012) and Diefendorf and Freimuth (2017). Furthermore,
when considering the application and preservation of molec-
ular proxies we restricted ourselves to topsoils as archives
(i.e., surface soil horizons or A horizons as defined by the
FAO in the Guidelines for Soil Description; FAO, 2006). 2.2.1
Wax lipids Many studies have indicated that the clear genetic control
of leaf wax genesis leads to a significant and meaning-
ful difference in their composition (Shepherd et al., 1995;
Shepherd and Griffiths, 2006). For instance, prompted by
the early work in this area (e.g., Eglinton et al., 1962a, b;
Herbin and Robins, 1968, 1969), Maffei performed an ex-
tensive evaluation of the n-alkane patterns in several hun-
dred plant species belonging to the Poaceae, Apiaceae, Bras-
sicaceae, Fabaceae, Cactaceae, Pinaceae, Lamiaceae, Bor-
aginaceae, Verbenaceae, Solanaceae, and Scrophulariaceae
(Maffei, 1994, 1996a, b; Maffei et al., 1997, 2004). These
studies were replenished by those on Styracaceae (Li et al.,
2013), Moraceae (Sonibare et al., 2005), and Clusiaceae
(Medina et al., 2004, 2006). Further, Dove et al. (1996) de-
scribed the alkane diversity among a grassland plant commu-
nity, which enables the tracing of the diet of grazing animals
due to the different alkane compositions of the plants. Re-
cently, Mueller-Niggemann and Schwark (2015) were able
to differentiate rice from alternating crop plants based on
their n-alkane patterns. The results support the chemotaxo-
nomic discriminatory power of n-alkane patterns at family,
subfamily, and tribal level, which has been further exam-
ined by Diefendorf and Freimuth (2017). Examining plant
n-alkane and n-alcohol distribution of 37 C4 grasses, Rom-
merskirchen et al. (2006) also found that chemotaxonomic
differentiation was possible at the subfamily level. Mon-
grand et al. (2001) examined the fatty acid composition of 2.1
Definition Source-related variability in molecular proxies pertains to in-
traspecies variation in the abundance of the molecules that
are used as a proxy. Such variability entails (i) variation in
the relative abundance of individual compounds that together www.soil-journal.net/3/211/2017/ SOIL, 3, 211–234, 2017 B. Jansen and G. L. B. Wiesenberg: Plant-derived lipid molecular proxies: opportunities and limitations 216 the leaves of over 137 species of gymnosperms belonging
to 14 families collected from different locations in France. They found a taxonomically meaningful clustering into four
main groups, with the highest discriminatory power in the
Pinaceae at the genus level (Mongrand et al., 2001). Ad-
ditionally, Wiesenberg and Schwark (2006) determined dif-
ferences in the fatty acid composition between temperate
C3 and C4 crops. Within the same Brassica species of kale
and swede, Shepherd et al. (1995) observed a difference in
the chain length distribution of wax lipids between two geno-
types of the same species, which is indicative of genetic con-
trol through variation in the enzyme system. Also for the iso-
prenoids, a genetically driven discriminatory power related
to (groups of) plant species is attributed (Ohsaki et al., 1999;
Jansen et al., 2007). loch (1973) also observed variation in the leaf waxes of
several Triticum species with age. In particular, the whole
plant n-alkane predominance shifted from C31 at 24 days af-
ter germination to C29 at 100 days after germination (Tul-
loch, 1973). Furthermore, Wiesenberg et al. (2004, 2012) and
Wiesenberg and Schwark (2006) observed changes in the n-
alkane and n-fatty acid compositions of a variety of temper-
ate crop species with plant age. Other publications reported
seasonal variations in the n-alkane composition for a variety
of pasture and crop plants (Dove et al., 1996; Hellgren and
Sandelius, 2001; Moseley, 1983; Shelvey and Koziol, 1986)
and various trees (Prasad and Gülz, 1990; Gülz et al., 1991;
Gülz and Muller, 1992; Gülz and Boor, 1992). Variations in
the n-alkane composition could be observed during the grow-
ing season among all investigated plants, but general trends
of increasing or decreasing chain length and n-alkane con-
tents have not consistently been determined. The n-alcohol
predominance also varied but to a much smaller extent, not
affecting the predominance of a specific n-alcohol (Tulloch,
1973). Esters gradually showed an increase in esters of trans
2,3-unsaturated C23 and C24 acids with plant age (Tulloch,
1973). 2.1
Definition The variation was related to the development of the
plant, in particular that of flag leaves and sheets between
55 and 66 days (Tulloch, 1973). The chemotaxonomic potential of wax lipids as just de-
scribed has been exploited to reconstruct past vegetation his-
tory from wax lipids preserved in soil archives (e.g., Bull et
al., 1999; Zech et al., 2009a; Jansen et al., 2013). Such re-
constructions often focus on the use of shifts in the ratios of
certain dominant higher chain length n-alkanes, fatty acids,
and n-alcohols representative of a shift in the dominant veg-
etation over time (Jansen et al., 2010; Gocke et al., 2016;
Wiesenberg et al., 2015). In a few instances, the entire suite
of higher chain length n-alkanes and n-alcohols (Jansen et
al., 2013) or n-alkanes and fatty acids has also been used
(Wiesenberg et al., 2015). In contrast to previous studies, Li et al. (1997) studied
the influence of ontogeny on leaf wax lipids (n-alkanes, n-
aldehydes, n-alcohols, esters, β-diketones, flavonoids, and
triterpenoids) in several Eucalyptus species of the subgenus
Symphyomyrtus on Tasmania and found no significant ef-
fect of ontogeny on leaf wax composition, which they found
to clearly and consistently differ between species (Li et al.,
1997). Eglinton et al. (1962a) also observed that the n-alkane
composition of the leaf waxes of 74 species of Crassulaceae
from the Canary Islands showed no appreciable variation
with respect to leaf position, age, size, or specimen. Further,
Bush and McInerney (2013) found no influence of canopy
position or sampling time on the n-alkane patterns of mature
leaves from 24 tree species. g
However, in addition to other issues such as those dis-
cussed in the other sections of this review, an important issue
when exploiting the chemotaxonomic potential of leaf wax
lipids is the phenotypic plasticity of the genetic variability
in leaf wax lipid patterns found and the implications thereof
for the stability of the patterns observed. Maffei et al. (2004)
concluded that phenotypic plasticity may overcome genetic
variability, particularly when plant developmental stages are
considered along with abiotic and biotic stress conditions. Several plant physiological studies have focussed on wax
lipid composition related to plant life stage and report dif-
ferent results. Avato et al. 2.1
Definition (1984) found that where the rela-
tive contribution of n-fatty acids, n-alcohols, and n-alkanes
differed between Sorghum seedlings and mature leaves, the
chain length distribution within a component class remained
the same for the n-alkanes and n-alcohols. Giese (1975) ob-
served a difference in the homologue dominance of n-alkanes
between leaves of seedlings and mature barley plants. Herbin
and Robins (1969), Dyson and Herbin (1970), Baker and
Hunt (1981), and Zhang et al. (2004) also identified increas-
ing chain length dominance of leaf wax alkanes with in-
creasing leaf age. However, the averaging of sampling over
leaves of different ages, positions, and other characteristics
within a stand of trees did allow for distinction from other
stands, indicating that interspecies variation was larger than
intraspecies variation (Dyson and Herbin, 1970). Baker and
Hunt (1981) observed differences between the adaxial and
abaxial parts of leaves for some of the plant species. Tul- SOIL, 3, 211–234, 2017 2.2.2
Cutin and suberin monomers Cutin and suberin monomers are mainly used as proxies to
distinguish leaf from root input in soils (Schreiber et al.,
1999; Bull et al., 2000; Mendez-Millan et al., 2011) or as a
proxy for related phenomena such as the degree of bioturba-
tion in the topsoil (Nierop and Verstraten, 2004). Therefore,
the possible (onto)genetic effects on cutin and suberin com-
position are a concern if they were to alter the composition of
the polyesters to such an extent that the separation between
cutin and suberin is compromised. Some general observations in the literature are that long-
chain even-numbered C20–C30ω-hydroxy fatty acids and
α- and ω-alkanediotic acids mainly originate from suberin,
whereas shorter-chained C16 and C18ω-hydroxy fatty acids
mainly derive from cutin (Schreiber et al., 1999; Otto et al., SOIL, 3, 211–234, 2017 www.soil-journal.net/3/211/2017/ B. Jansen and G. L. B. Wiesenberg: Plant-derived lipid molecular proxies: opportunities and limitations 217 chain length distribution. Bondada et al. (1996) reported an
increase in absolute amounts of epicuticular wax produc-
tion by 69 % in the leaves of cotton (Gossypium hirsutum)
under water stress, which was confirmed by Hamrouni et
al. (2001), Koch et al. (2006), Kim et al. (2007), and Bet-
taieb et al. (2010) for neutral lipids of other plant species. However, Kim et al. (2007) found that water stress had
only a minor effect on chain length distribution. The rela-
tive contribution of different component classes to the wax
composition remained unchanged except for Brassica oler-
acea var. gongylodes at the highest relative humidity, which
showed an increased contribution of ketones and primary al-
cohols and a reduction of secondary alcohols and aldehydes
(Koch et al., 2006). Recently, Srivastava et al. (2017) deter-
mined that sustained effects of drought on plant lipid compo-
sition are commonly missing with few exceptions for peren-
nial plants. Thus, several months after exposure to drought
the lipid biosynthesis and composition of leaves is resilient. The existing data show that the general effects of drought on
plant lipid composition are difficult to infer. 2005; Mendez-Millan et al., 2011). However, several pub-
lications challenge the universal applicability of such gen-
eral observations, indicating instead that genetic variability
results in many exceptions to such general rules. For in-
stance, Hamer et al. (2012) found that ωC22:0, ωC24:0, and
ωC26:0 hydroxy fatty acids were not exclusively associated
with roots, but also occurred in the shoots of several species. 2.3.3
Effects of increased CO2 Changes in greenhouse gases such as CO2 have also been
discussed as influencing the lipid biosynthesis and thus the
lipid composition of plants. Short-term exposure of several
hours to elevated CO2 concentrations, e.g., during 13CO2 or
14CO2 labeling experiments, has little to no effect on lipid
composition, especially if sampling occurs several days after
labeling (Wiesenberg et al., 2009). In contrast, a long-term
rise in atmospheric CO2 concentration has been investigated
in laboratory or free-air carbon dioxide enrichment (FACE)
experiments (Ainsworth and Long, 2005). Although numer-
ous such experiments have been conducted in the meantime,
the investigation of lipid composition is limited. Greenhouse
experiments showed that elevated CO2 concentration affects
the relative composition of saturated and unsaturated fatty
acids in wheat plants (Williams et al., 1994, 1995, 1998). However, rising nitrogen fertilization and rising temperature
can lead to competing trends (Williams et al., 1995; Griepen-
trog et al., 2016). Although specific abundances of individ-
ual long-chain alkanes and alcohols changed under elevated
CO2 concentration, the overall lipid composition expressed
as ACL and CPI did not change (Huang et al., 1999). Never-
theless, concentration changes, like an increase in n-alkane
and n-alcohol abundances and a decrease in n-fatty acid
abundance, were determined under a rising CO2 concentra-
tion, whereas nitrogen fertilization led to a decrease in the ef-
fect (Huang et al., 1999). This was confirmed by Wiesenberg
et al. (2008a) for n-alkanes, n-fatty acids, and n-alcohols. In
some forest FACE and open-top chamber experiments, the
effects of elevated CO2 on plant lipid concentration were not
identified (Feng et al., 2010; Griepentrog et al., 2015), but
the 13CO2 labeling associated with the CO2 enrichment was In addition to the effect of temperature on lipid synthesis,
temperature can also influence lipids after production specif-
ically as a result of fire. This topic is addressed in Sect. 4.2. 2.3.1
Effects of temperature Increased solar radiation levels are generally reported to lead
to higher absolute amounts of waxes produced (Sanchez et
al., 2001; Shepherd and Griffiths, 2006). In addition, the
composition of the various component classes of wax lipids,
i.e., the relative contribution of n-fatty acids, n-alkanes, and
n-alcohols, has been reported to change. A shift towards
lower chain lengths within different component classes was
sometimes found (Shepherd and Griffiths, 2006). Thus, a
positive correlation of long-chain odd n-alkanes with temper-
ature was observed (Maffei et al., 1993; Zhang et al., 2004). Also, the abundance of membrane fatty acids with 16 and
18 carbons can change as a result of temperature (Maffei
et al., 1993; Williams et al., 1995; Matteucci et al., 2011). Under heat stress, the relative abundance of C16:0 fatty acid
was often found to increase, and the abundance of polyun-
saturated C18:3 fatty acid was found to decrease (Larkindale
and Huang, 2004; Bakht et al., 2006). Furthermore, effects of
temperature were observed for monoterpenes and sesquiter-
penes, with compounds like limonene and myrcene having a
close correlation with temperature, whereas others like 1,8-
cineol were not affected by temperature (Maffei et al., 1993). As a cause, different sensitivities of individual steps in the
genesis of the wax lipid components are assumed (Shep-
herd and Griffiths, 2006). However, results were found to
vary between different species and genotypes, indicating a
species- or genotype-related sensitivity to changes in irradi-
ation (Shepherd and Griffiths, 2006), whereas cold- or heat-
acclimated plants respond differently than those that are not
acclimated (Larkindale and Huang, 2004). Thus, a depen-
dency between temperature and lipid metabolism is widely
observed, but, especially in plants, other factors such as hu-
midity or greenhouse gas composition might coincide with a
larger effect on the overall lipid composition. 2.2.2
Cutin and suberin monomers In addition, ωC16:0 and ωC18:0 fatty acids were not exclusive
to the leaves, but also occurred in the roots of several species. www.soil-journal.net/3/211/2017/ 2.4
Other or combined genetic, ontogenetic, and/or
environmental effects Many studies considered the effects of geographical loca-
tion on wax amounts and/or composition without differen-
tiating between individual genetic or environmental causes. Again, the exact parameters investigated vary greatly be-
tween studies, as do the conclusions drawn. Cowlishaw et
al. (1983) examined the n-alkane, n-alcohol, n-aldehyde,
and ester composition of composite samples of four species
of the Poaceae Chionochloa, one of which was sampled at
three different environmental locations to investigate envi-
ronmental effects. They found distinct chain length patterns
that allowed for chemotaxonomic identification, and varia-
tion between the three sampling sites did not alter the dom-
inant chain length patterns for any of the component classes
(Cowlishaw et al., 1983). Similar observations were made by
Herbin and Sharma (1969) for the ω-hydroxy fatty acid com-
position of Pinus species from Asia, Europe, North America,
Central America, and the Caribbean. Kreyling et al. (2012)
described differences in the n-fatty acid and n-alkane com-
position of the same plant species originating from differ-
ent regions across Europe with different climatic conditions
that are most likely due to biosynthetic adaptation to the spe-
cific conditions. Piervittori et al. (1996) found that the dis-
tribution of C25, C27, C29, and C31 n-alkanes in the lichen
Xanthoria parietina varied significantly between two differ-
ent Piedmont valleys in Italy and within the valleys with al-
titude, reflecting a combined influence of elevation, water
availability, radiation, and temperature. For plaggen ecosys-
tems, Kirkels et al. (2013) also observed significant vari-
ability in the reported ratios of the dominant n-alkanes with
chain lengths C27, C29, C31, and C33 that is most likely at-
tributable to the causes examined here. However, in spite
of this they found meaningful clustering of the three dif-
ferent plant groups (grasses, shrubs, and trees), indicating
that the variability did not obliterate the power of distinction
(Kirkels et al., 2013). In a larger study based on 2093 obser-
vations from 86 sources of plant material, Bush and McIn-
erney (2013) concluded that the general observation that C27
and C29 n-alkanes are dominant markers for woody vegeta-
tion and C31 for graminoids does not rigorously hold true. At
the same time, C23 and C25 n-alkanes do seem to be robust
indicators of Sphagnum (Bush and McInerney, 2013) as ob-
served by Baas et al. (2000) and Pancost et al. (2002). 2.4
Other or combined genetic, ontogenetic, and/or
environmental effects Bush
and McInery (2013) indicated that the lack of rigor of the
mentioned proxies is likely caused by environmental condi-
tions as indicated by a shift in patterns across African savan-
nah and rain forest environments. The distinction between African savannah and rain for-
i
i
l
d
i
i Rommerskirchen et al. (2006) observed a generally higher
content of C31 and C33 n-alkanes and therefore a higher
ACL value in African C4 grasses with respect to C3 grasses
from the same area as a result of the genetic adaptation
of C4 grasses to warm, arid habitats. In addition, n-fatty
acid patterns have also been shown to vary with C3 and C4
metabolism, with C3 crops having relatively large propor-
tions of C24 n-fatty acid in leaves, stems, and roots compared
to C22 and C26 n-fatty acids in C4 crops (Wiesenberg and
Schwark, 2006). 2.3.2
Effects of humidity With respect to the effects of water stress and/or high hu-
midity, Shepherd and Griffith (2006) reported mixed re-
sults in their review with respect to absolute amounts and www.soil-journal.net/3/211/2017/ SOIL, 3, 211–234, 2017 B. Jansen and G. L. B. Wiesenberg: Plant-derived lipid molecular proxies: opportunities and limitations 218 Vogts et al. (2009) studied the leaves and sometimes whole
plants of 24 African rain forest and 45 savannah species. They found that as a result of environmental influence, in-
cluding temperature and aridity, the chain length distribu-
tions of the n-alkanes and n-alcohols of some species shifted
to different chain length predominance. The environmental
influences overshadowed a taxonomic distinction at the or-
der, family, or subfamily level (Vogts et al., 2009). Patterns
in grasses were more consistent and thus less dependent on
environmental factors (Vogts et al., 2009). As a result, in spite
of the environmental variability observed, Vogts et al. (2009)
found that by averaging lipid patterns within a given envi-
ronment a clear distinction between rain forest and savan-
nah plants can be made, with a dominance of C29 n-alkane
representative of the average rain forest plant signal and a
dominance of C31 n-alkane of the savannah plants and C4
savannah grasses. For the n-alcohols, C28 dominated on av-
erage for savannah plants, C30 for rain forest plants, and C32
for C4 savannah grasses (Vogts et al., 2009). used for tracing the turnover of lipids in soils as introduced
by Wiesenberg et al. (2008b). SOIL, 3, 211–234, 2017 3.2
Leaf versus root input 3.2
Leaf versus root input Conservative estimates calculate roots to represent 33 % of
global annual net primary productivity (Jackson et al., 1997),
whereas more recent studies highlight the fact that the con-
tribution of root-derived organic matter in soils can account
for > 70 % of total plant-derived carbon (Rasse et al., 2005). As a result, roots form a considerable input of organic mat-
ter in soils and are proposed to improve carbon storage in
soils (Kell, 2012). In addition, root input occurs to consider-
able depth in soils, ranging from an average depth of 0.5 m
in tundra biomes to 15.0 m in tropical grassland/savannah
(Canadell et al., 1996). Also in the temperate zone under
certain circumstances, such as the presence of nutrient-rich
fossil A horizons at depth, deep rooting can be very sig-
nificant (Gocke et al., 2016). However, on average the ma-
jority of root biomass appears to be incorporated in the top
0.3 m of the soil in most biomes, i.e., in the topsoil (Jack-
son et al., 1996). The ratio of root / shoot biomass input is
also very variable across biomes, ranging from an average
of 0.10 in cropland to 4.5 in deserts (Jackson et al., 1996). Table 2 presents an overview of the average maximum root-
ing depth, root biomass input in the first 0.3 m of the soil, and
the root / shoot biomass input for different biomes (see also
Fig. 1). Based on the current insights it seems prudent to explicitly
take the possibility of genetically and environmentally driven
variability in lipid patterns into account when considering the
use of lipids as molecular proxies. This can be achieved, for
instance, by considering plant species from the same climatic
zone as that in which the reconstruction takes place and by
mixing plant material from different life stages to obtain the
average molecular fingerprint to look for. 2.5
Conclusions and implications regarding
source-related variability Herbin and Robins (1969) concluded that there is a basic
genetic control on the composition of the wax components,
including the n-alkanes, of plant leaves. However, variable
factors associated with age and environment can be super-
imposed upon the specific pattern in some cases, while in
others the genetically controlled pattern appears to be stable
and unaffected by external influences (Herbin and Robins,
1969). Now, 48 years later, a much more extensive database
has been accrued, albeit with a large emphasis on leaf wax
lipids in general and n-alkanes in particular. Nevertheless,
the results are still equivocal. On the one hand, there is am-
ple evidence that genetically driven variability in leaf wax
lipid composition in principle leads to chemotaxonomically
meaningful clustering that can form the basis of the appli-
cation of leaf wax lipids as molecular proxies. On the other
hand, it is clear that both ontogeny and environmental fac-
tors can have a significant and sometimes dominant influence
on lipid composition like chain length distribution. Matters
are complicated by the fact that a lot of data with respect to
the effects of environmental stress originate from studies in
which plants were studied for a limited period of time (typ-
ically one growing season) and extreme conditions were ar- The distinction between African savannah and rain for-
est environments in general and C3 versus C4 vegetation in
particular have been the subject of more detailed research. www.soil-journal.net/3/211/2017/ SOIL, 3, 211–234, 2017 B. Jansen and G. L. B. Wiesenberg: Plant-derived lipid molecular proxies: opportunities and limitations 219 Table 2. Average maximum rooting depth, biomass–depth distri-
bution, and root / shoot ratios in different biomes (Canadell et al.,
1996; Jackson et al., 1996). tificially imposed. In contrast, the lipid signal from soil or
sediment archives as used in reconstructions typically repre-
sents a mixture of input of decades or longer from perennial
plants in various life stages, induced diversity from the fre-
quent changes of annual plants in managed ecosystems, and
the average of natural fluctuations in stress conditions during
that time period. 2.5
Conclusions and implications regarding
source-related variability Biome
Average
Average
Average
maximum
percentage
root /
rooting
of roots
shoot
depth [m]
in the
ratio
top 0.3 m
Boreal forest
2.0 ± 0.3
83
0.32
Cropland
2.1 ± 0.2
70
0.10
Desert
9.5 ± 2.4
53
4.5
Sclerophyllous shrubland and forest
5.2 ± 0.8
67
1.2
Temperate coniferous forest
3.9 ± 0.4
52
0.18
Temperate deciduous forest
2.9 ± 0.2
65
0.23
Temperate grassland
2.6 ± 0.2
83
3.7
Tropical deciduous forest
3.7 ± 0.5
70
0.34
Tropical evergreen forest
7.3 ± 2.8
69
0.19
Tropical grassland/savannah
15.0 ± 5.4
57
0.70
Tundra
0.5 ± 0.1
93
6.6 p
In general from what is known to date, the conclusion
seems justified that because of genetic and environmental in-
fluences there are no unique compounds or “golden ratios”
of different chain lengths of compounds that can always be
linked to certain plants under all circumstances. However,
there are many situations in which the influence of genetic
and environmental effects are small enough that they do not
prevent the use of plant lipids as molecular proxies. The cur-
rently available data do not allow for objective, quantitative
rules to be formulated in this respect. From the plant wax
components, the n-alkanes are the dominant class studied. In addition, research attention has focussed to a lesser ex-
tent on n-alcohols and n-fatty acids. The other wax compo-
nents, such as isoprenoids and ester-bound lipids, have re-
ceived hardly any research attention to date with respect to
source-related variability in the context of their use as molec-
ular proxies. Yet even for the n-alkane patterns in leaf waxes,
only a tiny portion of the dominant plant species on the planet
has been examined in detail for the effects of genetics and en-
vironment on their amounts and patterns. It is clear that much
more research is needed in this respect. 3.1
Definition Further, the transport, age, and probability of preservation as general
properties of lipids are given on the left side of the figure. Figure 1. Conceptual overview of different incorporation pathways of lipids in soils originating from different biological sources and an-
thropogenic contamination. The different sources are indicated by distinct colors and lines of the arrows. The line thickness is an estimated
significance of individual sources without providing a quantitative measure for different sources. Autochthonous sources are further distin-
guished by their significance in different soil depths or soil horizons. Further, the transport, age, and probability of preservation as general
properties of lipids are given on the left side of the figure. many cases the absolute amount of lipids present per mass
unit of root material is an order of magnitude or more lower
than on leaf material (Marseille et al., 1999; Zech et al.,
2011). In addition, with respect to the degree to which wax
lipid chain length patterns vary between the leaves and roots
of plant species, there appears to be quite some variability. In
general, the observed differences between roots and leaves of
the same species are reported to be of the same order of mag-
nitude as the differences between leaves of different species
(e.g., Jansen et al., 2006; Kirkels et al., 2013; Gocke et al.,
2014). when such wax-derived lipids are applied as molecular prox-
ies for vegetation cover in soil, root input can be an issue for
two reasons: (i) roots may contain a different wax lipid com-
position than leaves qualitatively and quantitatively, thereby
clouding the leaf signal (Jansen et al., 2006; Martelanc et
al., 2007) and (ii) young root input at depth may disrupt the
chronology of a reconstruction in time by overprinting the
originally present signal (Lavrieux et al., 2012; Gocke et al.,
2014). The main discussion with respect to the influence of root
input in wax-lipid-based environmental reconstructions from
soils therefore revolves around assessing the relative impor-
tance of root versus aboveground biomass input. Since plant
wax lipids reside on the outer parts of leaves and roots, rel-
ative surface area and bioproductivity are important. On a
global scale, root surface area is almost always calculated to
be higher than leaf surface area by more than an order of
magnitude in grasslands (Jackson et al., 1997). SOIL, 3, 211–234, 2017 3.1
Definition Here we discuss differences in the amount and composition
of molecules used as proxies, which is possible due to differ-
ent input pathways of such molecules to the soil. A schematic
representation of the different input routes of molecular prox-
ies into the soil is provided in Fig. 1. The emphasis lies on
potential effects for their use as molecular proxies. For a gen-
eral description of the different molecular origins of organic
matter in soil, the reader is referred to a dedicated review on
this topic by Kögel-Knabner (2002). If the molecules to be used as a proxy are present in both
the leaves and roots of plants, the possibility of root in-
put is also a factor that has to be considered depending on
the purpose of the proxy. In the case of cutin and suberin
monomers, root input does not cause interference as discern-
ing root from leaf input is the specific purpose of this molec-
ular proxy (Mendez-Millan et al., 2011). However, this may
be different for the wax lipids, i.e., n-alkanes, n-alcohols,
n-fatty acids, and isoprenoids, that have been found to oc-
cur in the leaves and roots of species at varying concentra-
tions (Jansen et al., 2007; Huang et al., 2011). Particularly www.soil-journal.net/3/211/2017/ SOIL, 3, 211–234, 2017 B. Jansen and G. L. B. Wiesenberg: Plant-derived lipid molecular proxies: opportunities and limitations 220 B. Jansen and G. L. B. Wiesenberg: Plant-derived lipid molecular proxies: opportunities and limitations Figure 1. Conceptual overview of different incorporation pathways of lipids in soils originating from different biological sources and an
thropogenic contamination. The different sources are indicated by distinct colors and lines of the arrows. The line thickness is an estimate
significance of individual sources without providing a quantitative measure for different sources. Autochthonous sources are further distin
guished by their significance in different soil depths or soil horizons. Further, the transport, age, and probability of preservation as genera
properties of lipids are given on the left side of the figure. Figure 1. Conceptual overview of different incorporation pathways of lipids in soils originating from different biological sources and an-
thropogenic contamination. The different sources are indicated by distinct colors and lines of the arrows. The line thickness is an estimated
significance of individual sources without providing a quantitative measure for different sources. Autochthonous sources are further distin-
guished by their significance in different soil depths or soil horizons. 3.4
Airborne input In addition to the in situ production and incorporation of soil
lipids, airborne input must be considered. The distance of the
airborne transport of larger constituents such as leaves can be
expected to be limited. However, smaller physical forms con-
taining lipids, such as aerosols and dust particles, can travel
substantial distances (Conte and Weber, 2002), thus caus-
ing the input of alien molecules that may influence the lo-
cal signal. This is of special importance when airborne sedi-
ments with a low content of organic matter are investigated,
as in these environments low inputs of foreign organic mat-
ter can significantly influence the molecular proxies. Liu et
al. (2007) showed that the δ13C signature of sediment or-
ganic carbon in loess deposits on the western Chinese Loess
Plateau corresponds to that of dust sources instead of the lo-
cal vegetation. In a study of marine sediment cores along the
southwestern African continental margin, Rommerskirchen
et al. (2003) revealed that the aerosol-derived input of higher
chain length n-alkanes and n-alcohols provides a significant
signal, the δ13C signal of which corresponded well with con-
tinental C3 and C4 plant distribution and fossil pollen input
when prevailing wind patterns were taken into account. How-
ever, in this case, in contrast to vegetated soils, there was no
in situ input from higher plant vegetation. For wax lipids, n-alkanes, n-alcohols, and n-fatty acids
with longer chain lengths (> C20) and a distinct odd-over-
even (n-alkanes) or even-over-odd (n-alcohols and n-fatty
acids) chain length predominance are generally considered
to be higher plant derived, whereas shorter chain length ho-
mologues are considered to be predominantly of microbial
origin (Eglinton et al., 1962a; Dinel et al., 1990). More-
over, with the exception of an abundance of C16 and C18 n-
alcohol and n-fatty acid, such microbial lipids are described
to lack a specific chain length predominance (Stevenson,
1994; Lichtfouse et al., 1995). However, several researchers
challenge the observation that higher chain length lipids
in soils are exclusively of higher plant origin. Microorgan-
isms have been shown capable of synthesizing higher chain
length straight-chain lipids, albeit usually to a limited extent
(Ladygina et al., 2006; Nguyen Tu et al., 2011). Jambu et
al. (1978) indicated that while chain lengths > C20 in soils
are predominantly plant derived, particularly in acidic soils,
fungi may contribute such lipids as well. Furthermore, Mar-
seille et al. 3.3
Microbial input With respect to cutin and suberin monomers, in situ gene-
sis in soils through the microbial transformation of other pre-
cursor molecules can be an issue. For instance, the oxidation
of free fatty acids could be a source of ω-hydroxy fatty acids,
whereas the microbial β-oxidation of unsaturated fatty acids
and/or mid-chain hydroxy fatty acids may be a source of α-
and ω-alkanediotic acids, thus clouding the cutin or suberin
signal (Naafs et al., 2004b). In general, microbial biomass can be a significant source of
soil organic matter, with up to 40 % transformed to nonliv-
ing soil organic matter, but it is turned over much faster than
plant residues (Miltner et al., 2012). Focusing specifically on
lipids, isotopic studies show that 90 % of fatty acids of micro-
bial origin are turned over rapidly after cell death, whereas
the majority of biomass-derived residual bulk C was stabi-
lized in the nonliving soil organic matter pool (Kindler et
al., 2009). In spite of the potentially shorter residence time,
concurrent faster production makes microorganism-derived
molecules a factor to consider when applying molecular
proxies in soils, except when such proxies are used to study
microbial input. 3.1
Definition However, in The concurrent influence of such various quantitative and
qualitative factors makes the impact of root input a complex
issue that is still the subject of scientific debate (Wiesenberg
and Gocke, 2013). Given that different factors will have a
highly variable influence in different situations, no general
conclusion can be drawn. In some situations, the influence of
roots as an input pathway of extractable lipids to be used as SOIL, 3, 211–234, 2017 www.soil-journal.net/3/211/2017/ B. Jansen and G. L. B. Wiesenberg: Plant-derived lipid molecular proxies: opportunities and limitations 221 ally does not seem to be a major factor compared to direct-
plant-derived input in the topsoil (Jansen and Nierop, 2009;
Bai et al., 2009). In contrast, for steroids and triterpenoids
such as camposterol, stigmasterol, and lupeol, microbial in-
put in soils can be considerable (Naafs et al., 2004a). As
another example, arbuscular mycorrhizal fungi-derived β-
sitosterol is by far the most abundant sterol identified in soils
(Grandmougin-Ferjani et al., 1999). a molecular proxy may be limited (Quenea et al., 2006). In
others, root input may be dominant (Van Mourik and Jansen,
2013). In addition, the relative degree of influence may vary
greatly with depth, leading to the concurrent presence of leaf-
lipid-dominated and root-lipid-dominated zones at different
depths in the same profile (Angst et al., 2016). 3.4
Airborne input (1999) observed an abundance of C25 and C27
n-alkanes that they also attribute to in situ production by
fungi. This was confirmed for an agricultural soil by Que-
nea et al. (2006), who observed old-forest- and fungi-derived
odd long-chain alkanes based on compound-specific isotope
analysis and lipid distribution patterns. Possible pathways of
in situ genesis of n-alkanes in soils are the reduction of n-
alkenes and n-alcohols, the decarboxylation of bacterial n-
fatty acids, and the degradation of biopolymers containing
aliphatic side chains (Lichtfouse et al., 1998a). Nevertheless,
based on the large number of studies in which typical higher-
plant-derived patterns of lipids are reported and used in soils
(Table 1), including indicative ACL and CPI values, micro-
bial input of longer chain length straight-chain lipids gener- Aerosol studies above plant canopies revealed a certain re-
lationship of the plant wax composition of the present plants,
but significant differences from the biomass were observed
for n-alkanols and n-alkanes (Conte et al., 2003). While the
wax molecular composition was not directly linked between
biomass and aerosol, the compound-specific isotope compo-
sition (δ13C) revealed a closer link to both. For Bermuda
aerosols it could be shown that the aerosol compound-
specific isotope composition of n-alcohols and n-fatty acids
reflects the plant wax compound-specific isotope composi-
tion and the course of the bioproductivity during the different
seasons of the years (Conte and Weber, 2002). SOIL, 3, 211–234, 2017 www.soil-journal.net/3/211/2017/ B. Jansen and G. L. B. Wiesenberg: Plant-derived lipid molecular proxies: opportunities and limitations 222 In a study of PM10 aerosols collected during a winter sea-
son in Baoij, China, Xie et al. (2009) found concentrations
of C21–C33 n-alkanes in the 10–600 ng m−3 range as a result
of intensive coal burning in the region. In a 2-year study of
PM10 and PM2.5 aerosols in urban sites in Nanjing, Wang
et al. (2006) observed C21–C33 n-alkanes present in the 10–
100 ng m−3 range. Concentrations of C21–C35 n-alkanes in
PM10 aerosols in urban sites in Beijing sampled in all sea-
sons were even lower (Zhou et al., 2009). In this study n-fatty
acids and hopanes were also considered, but were found in
small concentrations that together with the n-alkanes consti-
tuted ca. 3 % of the total organic matter in the aerosols (Zhou
et al., 2009). 4
Transformations and turnover in soil Transformations and the turnover of soil organic matter are
an important study area in their own right (Kögel-Knabner,
2002; Von Lützow et al., 2008). Important in the context of
the application of molecular proxies is the recent paradigm
shift to the attribution of external factors as drivers of SOM
turnover rates as opposed to inherent recalcitrance related to
molecular structure (Schmidt et al., 2011; Lehmann and Kle-
ber, 2015). Coupled to this are indications that the micro-
bial recycling of organic matter upon entering the soil de-
couples the molecules from their biological sources (Miltner
et al., 2012; Gleixner, 2013). Here, we focus on the effects
of transformation and the differences in the degradation of
molecules in soils for their use as molecular proxies. This
includes transformations during the stages of senescence or
litter, and it covers attempts to estimate the successive degra-
dation processes of organic matter occurring after burial until
the stages of long-term preservation (see also Fig. 1). Trans-
formation processes can also include processes that affect
the detectability of a molecule used as a proxy, for instance
a transformation from the extractable to the non-extractable
lipid fraction as a result of chemical alterations or interac-
tions with the mineral phase (e.g., Almendros et al., 2001). A
special case is the influence of fire on SOM, including molec-
ular proxies, as reviewed by González-Pérez et al. (2004). 3.4
Airborne input In all studies, the straight-chain lipid patterns
lacked the odd-over-even chain length predominance typical
of higher plants (Wang et al., 2006; Xie et al., 2009; Zhou et
al., 2009). Nevertheless, in a large survey a clear odd-over-
even chain length predominance was found in spite of such
potentially intense aerosol-derived input (Rao et al., 2011). This indicates that even in areas under large aerosol deposi-
tion, as in the case of the intensive anthropogenic pollution
associated with fossil fuel burning, the effect of aerosol de-
position on n-alkane patterns in the soil is limited as a result
of the large in situ input via the roots and leaves of the local
vegetation. bination of plant groups responsible for the original lipid in-
put by treating leaves and roots explicitly as separate entities
(Jansen et al., 2010). This might form a starting point to dis-
entangle leaf- and root-derived lipid input. Although aerosol studies so far have provided the useful
information that plant wax components are transported via
aerosols to remote places, other factors like degradation dur-
ing transport and the integration of regional vegetation pat-
terns may hamper direct source-to-sink relationships of air-
borne molecular markers. Nevertheless, the overall impact
of aerosol-borne molecules on molecular-proxy-based recon-
structions seems to be limited whenever the total abundance
in the soil is high. SOIL, 3, 211–234, 2017 3.5
Conclusions and implications regarding
input-pathway-related variability The diversity of input pathways offers both opportunities
and limitations for the use of molecular proxies. Oppor-
tunities arise when different sources can be elucidated us-
ing molecular proxies. Examples are the differences in the
molecular composition of leaf and root waxes as used to
differentiate between their respective influences or when
aerosol-associated lipids are used for the source apportion-
ment of terrestrial plant input in terrestrial or marine sed-
iments. This can help budget the organic matter input of
different sources and thus improve (paleo)environmental in-
terpretations and reconstructions. Limitations are imposed
when input through multiple pathways clouds the linkage of
a (set of) molecule(s) to a certain source for which it is to
serve as a proxy, for instance when linking a suite of straight-
chain lipids to a particular group of plants at a certain site. When looking at the application of molecular proxies in soils,
the assessment of the influence of root-derived input is a par-
ticular challenge that is not always acknowledged. The sig-
nificance of root-derived organic matter in soils and terres-
trial sediments has been neglected for decades and has only
recently been highlighted (Rasse et al., 2005; Rumpel and
Kögel-Knabner, 2011). More research attention is needed to
pinpoint how large the possible interferences are and what
the potential can be to compensate for them, e.g., through
modeling approaches. For instance, the VERHIB model was
designed to unravel the mixed n-alkane, n-alcohol, and/or n-
fatty acid signal observed in soils into the most likely com- All of the attempts dealing with the incorporation and
preservation of organic matter involve different assumptions
and entail different problems in terms of uncertainties. Thus,
in the dependency of the environmental conditions, assump-
tions that are relevant for the incorporation and burial of or-
ganic matter play a major role, as should the different aspects
of degradation and preservation. However, much uncertainty
currently exists regarding the influences of individual envi-
ronmental and genetic factors in degradation and preserva-
tion. Therefore, the following paragraphs only provide the
first insights into tackling these issues, which need further
attention in future research projects. Molecular transformations and variations thereof in
molecular proxies mostly complicate the application of www.soil-journal.net/3/211/2017/ SOIL, 3, 211–234, 2017 B. Jansen and G. L. B. Wiesenberg: Plant-derived lipid molecular proxies: opportunities and limitations 223 More in general, Mambelli et al. 3.5
Conclusions and implications regarding
input-pathway-related variability (2011) observed root lit-
ter, including biomarkers, to be selectively preserved with
respect to needle litter, which was confirmed by Mendez-
Millan et al. (2010a, b) for maize and wheat roots ver-
sus shoots. Using isotopic signatures, Mendez-Millan et
al. (2011) were able to quantify and subsequently com-
pensate for such differences in turnover rate. This further
emphasizes the significance of root-derived organic matter
for turnover determinations as discussed by Wiesenberg et
al. (2004). In other words, the relative abundance of roots and
the uncertainties in terms of root-related overprint in the rhi-
zosphere and rhizosphere extension entail large uncertainties
and strong differences between different plant species and
environmental settings, especially at a molecular level. Fur-
ther research is required to enable extrapolations to or across
ecosystem scales. molecular proxies. However, in some instances they may
also offer opportunities. For instance, n-alkanes can be de-
graded to n-methyl ketones through β-oxidation (Chaffee
et al., 1986; Amblès et al., 1993), which can be used to
assess and trace n-alkane degradation in soils (Jansen and
Nierop, 2009). Similarly, the presence of certain seco acids
formed through the A-ring opening of 3-oxytriterpenoids un-
der anaerobic conditions may be used as a proxy for the
occurrence of such anaerobic episodes (Jaffe et al., 1996),
e.g., under stagnant water conditions. 4.1
Differences related to incorporation pathway The incorporation pathway (Fig. 1) may influence the sub-
sequent turnover of molecular proxies. This includes (differ-
ences in) degradation during senescence and/or litter degra-
dation stages, e.g., due to different input shapes (like root
versus leaf), that offer a different degree of physical protec-
tion. It also includes alterations induced by fire prior to or
upon incorporation of organic matter into the soil. With respect to the effects of fire, the burning of litter or
biomass can release additional extractable lipids (González-
Pérez et al., 2004). In addition, fire has been reported to alter
the chain length distribution of n-alkanes and n-fatty acids,
shifting it towards shorter chain lengths (González-Pérez et
al., 2004; Wiesenberg et al., 2009; Knicker et al., 2013). The
composition of terpenoids can also be influenced, resulting
in the preferential degradation of those with the lowest ther-
mal stability (González-Pérez et al., 2004). All such pro-
cesses potentially adversely affect the application of molec-
ular proxies to an extent that depends on the frequency and
intensity of fires. At the same time, fire events may also offer
opportunities. For instance, the thermal alteration of animal
fats in fireplaces may produce specific n-alkane / n-alkene
doublets preserved in the soil, the presence of which can
be used to reconstruct human fire usage in an archaeologi-
cal context (Lejay et al., 2016). p
p
g
In a study of Ginkgo biloba leaf wax lipids during the
senescence and litter stages, Nguyen Tu et al. (2003) found
limited degradation that did not affect the dominant chain
lengths of alkyl molecular proxies. When comparing differ-
ent classes of wax lipids they found the n-alkanes to be the
most resistant to degradation, followed by the n-fatty acids
and then the n-alcohols (Nguyen Tu et al., 2003). More gen-
erally in a study of grassland and forest soils, Otto and Simp-
son (2005) determined that the characteristic patterns of wax
lipids and isoprenoids were preserved throughout the stages
between fresh plant material and soil organic matter. They
also determined preferential enrichment of suberin with re-
spect to cutin monomers, in particular in one of the grass-
land soils (Simpson et al., 2008). This indicated, for exam-
ple, that the former is embedded in woody tissue, while the
latter is exposed on leaf surfaces (Simpson et al., 2008) (see
also Sect. 4.3.3). 4.2
Differences between different soil compartments (2004) and Flessa et
al. (2008) observed longer turnover rates of SOM in smaller
size fractions. Clemente et al. (2011) studied the preserva-
tion of long-chain aliphatic compounds in three soils with
similar clay mineralogy but different carbon contents and
standing vegetation. Regardless of these differences, they too
found the aliphatic compounds to be preferentially preserved
in the silt and clay fractions and again linked this to strong
interactions with the present clay minerals. In a recent study,
Griepentrog et al. (2015, 2016) confirmed the higher resi-
dence time of organic matter in small size density fractions
when compared to macroaggregates as a result of interac-
tion with the mineral phase. This implies an improved preser-
vation of organic matter associated with higher density and
thus mineral association when compared to organic matter
associated with lower density. However, physical fraction-
ation techniques such as particle and density fractionation
have the potential of creating analytical artifacts, especially
when molecular proxies are investigated. In addition, occlu-
sion or strong adsorption in the smallest mineral fractions
might hamper the extraction and analysis of the proxy in
question. magnitude between forest- and grass-derived molecules after
land use change have been observed as a result of saturation
of the adsorption sites on the mineral phase (Hamer et al.,
2012). In addition to heterogeneity in the effects of interactions
with the mineral phase on the preservation of molecular prox-
ies, analytical artifacts cannot be completely excluded when
physical and chemical fractionation techniques are applied
to separate particle size or density fractions. To date, system-
atic investigations addressing these issues are lacking, which
hampers the drawing of general conclusions with respect to
processes that are relevant, e.g., under different climates and
for different soil mineralogical composition. 4.2
Differences between different soil compartments When soils are used as archives of molecular proxies, mostly
bulk samples are used and replication per horizon or strati-
graphic layer is often limited or absent. However, several
studies indicate that the preservation of molecules used
as proxies can differ between different soil compartments
(Flessa et al., 2008; Clemente et al., 2011; Griepentrog et al.,
2014). Depending on the research question, this may pose
a problem; for instance, it might obscure chronology when
molecules are used as proxies to reconstruct changes over
time. When looking at bulk organic matter in soils, Rasse et
al. (2005) estimated that the main residence time of root-
derived organic matter is on average 2.4 times that of shoot-
derived organic matter. When comparing cutin and suberin
monomers, Andreetta et al. (2013) described the selective
preservation of leaf-derived monomers in the more acidic
and dryer soil, while in the more fertile soil root-derived
monomers were preferentially preserved. They attributed the
former to inhibited microbial degradation due to drought and
acidity and the latter to protection within aggregates. In an-
other study, small differences in the degradation of the same
n-alkanes derived from different plants were found, with a
slower degradation of n-alkanes derived from more woody
roots (Nierop and Jansen, 2009), although lipids were gener-
ally well preserved. Killops and Frewin (1994) reported that
the persistence of plant cuticles protected their composite
isoprenoids from degradation in mangrove sediments. Sim-
ilar preservation in soils is also perceivable. Lichtfouse et al. (1998b) showed that straight-chain lipids
can become encapsulated in larger organic macromolecules,
thus being protected against degradation. In addition, phys-
ical protection in (the micropores of) aggregates and/or
through association with clay minerals has been identified
as an important pathway for the stabilization of soil organic
matter in general, including molecules used as molecular
proxies (Tonneijck et al., 2010). Using bulk and compound-
specific δ13C analysis, Cayet and Lichtfouse (2001) showed www.soil-journal.net/3/211/2017/ SOIL, 3, 211–234, 2017 B. Jansen and G. L. B. Wiesenberg: Plant-derived lipid molecular proxies: opportunities and limitations 224 that plant-derived n-alkanes in a soil under maize cultivation
varied in average age per particle size fraction, with the C31
n-alkane from the 200–2000 µm fraction being significantly
younger than that from the 50–200 and 0–50 µm fractions. A general trend of preferential preservation in smaller size
fractions, in particular the clay fraction, is also reported in
other studies. For instance, Quenea et al. 4.3
Selective preservation within or between classes of
molecules (1995) in which they observed differences in
the preservation of SOM derived from tropical pastures com-
pared to the preceding native savannah vegetation. They at-
tributed this effect to a difference in interactions with the
mineral phase, leading to the physical protection of SOM
and the molecular proxies contained therein. Similarly, dif-
ferences in the turnover rates of approximately 1 order of Furthermore, the effects of size or density fractions of the
soil on the preservation of organic matter, including molecu-
lar proxies, are not uniform. For instance, Höfle et al. (2013)
found size- and density-fraction-related organic matter stabi-
lization to be much less pronounced in the active upper layer
than in the deeper soil horizons. This points to the selective
preservation of organic matter in the deeper soil because of
more extensive aggregation and organo-mineral association. In a study of volcanic ash soils, Stewart et al. (2011) did
not find differences in the preservation of bulk SOM in gen-
eral or lipids in particular between different size fractions. They attributed this lack of differentiation to the presence of
a large proportion of SOM that was not associated with min-
eral components as these were already saturated with previ-
ously incorporated soil organic matter (Stewart et al., 2011). An important issue with respect to the application of
straight-chain lipids as molecular proxies is also the prefer-
ential degradation of certain chain lengths within a certain
class of molecules, as molecular ratios of various (higher)
chain lengths are often used as proxies for certain vegetation
types (see Sect. 2). This issue is addressed in the following
sections. In general a combination of physical protection and sorp-
tive preservation seems to be responsible for the observed
differences (or lack thereof) in the preservation of organic
molecules in soils between different size or density fractions. This is corroborated amongst others by a study by Guggen-
berger et al. (1995) in which they observed differences in
the preservation of SOM derived from tropical pastures com-
pared to the preceding native savannah vegetation. They at-
tributed this effect to a difference in interactions with the
mineral phase, leading to the physical protection of SOM
and the molecular proxies contained therein. Similarly, dif-
ferences in the turnover rates of approximately 1 order of SOIL, 3, 211–234, 2017 4.3
Selective preservation within or between classes of
molecules The turnover rates of molecular proxies not only vary be-
tween different compartments, but may also vary within
the same compartment between and even within differ-
ent (classes of) molecules (Dinel et al., 1990; Bull et al.,
2000; Amelung et al., 2008). For instance, Feng and Simp-
son (2007) found preferential enrichment of straight-chain
lipids and cutin and suberin monomers with increasing
depth with respect to bulk SOM. In contrast, in a study
of grain-maize and silage-maize cropped soils, Wiesenberg
et al. (2004) found turnover times in the sequence bulk
SOM > n-alkanes > n-fatty acids, with rate differences that
varied substantially between the two cultivations. The dif-
ferences could be related to the different biomass input on
the one hand and large amount of lignite dust and the low
biomass input on the other hand, thus hampering degrada-
tion at this site. The faster turnover of n-fatty acids than n-
alkanes was also confirmed by Wiesenberg et al. (2008a) and
Griepentrog et al. (2015, 2016). This may also offer opportu-
nities to apply such differences between molecular classes
and their response to external factors to trace transforma-
tions and input of organic matter in soils (Feng and Simpson,
2007). q
Furthermore, the effects of size or density fractions of the
soil on the preservation of organic matter, including molecu-
lar proxies, are not uniform. For instance, Höfle et al. (2013)
found size- and density-fraction-related organic matter stabi-
lization to be much less pronounced in the active upper layer
than in the deeper soil horizons. This points to the selective
preservation of organic matter in the deeper soil because of
more extensive aggregation and organo-mineral association. In a study of volcanic ash soils, Stewart et al. (2011) did
not find differences in the preservation of bulk SOM in gen-
eral or lipids in particular between different size fractions. They attributed this lack of differentiation to the presence of
a large proportion of SOM that was not associated with min-
eral components as these were already saturated with previ-
ously incorporated soil organic matter (Stewart et al., 2011). In general a combination of physical protection and sorp-
tive preservation seems to be responsible for the observed
differences (or lack thereof) in the preservation of organic
molecules in soils between different size or density fractions. This is corroborated amongst others by a study by Guggen-
berger et al. 4.3.1
Straight-chain lipids Moucawi et al. (1981a) reported decreasing degradation rates
with larger chain lengths for n-alkanes in soils, which was
confirmed by Lichtfouse et al. (1998a), who determined a
higher resistance of long straight-chain lipids in soil com-
pared to their shorter-chain counterparts. However, such
preferential degradation was found in agricultural and acidic www.soil-journal.net/3/211/2017/ SOIL, 3, 211–234, 2017 B. Jansen and G. L. B. Wiesenberg: Plant-derived lipid molecular proxies: opportunities and limitations 225 soils in the absence of Fe(OH)3 (Moucawi et al., 1981a;
Lichtfouse et al., 1998a). Similar results were found for
other lipid classes as well (Moucawi et al., 1981b). More re-
cently, several authors have also indicated that such preferen-
tial degradation can occur in other soils (Jansen and Nierop,
2009; Cui et al., 2010). However, the extent of the effect calls
into question the suitability of the compounds as molecu-
lar proxies. For instance, Jansen and Nierop (2009) found
the overall effect of preferential degradation on higher-plant-
derived n-alkane patterns in soils to be small and not of in-
fluence for their use as a vegetation proxy. Similarly, Lei et
al. (2010a, b) determined that in spite of strong evidence of
microbial degradation, the relative abundance of long-chain
n-alkanes could still be used to distinguish coniferous from
broadleaf tree input in soils. cutin and suberin to be more resistant to degradation than
free lipids residing in the same particle size fraction. In a study of hydrolysable lipids using compound-specific
13C analysis, Feng et al. (2010) described the mean turnover
times for cutin- and suberin-derived ester-bound lipids of 32–
34 years. While slower than for bulk soil organic matter in
this system, it was much shorter than anticipated, leading
them to conclude that a large portion of cutin- and suberin-
derived compounds reside in the non-hydrolysable fraction
(Feng et al., 2010). As mentioned earlier (Sect. 4.1), Simpson et al. (2008)
observed the preferential enrichment of suberin monomers
with respect to cutin monomers, which was confirmed by
Mendez-Millan et al. (2010b). In addition to the physical
location of suberin versus cutin as a potential cause, Simp-
son et al. (2008) suggested a higher resistance of suberin to
degradation than cutin owing to a larger content of pheno-
lic units in the former. Mendez-Millan et al. (2010b) argued
that microbial degradation, potentially influenced by the ac-
cess to degradation sites, is another factor influencing the
slower turnover of suberin versus cutin monomers. 4.4
Conclusions and implications regarding differences
in transformations and turnover of molecular proxies
in soils Although available data are limited, it is clear that the degra-
dation of organic matter at a molecular level in terrestrial
archives such as soils, paleosols, and sediments can signif-
icantly influence the applicability of molecular proxies. As a
result it seems useful to explore the possibility of a correction
to improve the determination of paleovegetation, vegetation
shifts, and other paleoenvironmental information like pale-
otemperature and pH. The number of published approaches
to compensate for the influence of degradation on paleoenvi-
ronmental reconstructions is still small. Zech et al. (2009a)
provided a simple two-end-member model approach to im-
prove paleovegetation reconstruction based on the molecu-
lar ratios of different long-chain n-alkanes (C27–C33). As-
suming that forest vegetation is dominated by n-C27 alkane
and grass vegetation by n-C31 and n-C33 alkanes, high rel-
ative contributions of the respective homologues of the as-
sumed source vegetation are used as end-members. At the
same time, the source vegetation is typically characterized by
high odd-over-even predominance of long-chain n-alkanes. On the other hand, soils reveal a low odd-over-even predom-
inance and the abovementioned molecular ratios with smaller
differences between the different vegetation types. In theory,
the degradation continuum from the plant leaves to the soils
of the respective vegetation type thus enables the identifica- In addition, 15 sterols are transferred both aerobically and
anaerobically to 5α- and 5β-stanols (De Leeuw and Baas,
1986), which are reported to persist much longer in soils than
their precursors (Bull et al., 2000). Simpson et al. (2008) sug-
gest using the ratio of precursor sterols to their stanol and
stanone degradation products as a measure of their degree of
degradation. 4.3.1
Straight-chain lipids Regard-
less of the mechanism, the general difference in root versus
aboveground biomass-derived suberin and cutin monomers
and their individual turnover would clearly influence the ap-
plication of the cutin / suberin monomer ratio as a proxy for
leaf versus root input. Within the group of straight-chain lipids, the overall degra-
dation rates of subclasses have been found to vary depending
on soil physicochemical properties. For instance, n-alkanes
have been reported to be better preserved in alkaline soils,
whereas n-fatty acids accumulate in more acidic soils (Simp-
son et al., 2008). 4.3.2
Isoprenoids Isoprenoids are reported to have varying turnover rates un-
der both oxic and anoxic conditions in soils (Jaffe et al.,
1996; Amelung et al., 2008). Generally, sterols, diterpenes,
and pentacyclic, triterpenes are reported to be turned over
rapidly compared to straight-chain lipids in grassland and
forest soils, hindering their application as molecular prox-
ies for their sources (Bull et al., 2000; Naafs et al., 2004b;
Jansen et al., 2007). However, Otto and Simpson (2005) ob-
served the exact opposite trend, indicating a strong environ-
mental control on the relative transformation rate of differ-
ent classes of components. In an incubation study of de-
rived triterpenols, Koch et al. (2005) highlighted marked dif-
ferences between the degradation rates of individual triter-
penols, leading to a sharp relative increase in the proportion
of taraxerol with respect to the other triterpenols. 5 4.4
Conclusions and implications regarding differences
in transformations and turnover of molecular proxies
in soils SOIL, 3, 211–234, 2017 5
General conclusions (2010) who also used long-chain n-alkane ratios and
the odd-over-even predominance of alkanes for their correc-
tion. While Zech et al. (2009a) used correlations and then
graphically based reconstructions, Buggle et al. (2010) used
a calculation-based approach. The degradation in the contin-
uum from recent soils is taken as an analogy and the slope
of the regression line is multiplied with the odd-over-even
predominance and the addition of the intercept of a long-
chain n-alkane ratio in the cross plot of the ratio with the
odd-over-even predominance. By moving the regression line
to an ancient sample set, the end of the regression line yields
the former topsoil value of the molecular ratio and odd-over-
even predominance. Variation in the corrected long-chain n-
alkane ratio enables the assessment of fluctuations in pale-
ovegetation. Both of the mentioned approaches rely on the general dif-
ferentiation of grass versus forest vegetation based on long-
chain n-alkane composition. As mentioned above, such a
clear distinction of vegetation types exclusively based on
compounds deriving from one compound fraction, such as n-
alkanes, might be hampered by various factors such as vari-
ability within and between plant species, thus leading to sim-
ilar composition of, for example, n-alkanes from coniferous
trees and grass plants (Maffei, 1996b; Maffei et al., 2004). Thus, such simple approaches might be appropriate only in
very well-defined settings in which independent records such
as pollen data confirm the composition of specific plant as-
semblages determined by molecular proxies. The expansion of approaches like the ones mentioned
here to a broader range of molecular proxies is required to
receive a more complete picture and to acknowledge the
different turnover and degradation of different substance
classes. However, the availability of datasets on plant and
soil chemical composition for substance classes other than
the n-alkanes is quite limited, hindering such expanding ap-
proaches. Thus, further surveys are required for molecular
proxies other than n-alkanes for a high diversity of plants and
soils from different climates. Afterwards, combined stud-
ies of more than one substance class will enable improved
paleoenvironmental reconstructions, whereas cross-checking
with other nonmolecular proxies, e.g., fossil pollen data,
might be essential, especially if the paleorecord is targeted. 5
General conclusions In this review we considered the three most important con-
straining factors for the application of molecular proxies in
soil science: (i) variability in the molecular composition of
plant-derived organic matter as a result of genetic or life stage
variations or external environmental factors; (ii) variability
in (the relative contribution of) input pathways into the soil;
and (iii) the transformation and/or (selective) degradation of
(some of) the molecules once present in the soil. From the
various studies within and outside of soil science over the
last decades, the following general picture emerges. All con-
straining factors considered can have a significant influence
on the applicability of molecular proxies in soil science. The
degree of influence of the constraining factors strongly de-
pends on the type of molecular proxy and the environmental
context in which it is applied. In addition, the research ques-
tion to be addressed through the application of the molecu-
lar proxy has a strong influence. A factor that poses a con-
straining factor in one study might offer an opportunity in
another. For instance, the fire-induced alteration of biomass
may release lipids to the soil that potentially confound their
chemotaxonomic application, but may offer opportunities for
the reconstruction of the occurrence of human-induced fire
in an archaeological context. Recently, the first modeling
approaches to potentially compensate for some of the con-
straining factors, specifically variability in input pathways
and the degradation of molecular proxies once in the soil,
have started to emerge. Based on the above discussion, we
strongly recommend that the potential constraining factors
always be explicitly considered whenever studies are planned
in which molecular proxies in soils play a role. This review
may serve as a starting point for gathering the necessary in-
formation to decide which constraining factors may play a
role and how they can best be addressed. At the same time,
it became clear from the available literature that much infor-
mation about the mentioned constraining factors is still lack-
ing. In particular for molecular classes other than n-alkanes,
systematic information is often very scarce. We therefore
strongly appeal to the soil scientific community to address
this knowledge gap. Our review may also serve as a starting
point in this respect with future applicability in soil science
and in paleopedology. A slightly different approach was established by Buggle
et al. 4.3.3
Cutin and suberin monomers Bull et al. (2000) observed different degradation rates for dif-
ferent components within the classes of free and ester-bound
lipids, depending on soil chemical and physical composition. However, Otto and Simpson (2006) found the degradation of
cutin and suberin to take place without preference for spe-
cific constituents. In general, Quenea et al. (2004) described www.soil-journal.net/3/211/2017/ SOIL, 3, 211–234, 2017 B. Jansen and G. L. B. Wiesenberg: Plant-derived lipid molecular proxies: opportunities and limitations 226 but also to acknowledge and identify the degradation of or-
ganic matter at a molecular scale. tion of the degradation intensity of an unknown sample if the
sample is mainly influenced by a single vegetation. If the un-
known sample does not plot on the degradation continuum
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General conclusions The extrapolation of such approaches to different environ-
mental and climatic settings might also be limited as the ef-
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factors influence the degradation of organic matter as dis-
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are required to assess not only palaeoenvironmental changes, Data availability. No data sets were used in this article. Data availability. No data sets were used in this article. Competing interests. The authors declare that they have no con-
flict of interest. www.soil-journal.net/3/211/2017/ SOIL, 3, 211–234, 2017 B. Jansen and G. L. B. Wiesenberg: Plant-derived lipid molecular proxies: opportunities and limitations 227 B. Jansen and G. L. B. Wiesenberg: Plant-derived lipid molecular proxies: opportunities and limitations Bakht, J., Bano, A., and Dominy, P.: The role of abscisic acid and
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Analysis of Plant Growth and Gallic Acid Content for Cavendish Banana (Musa acuminata) Shoot Culture with Bubble Column Bioreactor
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Journal of Integrated and Advanced Engineering
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Journal of Integrated and Advanced Engineering
(JIAE)
Vol. 2, No. 1, March 2022: 33-44
http://asasijournal.id/index.php/jiae
http://doi.org/10.51662/jiae.v2i1.37 Journal of Integrated and Advanced Engineering
(JIAE)
Vol. 2, No. 1, March 2022: 33-44
http://asasijournal.id/index.php/jiae
http://doi.org/10.51662/jiae.v2i1.37 Journal of Integrated and Advanced Engineering
(JIAE)
Vol. 2, No. 1, March 2022: 33-44
http://asasijournal.id/index.php/jiae
http://doi.org/10.51662/jiae.v2i1.37 Analysis of Plant Growth and Gallic Acid Content
for Cavendish Banana (Musa acuminata) Shoot
Culture with Bubble Column Bioreactor Nunung Nurhayati, Rizkita Rachmi Esyanti*, Khalilan Lambangsari
1Department of Bioengineering, School of Life Sciences and Technology, Institut Teknologi Bandung, Indonesia Keywords:
Bubble Column Bioreactor;
Cavendish Banana;
Gallic Acid;
Tissue Culture; Abstract Abstract
Cavendish banana (Musa acuminata) is one of the most important fruits in the
world. Cavendish shoots tissue culture using bubble column bioreactor can be a
solution to produce high yielding plantlet and gallic acid due to the aeration with
minimum shear stress. In this study, the average growth rate, presence of gallic
acid, and antioxidant activity (IC50) in the bubble column bioreactor (200 mL
capacity) with the aeration rates of 1 mL/s and 2 mL/s using Murashige & Skoog
half-strength liquid medium supplemented with 0.5 ppm gibberellic acid will be
analyzed. The aeration system used was atmospheric air. The leaves and stems
were extracted by maceration using 96% ethanol solvent (1:10 (w/v)). A qualitative
phenolic test with FeCl3, thin layer chromatography, and antioxidant test with 2,2-
diphenyl-1-picrylhydrazyl was carried out. The average growth rate in the
bioreactor were 0.22 ± 0.001 g/day (1 mL/s) and 0.21 ± 0.001 g/day (2 mL/s). All
the leaf and stem extracts showed positive results for the phenolic test, but the
presence of gallic acid could not be detected clearly by thin-layer chromatography. The IC50 values in aeration rates of 1 mL/s and 2 mL/s of the leaves were 41.35
and 79.54 μg/mL, respectively, while the stems were 51.87 and 104.94 μg/mL,
respectively. It could be concluded that the growth of the banana plantlet and the
production of antioxidants in the bubble column bioreactor was higher in aeration
rate of 1 mL/s than 2 mL/s. Article History:
Received: January 25, 2022
Revised: March 12, 2022
Accepted: March 17, 2022
Published: March 30, 2022 Corresponding Author:
Rizkita Rachmi Esyanti
Bioengineering Department,
Institut Teknologi Bandung,
Indonesia
Email: rizkita@sith.itb.ac.id This is an open access article under the CC BY-SA license N. Nurhayati et al., Analysis of Plant Growth and Gallic Acid Content for Cavendish Banana … This is an open access article under the CC BY-SA license INTRODUCTION Cavendish banana (Musa acuminata) is one of the most important commercial fruits in
the world. This banana is very popular and has become the most widely traded globally,
including in Indonesia and Malaysia [1]. Based on Statistics Indonesia (BPS), banana
production in Indonesia generally reached 7,280,658 tons in 2019, and it took as the first
position for the fruits category [2]. Cavendish banana belongs to the Musaceae family with
AAA triploid [3]. The fruit of Cavendish banana has a distinctive shape and taste as well as
contains complete nutrients, such as carbohydrates, fiber, potassium, calcium, manganese,
and vitamins, so it is very popular with people in various regions [4]. Apart from the fruit,
other parts of the banana plant, particularly the leaves and stems (pseudo-stems), have
much medicinal use [5]. In addition, Cavendish banana plants can also widely adapt to
various conditions in tropics and subtropics [6]. The production of superior seeds using tissue culture can increase productivity and so to
overcome the high demand for Cavendish bananas [7]. Tissue culture is an in vitro plant
propagation that is carried out under controlled conditions by utilizing tissue and the
totipotency of plants to produce large numbers of plant clones in a short time [8, 9, 10]. Compared to conventional methods, the advantages of tissue culture techniques include
similarity of genetic characteristics as mother plants, disease-free, faster growth, and faster 33 Journal of Integrated and Advanced Engineering (JIAE)
Vol. 2, No. 1, March 2022: 33-44 Journal of Integrated and Advanced Engineering (JIAE)
Vol. 2, No. 1, March 2022: 33-44 maturation process [11]. On the other hand, tissue culture is also very beneficial for
production of certain metabolites, because propagation can be adapted to the tissue with
the highest content of the desired metabolite so that the extraction process and metabolite
acquisition will be more efficient [11]. maturation process [11]. On the other hand, tissue culture is also very beneficial for
production of certain metabolites, because propagation can be adapted to the tissue with
the highest content of the desired metabolite so that the extraction process and metabolite
acquisition will be more efficient [11]. The use of bioreactors in tissue culture can also optimize the growth process and
accumulation of secondary plant metabolites. One of the potential bioreactors to be
developed is the bubble column bioreactor. METHOD
Materials In this study, Cavendish banana explants were obtained from the School of Life Sciences
and Technology inventory, Bandung Institute of Technology, Ganesha Campus, with the
characteristic of about 3-4 leaves and was approximately one month after the initiation
process. The chemicals used in this study were Murashige & Skoog (MS) medium
(PhytoTech LABS), sucrose, Gibberellic Acid (GA3) (HIMEDIA), ethanol (Merck, 96%),
FeCl3 (Smart-Lab), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Smart-Lab). Medium Preparation The pH condition in each medium was set at 5.8. Then, the medium was sterilized by
autoclaving at 121℃ for 15 minutes. N. Nurhayati et al., Analysis of Plant Growth and Gallic Acid Content for Cavendish Banana … INTRODUCTION A bubble column bioreactor is a multiphase
cylindrical reactor with a submerged culture system equipped with a bubble gas spout into
the liquid system or liquid-solid suspension [12]. The bubbles are used to mix, circulate,
and aerate nutrients in medium culture with minimal shear stress not to damage the culture
[13]. This causes high gas transfer and nutrient transfer rates [14]. In addition, the use of
liquid media in bioreactors also has several advantages, such as renewing the air during
cultivation and higher plantlet growth rates and lower production costs compared to solid
media [15]. The Cavendish banana plant can also be developed through the biorefinery concept by
utilizing its metabolite content. Parts of the banana plant other than fruit, such as leaves
and stems, which are often only used as low-value products or simply thrown away,
contain polyphenol compounds in the form of gallic acid, which has the pharmacological
and economic potential [16]. Gallic acid (3,4,5-trilhydroxybenzoic acid) is a plant
polyphenol found in tea, berries, grapes, and other fruits that is cytotoxic for many types of
tumor cells and is used as a chemoprevention drug in cancer treatment [17]. Gallic acid
affects several pharmacological and biochemical pathways, making it known as a strong
antioxidant, anti-inflammatory, antimutagenic and anticancer compound [17]. Sagrin &
Chong [18] reported that banana leaves have a quite high gallic acid content, which is
around 2.7% (w/w). In banana stems and humps, a small number of gallic acid and other
phenolic compounds have also been found [19, 20, 21]. Therefore, banana plants offer a
great opportunity as an affordable source for producing bioactive compounds. This study aims to compare the plant growth, the presence of gallic acid, and the
antioxidant activity of Cavendish banana shoots (Musa acuminata) in bubble column
bioreactor at various aeration rates of 1 mL/s dan 2 mL/s. Shoot Culture in Bubble Column Bioreactor The acclimatized Cavendish banana shoot cultures were cultivated in a bubble column
bioreactor with 200 mL medium. The medium used was the same as in the control system. The initial weight (g) and height (cm) of the explants were measured. Then, the air for
aeration was transferred to the system using an air pump. A flow meter sets Aeration rates at 1
mL/s and 2 mL/s. This system was operated for 14 days at room temperature with a lighting
period of 24 hours. Cavendish Banana Shoot Subculture and Acclimatization The method used has been optimised in the School of Life Sciences and Technology
laboratory for four years. The medium used in the subculture process was a solid growth
medium consisting of full-strength Murashige & Skoog (MS) medium, 30 g/L sucrose, 1 ppm 34 p-ISSN: 2774-602X e-ISSN: 2774-6038 Gibberellic Acid (GA3), and 8 g/L agar. Subcultures were performed three times within six
weeks. First, the newly formed shoots were ready to be acclimatised in a liquid medium
consisting of half-strength MS and sucrose 20 g/L without hormones. Banana cultures were
then incubated at room temperature and 24-hour lighting for one week. Shoot Culture in Thin-Layer System y
y
The acclimatized Cavendish banana shoot cultures were cultivated in a thin-layer system
with 25 mL medium. The medium used was half-strength MS, 20 g/L sucrose, and 0.5 ppm
GA3. The initial weight (g) and height (cm) of the explants were measured. The culture was
incubated on a shaker with a rotation speed of 60 rpm at room temperature and a 24-hour
lighting period for 14 days. The experiment was conducted in triplicate. Plant Growth and Medium Analysis The final weight (g) and height (cm) of the explants were measured after cultivating. The
average growth rate of plant height and biomass were calculated as the difference between the
initial and the final conditions, then divided by the cultivation time. Sucrose levels and
conductivity in the cultivation medium were measured using a refractometer and conductivity
meter, respectively. N. Nurhayati et al., Analysis of Plant Growth and Gallic Acid Content for Cavendish Banana … Determination of Phenolic Compound The determination of phenolic compounds in banana leaf and stem extracts was carried out
with a qualitative test using FeCl3 [24, 25, 26]. A total of 1 mL of each extracted sample was
taken and one drop of 5% FeCl3 solution was added. Gallic acid solutions of 0,01 M and 0,5
mM were used as controls. The color change from brownish yellow to dark green indicates
the presence of phenolic compounds in the extract, as shown in Figure 1. (3) Subsequently, 5% FeCl3 solution was sprayed onto the plate surface to detect phenolic in
the obtained stains. The existence of gray or blackish gray dots confirmed the presence of
phenolic in the extracts [25]. Determination of Antioxidant Activity An antioxidant test was carried out based on Ayoola et al. [5]. A total of 2 mL of banana
leaf and stem extract at various concentrations (100, 50, and 25 μg/ml) and control were
added to 3 mL of 0,1 mM DPPH solution. The solution mixture was vigorously stirred and
incubated in the dark for 30 minutes at room temperature. The absorbance of the solution was
measured using a spectrophotometer at 517 nm wavelength. The degree of color changes of
the DPPH from purple to clear yellowish indicates the efficiency of the extract's free radical
molecule reforming activity. The DPPH inhibition was calculated using (4). (4) Determination of Moisture Content and Extract Yield The leaves and stems of plantlets in the control and bioreactor after cultivating were then
separated, and each was weighed as Mfresh (g). The plantlets were dried in an oven at 105℃
for 15 hours [23]. Further, each part of the dry plantlet was weighed as Mdry (g). The moisture
content was determined using (1) [23]. (1) (1) (1) Dried plant parts were then crushed and extracted by the maceration method using 96%
ethanol (1:10 (w/v) for 24 hours [24]. Hereafter, the solvent was evaporated using a rotary
vacuum evaporator for 10-15 minutes to obtain the crude extract weighed as Mextract (g) and
the yield was determined using (2) [24]. (2) (2) Then, the extract was dissolved in 96% ethanol and stored at -4℃ for further analysis. Nurhayati et al., Analysis of Plant Growth and Gallic Acid Content for Cavendish Banana … 35 Journal of Integrated and Advanced Engineering (JIAE) g
Vol. 2, No. 1, March 2022: 33-44 Phenolic compound
Blue/green/black complex
Figure 1. The reaction of Phenolic Compound with FeCl3 Phenolic compound Blue/green/black complex Figure 1. The reaction of Phenolic Compound with FeCl3 Thin Layer Chromatography Analysis Thin-layer chromatography (TLC) method was carried out based on Sonam et al. [25] with
a modified TLC plate of silica gel 60 F254 (Merck, Germany) 7 cm × 3 cm. Gallic acid
solutions were used as controls. First, the sample was applied to the silica plate in the lower
limit and eluted using ethanol:water (6:3) as the mobile phase. Then, the plate was incubated
until the solvent reached the upper limit of the plate. The Rf value is calculated using (3). N. Nurhayati et al., Analysis of Plant Growth and Gallic Acid Content for Cavendish Banana … Subculture and Acclimatisation Subculture and Acclimatisation The cultivation of Musa acuminata shoot cultures in solid Murashige & Skoog (MS)
medium with 0.5 and 1 ppm of Gibberellic Acid (GA3) hormones for four weeks aimed to
prepare Cavendish banana explants that will be cultivated in the thin-layer system, as a role of
the unaerated system, and bubble column bioreactor. After four weeks, banana explants on
GA3 medium showed shoot elongation but did not differ much from each other, as depicted in
Figure 2a. Giberellic acid (GA3) is a growth regulator which has an important role in 36 p-ISSN: 2774-602X e-ISSN: 2774-6038 inducing shoot formation, promoting shoot elongation, and facilitating single node separation
of plants [8, 27, 28]. inducing shoot formation, promoting shoot elongation, and facilitating single node separation
of plants [8, 27, 28]. Acclimatization of Cavendish banana shoots was carried out in a thin-layer system
containing a half-strength MS liquid medium without hormones. It aimed to adapt the
explants in a liquid medium before being cultivated into the treatment used, thus enabling an
optimal response of banana plants after being cultured in the treatments [29]. After seven days
of the process, the explants showed normal morphology and could be further used in a thin-
layer system, which will be called TLS, and bubble column bioreactor as shown in Figure 2b. Visual Analysis Both control and bioreactor systems used half-strength MS liquid medium and 0.5 ppm of
GA3 for 14-day cultivation, as shown in Figure 2c and Figure 2d. Visually, some explants in
TLS showed hyperhydricity, where some of the leaves were light brown and died or abscised,
as depicted in Figure 3b. Hyperhidricity is a physiological disorder in plant tissue cultured in
vitro due to containing too much water [30]. In TLC, the liquid environment constantly
soaking the explants will drastically reduce transpiration. As a result, the water absorbed from
the medium is not transpired sufficiently and will accumulate in the intercellular tissue [30]. While in the bubble column bioreactor treatment, both flow rate variations resulted in a
normal explant appearance after 14 days, where the leaves were green, and the stems were
healthy, as shown in Figure 3c and Figure 3d. N. Nurhayati et al., Analysis of Plant Growth and Gallic Acid Content for Cavendish Banana … Plant Growth Rate The growth rate of Cavendish banana (Musa acuminata) in the bubble column bioreactor
treatment was higher than the TLS. The highest increase in plant height was obtained in the
flow rate variation of 1 mL/s, so as resulted in the largest average height growth rate of 0.04 ±
0.009 cm/day as listed in Table 1. The largest average biomass growth rate was also obtained
in the flow rate variation of 1 mL/s, which was 0.22 ± 0.001 g/day, while the lowest was in
the TLS. a. b. c. d. Figure 2. Shoot Culture of Cavendish Banana in a. Solid Medium, b. Acclimatization, c. TLS, d. Bubble Column
Bioreactor c. d. b. a. d. b. c. a. c. 2. Shoot Culture of Cavendish Banana in a. Solid Medium, b. Acclimatization, c. TLS, d. Bubble Colum
Bioreactor a. Before
b. TLS
c. 1 mL/s Rate
d. 2 mL/s Rate
After
Figure 3. Visual observation of Cavendish Culture in TLS and Bubble Column Bioreactor Treatment a. Before d. 2 mL/s Rate c. 1 mL/s Rate
After b. TLS
c d. 2 mL/s Rate b. TLS c. 1 mL/s Rate
After a. Before 1 mL/s Ra
After After Figure 3. Visual observation of Cavendish Culture in TLS and Bubble Column Bioreactor Treatment N. Nurhayati et al., Analysis of Plant Growth and Gallic Acid Content for Cavendish Banana … 37 Journal of Integrated and Advanced Engineering (JIAE)
Vol. 2, No. 1, March 2022: 33-44 Journal of Integrated and Advanced Engineering (JIAE)
Vol. 2, No. 1, March 2022: 33-44 Table 1. Average Growth Rate of Cavendish Banana in Bubble Column Bioreactor
Treatments
Δ Plant Height
(cm)
Δ Biomass
(g)
Average Growth Rate of
Plant Height
(cm/day)
Average Growth Rate
of Biomass
(g/day)
Flow rate of 1 mL/s
0,53 ± 0,125
3,02 ± 0,005
0,04 ± 0,009
0,22 ± 0,001
Flow rate of 2 mL/s
0,45 ± 0,250
2,94 ± 0,005
0,03 ± 0,025
0,21 ± 0,001 Table 1. Average Growth Rate of Cavendish Banana in Bubble Column Bioreactor In the bubble column bioreactor, the lower flow rate will result in the minimum shear
stress generated by bubble gases in the bioreactor. As a result, the medium's rate of gas
transfer and nutrient transfer will increase [14]. Therefore, biomass production is higher than
at higher flow rates [14]. Plant Growth Rate Treatment in flow rate of 1 mL/s resulted in the highest increase in
biomass growth rate compared to flow rate of 2 mL/s and TLS, which was 1.9 times greater
than the initial average weight of 3.1 g in 14 days. This result is further supported by Esyanti
et al. [29], who obtained a 1.8-fold increase in Aquilaria malaccensis shoot culture biomass
using a small flow rate variation of 0.42 mL/s. The shoot growth rate depends on the
medium's surface area in direct contact [31]. The bubble column bioreactor has a high
probability of direct contact between the shoot surface area and the medium. Still, the shoots
are not completely soaking in the liquid culture system, so it takes more energy to transfer
nutrients to the meristematic tissue that is in direct contact with the medium [31]. In addition,
the bubble column bioreactor also uses non-metallic agitation, which reduces shear stress and
provides a continuous supply of oxygen. Nevertheless, the oxygen content in the medium
from all treatments was not measured because it is implied that the increase of aeration rate
will increase the rate of oxygen absorption in the medium [32][33]. N. Nurhayati et al., Analysis of Plant Growth and Gallic Acid Content for Cavendish Banana … Moisture Content and Extract Yield Figure 4 shows the results that all samples of Cavendish banana leaves and stems from
each treatment had a moisture content of more than 90%. In general, banana leaves have a
lower moisture content than banana stems. These results are in accordance with the research
of Costa et al. [39] and Ramdhonee & Jeetah [23], who found that the moisture content in
banana (Musa spp.) leaves and stems was 93.95% and 96.3%, respectively. Furthermore, the
highest moisture content was observed in the sample of the TLS, while the lowest was in the
bubble column treatment with a flow rate of 1 mL/s. This shows that in TLS, banana plants
hold up more water than in the bioreactor treatment. The percentage yield of crude ethanol extract in all treatments has various values between
14.89%-21.07%, as shown in Figure 4. Based on Ayoola et al. [5], the ethanol extract of
banana leaves (Musa spp.) ranged from 6.15% to 42.04%. In general, stem samples at a flow
rate of 1 mL/s produced the highest extract yields among all samples, while leaf samples'
highest result was obtained at a flow rate of 2 mL/s. A high aeration rate is beneficial for
accelerating oxygen transfer into the bioreactor, where the process increases the accumulation
of secondary metabolites and cell growth [40, 41, 42]. Medium Analysis The Change of Conductivity, pH, and Sucrose in Medium
Treatments
Δ Conductivity
(mS)
Δ pH
Δ Sucrose Level
(g/L)
Flow Rate of 1 mL/s
0,05 ± 0,03
1,9 ± 0,10
0,33 ± 0,21
Flow Rate of 2 mL/s
0,23 ± 0,23
1,3 ± 0,00
0,12 ± 0,10
Figure 4. Moisture Content and Extract Yield of Cavendish Leaves and Stems Table 2. The Change of Conductivity, pH, and Sucrose in Medium
Treatments
Δ Conductivity
(mS)
Δ pH
Δ Sucrose Level
(g/L)
Flow Rate of 1 mL/s
0,05 ± 0,03
1,9 ± 0,10
0,33 ± 0,21
Flow Rate of 2 mL/s
0,23 ± 0,23
1,3 ± 0,00
0,12 ± 0,10
Figure 4. Moisture Content and Extract Yield of Cavendish Leaves and Stems Figure 4. Moisture Content and Extract Yield of Cavendish Leaves and Stems N. Nurhayati et al., Analysis of Plant Growth and Gallic Acid Content for Cavendish Banana … Medium Analysis y
TLS treatment resulted in the highest decrease of electrical conductivity (EC) than others,
which was around 55%. EC is expressed in mS (milisiemens) and generally increases when
the ion concentration in the medium is high [35]. According to Esyanti et al. [34], the
decrease in the EC medium occurred because of nutrients absorption by the shoots for its
growth. However, the results of this study are not by the statement, where TLS, which has the
highest decrease in conductivity, produces the lowest growth rate of biomass. This allows for
the influence of the remaining sucrose content in the final medium, where the EC of the
medium is changed in parallel with the consumption of sucrose [31]. All treatments showed a decrease in the final medium pH after 14 days from the initial pH
conditions, as listed in Table 2. The highest decrease was resulted in a flow rate variation of 1
mL/s, from 5.8 to 3.9. The decrease in pH indicates the consumption of nutrients in the form
of ions by plants. Therefore, the pH conditions will affect the absorption of nutrients in the
medium and regulate salt formation [8]. In media containing NO3- and NH4+ with an initial
pH of 5-6, the uptake of NH4+ was preferred and caused the pH to drop during the initial
growth of the culture [8]. All treatments generally showed a decrease in the final sucrose level, as shown in Table 2. The decrease in medium sucrose was exceedingly small in bubble column bioreactor
treatment flow rate variations. At the same time, TLS resulted in the greatest decrease of
sucrose level in the medium. Sucrose is a crucial factor for the growth of plant biomass on in
vitro culture [29]. The decrease in sucrose at the end of the treatment indicated the use of
carbon sources by plants. Sucrose acts as an energy source for growth, biosynthesis, and other
metabolic processes [36, 37, 38]. The bubble column bioreactor was reported to have the most
effective bioconversion compared to the TIS-RITA® bioreactor and thin-layer culture, with
the lowest sucrose consumption, at 20%. In contrast, thin-layer consumed the highest sucrose,
at 40% among the three [31]. 38 p-ISSN: 2774-602X e-ISSN: 2774-6038 Table 2. Phenolic Test Stem
TLS
Gallic
acid
Leaf
TLS
Leaf
1 mL/s
Stem
1 mL/s
Leaf
2 mL/s
Stem
2 mL/s
Gallic Acid
Leaf
TLS
Stem
TLS
Leaf
1 mL/s
Leaf & Stem
2 mL/s
Stem
1 mL/s Before
After
Figure 5. Result of Phenolic Test on Cavendish Leaf and Stem Extracts with FeCl3
Stem
TLS
Gallic
acid
Leaf
TLS
Leaf
1 mL/s
Stem
1 mL/s
Leaf
2 mL/s
Stem
2 mL/s
Gallic Acid
Leaf
TLS
Stem
TLS
Leaf
1 mL/s
Leaf & Stem
2 mL/s
Stem
1 mL/s Table 3. Result of Phenolic Test
Treatments
Phenolic Test Result
Leaf
Stem
Flow rate of 1 mL/s
+
+
Flow rate of 2 mL/s
+
+ This factor makes the color changes in the leaf samples tend to be darker than the stems
after the addition of FeCl3. The difference in color between the gallic acid solutions, which is
more blue-green, and the leaf samples may occur because of the presence of compounds other
than polyphenols, such as flavonoids and tannins, in the banana leaf samples [44]. A positive
result of the tannin test with FeCl3 is shown by a change in color to blackish green. Thin Layer Chromatography Thin layer chromatography (TLC) was performed to further determine the presence of
gallic acid in each extract. TLC was carried out using ethanol:water (6:3) as the mobile phase
and silica plate as the stationary phase [25]. Pure gallic acid solutions were used as
comparisons. Thin-layer chromatography is commonly used to identify bioactive compounds,
such as polyphenols, alkaloids, flavonoids, and tannins [25]. The stains produced by the
extract samples and gallic acid solutions were both very faint brown in color. Spraying of 5% FeCl3 solution was carried out to confirm that the stains obtained in the
TLC were phenolic compounds. Phenolic compounds in the ethanol extract will show a grey
spot on the TLC plate after being sprayed with FeCl3 [25]. The results showed that a pale grey
spot was observed in the leaf extract from the 2 mL/s flow rate treatment as well as in the
stem extracts from all flow rate variations with Rf values of 0.56 and 0.54, respectively after
FeCl3 spraying as depicted in Figure 6. However, the spots observed on the stems of the two
flow rates have different Rf values with pure gallic acid. In polar solvents, compounds with
higher Rf showed more polar extract properties than compounds with lower Rf values [25]. This allows the presence of other phenolic compounds than gallic acid contained in the
extract. Meanwhile, no grey spots were observed in 0.1 mM gallic acid solution and extracts
from the control treatment. This indicated that the leaf and stem extract of Cavendish banana
in all treatments contained a very small amount of gallic acid so that the presence of gallic
acid could not be detected by spraying FeCl3. N. Nurhayati et al., Analysis of Plant Growth and Gallic Acid Content for Cavendish Banana … Phenolic Test Determination of phenolic compound with FeCl3 was carried out as a first step to detect the
presence of gallic acid compounds in a crude extract of Cavendish banana leaves and stems. The formation of a blue-green or blue-black color in the solution indicates a positive result for
polyphenol compounds because phenol reduces Fe3+ to Fe2+, which causes a blue-black color
(Iron (III) hesacyanoferrate) [43][44]. Figure 5 shows the crude ethanol extract of the banana leaf sample was greenish in color,
while the stem extract had an orange to brownish color. However, Table 3 lists all extracts
showed positive results for the phenolic test because they produced a green/blue color after
being added with FeCl3. However, the extract from TLS produced a very faded green color. These results indicated that banana leaves and stems in the bubble column bioreactor contain
more phenolic compounds than TLS. All Musa spp. known to have a high amount of total
phenolic content in the leaves, stems, flowers, and fruit, with the highest phenolic found in the
leaves, which is about 64%, while the stem is only about 3% [44]. 39 Journal of Integrated and Advanced Engineering (JIAE)
Vol. 2, No. 1, March 2022: 33-44 Before
After
Figure 5. Result of Phenolic Test on Cavendish Leaf and Stem Extracts with FeCl3
Stem
TLS
Gallic
acid
Leaf
TLS
Leaf
1 mL/s
Stem
1 mL/s
Leaf
2 mL/s
Stem
2 mL/s
Gallic Acid
Leaf
TLS
Stem
TLS
Leaf
1 mL/s
Leaf & Stem
2 mL/s
Stem
1 mL/s Before
After
Figure 5. Result of Phenolic Test on Cavendish Leaf and Stem Extracts with FeCl3
Table 3. Result of Phenolic Test
Treatments
Phenolic Test Result
Leaf
Stem
Flow rate of 1 mL/s
+
+
Flow rate of 2 mL/s
+
+
This factor makes the color changes in the leaf samples tend to be darker than the stems
after the addition of FeCl3. The difference in color between the gallic acid solutions, which is
more blue-green, and the leaf samples may occur because of the presence of compounds other
han polyphenols, such as flavonoids and tannins, in the banana leaf samples [44]. A positive
esult of the tannin test with FeCl3 is shown by a change in color to blackish green. N. Nurhayati et al., Analysis of Plant Growth and Gallic Acid Content for Cavendish Banana … ACKNOWLEDGMENT The author would like to thank Banana Group for the research opportunity and LPIK ITB
for partially funded the research. Antioxidant Test 1, March 2022: 33-44 compounds than those at a flow rate of 2 mL/s and TLC. Cultures in bubble column
bioreactors produced metabolites like those of thin-layer cultures but in larger quantities due
to increased respiratory energy caused by the input of oxygen into the bioreactor [34]. In
addition, the higher antioxidant activity of the leaf extract than the stem in the bioreactor
treatment could be due to polyphenols generally accumulating in the epidermal and
subepidermal cells of the leaves and shoots [47]. CONCLUSION Shoot culture of Cavendish banana (Musa acuminata) in bubble column bioreactor has a
higher growth rate, phenolic content, and antioxidant activity than TLC. The average growth
rate of biomass at a flow rate variation of 1 mL/s and 2 mL/s respectively was 0.22 ± 0.001
g/day and 0.21 ± 0.001 g/da. The phenolic content in banana leaf and stem extracts in the
bubble column bioreactor was also higher than in the TLC. This result had implications for
the presence of higher gallic acid compounds. However, gallic acid in the extracts could not
be detected on thin layer chromatography because of the small amount of extract. The IC50
values for the banana leaf and stem extract were 41.35 and 51.87 g/mL for a flow rate of 1
mL/s; 79.54 and 104.94 g/mL for a flow rate of 2 mL/s. REFERENCES [1] S. S. Harith, N. H. Yasim, A. Harun, W. S. Omar & M. S. Musa, “Phytochemical screening, antifungal, and
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[14] M. N. Nurhayati et al., Analysis of Plant Growth and Gallic Acid Content for Cavendish Banana … Antioxidant Test Antioxidants have anti-radical properties with scavenging activity against free radicals,
which are useful for the treatment process in various diseases. Free radicals are the cause of
many diseases, such as neurodegenerative diseases, cancer, and AIDS. The DPPH method is a
sensitive method for determining the antioxidant activity of various plant extracts [22]. 40 p-ISSN: 2774-602X e-ISSN: 2774-6038 Figure 6. Result After Spraying Fecl3 on TLC Plate
Figure 7. IC50 Value of Cavendish Leaf and Stem Extracts
Gallic Acid
Leaf
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Gallic Acid
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Stem Figure 6. Result After Spraying Fecl3 on TLC Plate
Gallic Acid
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Modelling mixed-phase clouds with the large-eddy model UCLALES–SALSA
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Atmospheric chemistry and physics
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Correspondence: Jaakko Ahola (jaakko.ahola@fmi.fi) Received: 20 December 2019 – Discussion started: 28 January 2020
Revised: 9 July 2020 – Accepted: 23 July 2020 – Published: 14 October 2020 Abstract. The large-eddy model UCLALES–SALSA, with
an exceptionally detailed aerosol description for both aerosol
number and chemical composition, has been extended for ice
and mixed-phase clouds. Comparison to a previous mixed-
phase cloud model intercomparison study confirmed the ac-
curacy of newly implemented ice microphysics. A further
simulation with a heterogeneous ice nucleation scheme, in
which ice-nucleating particles (INPs) are also a prognostic
variable, captured the typical layered structure of Arctic mid-
altitude mixed-phase cloud: a liquid layer near cloud top and
ice within and below the liquid layer. In addition, the simu-
lation showed a realistic freezing rate of droplets within the
vertical cloud structure. The represented detailed sectional
ice microphysics with prognostic aerosols is crucially impor-
tant in reproducing mixed-phase clouds. et al., 2007; Stevens and Feingold, 2009; Morrison et al.,
2011a; Morrison, 2012; Li et al., 2013). ;
,
;
,
)
What we know about mixed-phase clouds is that by defi-
nition supercooled liquid droplets co-exist with ice crystals. Such clouds are frequent at temperatures between −10 and
−25 ◦C (Filioglou et al., 2019) but can be present from −35
to 0 ◦C and require specific microphysical and dynamical
conditions (Andronache, 2017). Ice crystals can form either
by homogeneous or heterogeneous freezing (the term nucle-
ation used also). At temperatures lower than −38 ◦C, liquid
droplets can freeze homogeneously without the need for ice-
nucleating particles (INPs). In heterogeneous ice nucleation,
freezing initiates from the surface of seed particles and can
occur at higher temperatures than homogeneous ice nucle-
ation. Droplet freezing processes are not yet fully understood
and quantified despite of decades of research (Phillips et al.,
2008; Atkinson et al., 2013; DeMott et al., 2011). Kiselev et
al. (2017) stated that ice formation on aerosol particles (het-
erogeneous ice nucleation) is a process of crucial importance
to Earth’s climate, but it is not understood at the molecular
level. However, in Morrison et al. (2011a) it is noted that
although many details of droplet freezing are poorly under-
stood, enough knowledge exists to draw first-order (ice wa-
ter path) conclusions. Furthermore, droplet freezing models
and even the representation of cloud structure often require
a resolution that is too detailed for large-scale models. Correspondence: Jaakko Ahola (jaakko.ahola@fmi.fi) For
instance, the structure of Arctic and mid-altitude clouds is
complex, with a layered structure with liquid near cloud top
and ice within and below the liquid layer (Curry et al., 1997;
Hobbs and Rangno, 1998; Pinto, 1998; Rangno and Hobbs,
2001; Zuidema et al., 2005; Shupe et al., 2006; Verlinde et
al., 2007; de Boer et al., 2009; McFarquhar et al., 2011; Mor- Modelling mixed-phase clouds with the large-eddy model
UCLALES–SALSA
Jaakko Ahola1, Hannele Korhonen1, Juha Tonttila2, Sami Romakkaniemi2, Harri Kokkola2, and Tom Jaakko Ahola1, Hannele Korhonen1, Juha Tonttila2, Sami Romakkaniemi2, Harri Kokkola2, and Tomi Raatikainen1
1Finnish Meteorological Institute, Helsinki, Finland
2Finnish Meteorological Institute, Kuopio, Finland 1
Introduction Clouds are known to have a prominent influence on the
hydrological cycle and the atmospheric radiation balance. While significant advances have been made in characteri-
sation of liquid-phase clouds, the microphysical processes,
especially heterogeneous ice nucleation, dynamics and ra-
diative effects of mixed-phase and ice clouds remain more
poorly constrained. This is mainly because of challenges in
obtaining representative observations and a lack of a detailed
enough representation of microphysics in climate and nu-
merical weather prediction models. Specific challenges are
known to be associated with aerosol–cloud interactions (Cox,
1971; Knight and Heymsfield, 1983; Curry, 1995; Solomon Atmos. Chem. Phys., 20, 11639–11654, 2020
https://doi.org/10.5194/acp-20-11639-2020
© Author(s) 2020. This work is distributed under
the Creative Commons Attribution 4.0 License. 2
Model description The UCLALES–SALSA (Tonttila et al., 2017) model con-
sists of two components: first, the widely used large-eddy
simulator UCLALES (Stevens et al., 1999, 2005), and sec-
ond, the aerosol bin microphysics model SALSA (Sectional
Aerosol module for Large-Scale Applications) (Kokkola
et al., 2008; Tonttila et al., 2017; Kokkola et al., 2018). UCLALES handles e.g. surface fluxes, transportation of mi-
crophysical prognostic variables and atmospheric dynamics
including turbulence. The previous version of UCLALES–
SALSA incorporated interactions between aerosols, clouds
and drizzle (Tonttila et al., 2017). Now we have extended the
model with a description for ice crystals. In this study, we
focus on how ice crystals and ice-nucleating particles (INPs)
interact with clouds while tracking sectional aerosol size dis-
tribution. Figure 1 illustrates the microphysical treatment of dif-
ferent hydrometeor classes and their size distributions in
UCLALES–SALSA. All four classes (aerosol, cloud and rain
droplets, and ice crystals) are tracked with a bin scheme. The
bin scheme offers the benefit of greater accuracy in simu-
lating interactions between hydrometeors of different sizes. Better accuracy is gained by dividing the size distribution
into bins. This also enables better flexibility as the shape of
the distribution is allowed to evolve. Bulk schemes provide
a simpler method and track one or several moments of the
size distribution, whereby the shape of the distribution is pre-
scribed. The disadvantage of the bin scheme is higher com-
putational cost compared to the bulk scheme. There is a growing number of studies focusing on examin-
ing the properties of mixed-phase or ice clouds by combining
observations and models, including large-eddy simulation
(LES) modelling and other cloud-resolving models (CRMs)
(Jiang et al., 2000; Klein et al., 2009; Morrison et al., 2011b;
Ovchinnikov et al., 2014; Andronache, 2017). Large-eddy
simulations are particularly attractive for modelling bound-
ary layer clouds since they offer a good compromise between
computational cost and accuracy in terms of model resolution
(Tonttila et al., 2017; Andronache, 2017). LES models ex-
plicitly solve the largest eddies in turbulent flows and use pa-
rameterisations for the smallest length scales. In atmospheric
applications they are usually coupled with cloud microphysi-
cal packages. Recent developments in the computational per-
formance of supercomputers have also made an explicit and
detailed description of aerosol–cloud–ice microphysical in-
teractions possible in LES modelling, allowing for the inves-
tigation of non-linear cloud phenomena, such as secondary
ice production and heterogeneous ice nucleation. J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA 11640 rison et al., 2011a). The lack of a proper calculation of ice
processes in climate models is seen in comparisons to obser-
vations of mid- and high-latitude mixed-phase clouds. These
models tend to underestimate the lifetime of such clouds
(Andronache, 2017). Better quantification of droplet freezing
processes is expected to narrow the gap between observations
and model results. we have now also extended this description for ice crystals. This sectional aerosol description allowed the implementa-
tion of a detailed heterogeneous freezing processes. First, the
model results are compared with previously published mod-
elling results. Finally, we demonstrate the benefits of this ap-
proach to handle heterogeneous freezing over more simpli-
fied aerosol–ice–cloud treatments. we have now also extended this description for ice crystals. This sectional aerosol description allowed the implementa-
tion of a detailed heterogeneous freezing processes. First, the
model results are compared with previously published mod-
elling results. Finally, we demonstrate the benefits of this ap-
proach to handle heterogeneous freezing over more simpli-
fied aerosol–ice–cloud treatments. Including a detailed aerosol description is vital in cloud-
resolving models. Scarcity of INPs is an important factor in
mixed-phase cloud lifetime and structure, since roughly one
in a million particles acts as an ice nucleus, and even these
particles might have highly different ice-forming activity at
different temperatures (Lebo et al., 2008; Morrison et al.,
2011a). Therefore, the loss of INPs along with precipitating
ice crystals limits cloud glaciation and dissipation (Rauber
and Tokay, 1991; Harrington et al., 1999; Avramov and Har-
rington, 2010). Describing this process is not possible with-
out a detailed description of aerosols, as is demonstrated in
a 1-D cloud model study by Morrison et al. (2005). The
significance of aerosols is shown in Filioglou et al. (2019)
wherein a high aerosol load was linked with a higher occur-
rence of mixed-phase clouds. Also, the Norgren et al. (2018)
study shows that there is less ice in polluted clouds. An-
dronache (2017) and Morrison et al. (2011a) provide com-
prehensive review resources for further details about mixed-
phase clouds. Published by Copernicus Publications on behalf of the European Geosciences Union. ublished by Copernicus Publications on behalf of the European Geosciences Union. 2
Model description Three of the hydrometeor classes, i.e. aerosol, cloud
droplets and ice, are further divided into parallel bins labelled
a and b as shown in Fig. 1. This division into a and b bins is
done to enable the tracking of externally mixed distributions
and to see how different particles affect clouds. For aerosol
particles, subrange 1a is an additional feature to describe
the nucleation mode. Otherwise, Aitken- and accumulation-
mode size ranges are sufficient to characterise cloud phenom-
ena. There are several LES models that solve cloud-related in-
teractions (Fridlind et al., 2012; Khain et al., 2004; Savre and
Ekman, 2015; Fu and Xue, 2017). In comparison to those
models, we present an LES model, UCLALES–SALSA, that
brings additional value with a more detailed aerosol descrip-
tion. UCLALES–SALSA explicitly resolves interactions be-
tween aerosols, ice crystals and cloud droplets with sectional
microphysics for all hydrometeors while keeping track of the
aerosol dry size distribution. The sectional description, espe-
cially for aerosols, is a clear asset of UCLALES–SALSA and The aerosol, cloud and ice crystal size distributions are dis-
cretised into the bins according to the dry aerosol diameter,
whereas the rain droplet size distribution is defined by the
wet diameter of the droplet. Identical 2a and 2b size bins are
used for aerosol, cloud droplets and ice. Such parallel bins
are useful for tracking aerosol development through cloud
activation, freezing and sublimation. Prognostic variables for Atmos. Chem. Phys., 20, 11639–11654, 2020 https://doi.org/10.5194/acp-20-11639-2020 11641 et al.: Modelling mixed-phase clouds with UCLALES–SALSA J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA Figure 1. Bin scheme of UCLALES–SALSA with newly implemented particles; see also Fig. 1 in Tonttila et al. (2017). heme of UCLALES–SALSA with newly implemented particles; see also Fig. 1 in Tonttila et al. (2017). Figure 1. Bin scheme of UCLALES–SALSA with newly implemented particles; see also Fig. 1 in Tonttila e – Deposition freezing is possible for dry insoluble aerosol
at subsaturated conditions (RH < 100 %). This is imple-
mented following Khvorostyanov and Curry (2000) and
additional parameters from Hoose et al. (2010). See also
Appendix C. each bin include aerosol number and the masses of all com-
pounds (water, sulfate, dust, organic carbon, sea salt, nitrate
and ammonium). J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA Coagulation is affected by
diffusion, especially aerosols, and by sedimentation, espe-
cially large particles. In a collision, bigger particles absorb
smaller particles. Interaction with radiation is implemented either with the
same four-stream radiative transfer solver (Fu and Liou,
1993) as in Tonttila et al. (2017) with an extension to in-
clude ice particles or parameterised as in Ovchinnikov et al. (2014). We used the latter method in our simulation. In the
parameterised radiation, the net upward long-wave radiative
flux is computed as a function of the liquid water mixing ra-
tio profile. The effect of interaction with radiation can be seen
in simulations: radiative cooling weakens after liquid water
path decreases below a specific value. Furthermore, UCLALES–SALSA was upgraded with mi-
nor bug fixes and improvements. For example, hygroscop-
icity is now calculated with κ-Köhler (Petters et al., 2006;
Petters and Kreidenweis, 2007) instead of the previously
used ZSR method (Zdanovskii–Stokes–Robinson method;
Zdanovskii, 1936; Stokes and Robinson, 1966). We ran UCLALES–SALSA for the three different simu-
lation setups investigated in the Ovchinnikov et al. (2014)
study: no ice (ICE0), average ice (ICE1) and high ice (ICE4)
number concentration. The ice number concentration is the
only variable that was changed between the evaluation sim-
ulations (0, 1 or 4 L−1). Liquid and ice water paths (marked
LWP and IWP from now on), i.e. column-integrated mass
values averaged over the horizontal domain, in these three
cases show how water is distributed between ice and liquid
phases depending on the ice crystal concentration. J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA 11642 If ice sublimates, the immersed aerosol nuclei are added back
to the aerosol population. large-scale forcings, radiation, cloud droplet freezing and ice
growth processes and sedimentation, and the nudging of hor-
izontal winds, potential temperature and water content above
the altitude of 1200 m. In the simulations ice processes were
excluded during the first 2 h, i.e. the spin-up period, to allow
the mixed-layer turbulence and the warm stratus cloud to de-
velop. After the spin-up, cloud droplets are allowed to freeze
until a specified target ice concentration is reached (Morrison
et al., 2011b). Ice shape is described with a mass-diameter
parameterisation so that ice can be considered spherical par-
ticles with low effective density (ρ = 84.5 kg m−3). Ice fall
speed is related to the maximum dimension, while capaci-
tance, which is used in the condensation equation, is modi-
fied from that of a sphere to C = D/π. Radiation and sedi-
mentation were parameterised similar to Ovchinnikov et al. (2014). For the sake of simplicity, coagulation was switched
off as in Ovchinnikov et al. (2014). Warm rain formation was
switched off, allowing for more straightforward model inter-
comparison. Also, the warm rain mass mixing ratios would
have been small due to a relatively small cloud droplet size
in the simulated case. Aerosol size distribution is given as
a sum of lognormal accumulation and coarse modes with
concentrations of 159×106 and 6.5×106 kg−1, mode mean
diameters of 0.2 and 0.7 µm, and geometric standard devi-
ations of 1.5 and 2.45, respectively. Aerosol is composed
of ammonium bisulfate. During the simulations this aerosol
size distribution provides on average 129 × 106 kg−1 cloud
droplets. These aerosol distribution parameters provide the
best fit to the measured distributions below the liquid cloud
layer (Earle et al., 2011; Ovchinnikov et al., 2014). Activation of aerosols to cloud droplets happens when RH
is over 100 % and aerosol wet diameter exceeds the critical
limit corresponding to the resolved supersaturation. At this
time, a certain proportion of activated aerosols (i.e. cloud
condensation nuclei, CCN) is moved to cloud droplet bins. Sedimentation is defined as before in Tonttila et al. (2017)
and now extended for ice particles. For simulations in this
study, a fall rate of ice particles is set as in Ovchinnikov et al. (2014). Coagulation is implemented the same way as before and
now also including ice particles. 2
Model description In UCLALES–SALSA, recently implemented processes
involving ice crystals are droplet freezing, deposition of wa-
ter vapour, sublimation, melting when T > 0 ◦C, coagulation
between different hydrometeors, sedimentation, and interac-
tions with radiation (see also Fig. 1). Most of these processes
are included in a similar way as in the previously published
version of UCLALES–SALSA (Tonttila et al., 2017). – Contact freezing is implemented in UCLALES–SALSA
following Hoose et al. (2010) so that first the coagu-
lation code is used to calculate collision rates between
dry particles and liquid droplets, and then the immersion
freezing code gives the freezing probability. Regarding the scope of this study, we describe droplet
freezing in higher detail. There are five mechanisms for
droplet freezing, and they are all currently implemented in
UCLALES–SALSA. – Condensation freezing is implemented as a part of im-
mersion freezing because these droplets can freeze dur-
ing the modelled condensational growth. – Immersion freezing is possible for aqueous droplets
that have an insoluble core, which in UCLALES–
SALSA is either dust (DU) or black carbon (BC). The
rate of heterogeneous germ formation in a supercooled
droplet of water or solution is calculated mostly fol-
lowing Khvorostyanov and Curry (2000), and addi-
tional parameters are from Jeffery and Austin (1997),
Khvorostyanov and Sassen (1998), Khvorostyanov and
Curry (2004), and Li et al. (2013). See also Appendix A. In our simulations (Sect. 3.3), only immersion freezing is
active. This is due to high temperatures, when homogeneous
freezing is not possible, when the mixing state of INPs leads
to aqueous droplets, and when deposition and contact freez-
ing are not feasible. Deposition of water, i.e. diffusion-limited condensation or
evaporation of water vapour, is defined for aerosol when rela-
tive humidity (RH) is over 98 % (equilibrium assumed other-
wise) and always for other hydrometeors. This is based on the
analytical predictor of condensation (APC) scheme by Jacob-
son (2005) and implemented following Tonttila et al. (2017)
(Eqs. 7 and 8). According to this definition, the particles
compete for the available water vapour. For solids, the con-
densation equation does not require Kelvin or Raoult terms. – Homogeneous freezing is possible for any aqueous
droplet with or without insoluble particles. This is ap-
plied to the model according to Khvorostyanov and
Sassen (1998). See also Appendix B. https://doi.org/10.5194/acp-20-11639-2020 Atmos. Chem. Phys., 20, 11639–11654, 2020 J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA https://doi.org/10.5194/acp-20-11639-2020 3.2
Sensitivity on ice concentration centrate on examining the properties of the ice microphysics
implementation. Motivated by simulated differences with the 4 L−1 ice con-
centration, we wanted to further investigate how sensitive
cloud glaciation is to changes in ice number concentration. In
addition to ICE1 and ICE4 simulations, we performed sim-
ulations in which the target ice number concentration was 2,
3, 5 or 6 L−1 (marked with ICE2, ICE3, ICE5 and ICE6, re-
spectively). Figure 3 depicts the LWP and IWP evolution in
all six UCLALES–SALSA simulations. The simulation time
was extended to 24 h in those cases in which cloud still exists
after 8 h (marked with a vertical line in Fig. 3). The simula-
tion time was not extended any further because we do not see
any major changes or trends in the last simulation hours. Second, Fig. 2b and c present the LWP and IWP time
series when the target ice number concentration is 1 L−1,
marked with ICE1. Again, LWP in UCLALES–SALSA
matches the other models well, being at the lower end
of the intermodel spread. As expected, the LWP growth
rate is lower than in the ICE0 simulation, as some of
the water vapour condenses onto ice crystals. Furthermore,
IWP matches well, especially with other bin models in the
Ovchinnikov et al. (2014) study. y
Third, Fig. 2d depicts LWP time series with an ice num-
ber concentration of 4 L−1, which can be regarded as high
ice concentration and is marked with ICE4. After spin-up,
LWP has a decreasing trend since the ice number concentra-
tion is so high that it consumes much of the water vapour. Subsequently, IWP in Fig. 2e increases rapidly after the
spin-up and in UCLALES–SALSA reaches its peak value of
15.7 g m−2 just before 4 h of simulation. It then decreases to
a value of 9.4 g m−2 at the end of the simulation. The re-
duction of IWP is caused by ice crystal precipitation at the
surface and evaporation below the cloud. Figure 3 shows that when the ice number concentration
is set to a higher value, LWP decreases faster and cloud
glaciates sooner. In simulations ICE4, ICE5 and ICE6, the
cloud dissipates totally after glaciation. The cloud glaciation
happens because water vapour condenses on the ice crystals
at the expense of the cloud droplets. 3.2
Sensitivity on ice concentration In simulations ICE1,
ICE2 and ICE3, IWP stabilises to values of approximately
6.5, 10 and 12 g m−2, respectively, towards the end of the
simulation. Compared to the model results in Ovchinnikov et al. (2014), IWP in UCLALES–SALSA declines faster after the
peak IWP has been reached in ICE4. One reason for this is
that dry particle size is tracked in UCLALES–SALSA, and
this seems to have an important effect on ice crystal sedimen-
tation. Namely, sedimentation velocities and particle mixing
(flux divergency) are calculated here for the dry size bins
rather than bins tracking ice particle size. This reduces parti-
cle flux, especially in the lowest 200 m, leaving more parti-
cles there to evaporate. Evaporative cooling leads to a surface
inversion which prevents the mixing of moist surface air. As
such, the higher sensitivity to INP concentration is partly re-
lated to the initial conditions of the ISDAC case study. The
other reason is related to the model-dependent technical de-
tails. Our test simulations (not shown) indicate that chang-
ing model options, such as flux limiter method, impact IWP
and LWP so that the gap between UCLALES–SALSA and
the other models decreases. In Ovchinnikov et al. (2014) it
was also stated that when the ice number concentration gets
higher the differences between models are more caused by
discrepancies in microphysics than in cloud dynamics. This
underlines the sensitivity and significance of microphysics. From Fig. 3 we can also see that LWP still increases dur-
ing the first 8 h with ICE2 but not anymore with ICE3. With
ICE1, the water paths of the cloud are very stable after 8 h of
simulation. LWP decreases about 2 g m−2, reaching a value
of 44 g m−2 at the end of the simulation. IWP is around
7 g m−2 at the end of the simulation. LWP values for ICE2
and ICE3 simulations are around 22 and 18 g m−2, and IWP
values are 10 and 12 g m−2 at the end of the simulation, re-
spectively. These are close to ICE4 simulations presented in
Ovchinnikov et al. (2014), and this illustrates the fine balance
between co-existing liquid and ice phases. These results show how sensitive the mixed-phase cloud
is to ice number concentration either by showing how fast
the cloud glaciates or when balance is reached. 3.1
Model evaluation The model performance is evaluated by simulating a well-
documented mixed-phase cloud case from the Indirect and
Semi-Direct Aerosol Campaign (ISDAC) Arctic study (Mc-
Farquhar et al., 2011). This observation case has been used
before for comparisons to LES models (e.g. Savre and Ek-
man, 2015; Fu and Xue, 2017). Ovchinnikov et al. (2014)
presented an intercomparison of 11 LES models for this same
case, in which initial profiles were based on aircraft obser-
vations in the mixed layer (Flight F31) and idealisation of
a sounding on 26 April 2008 at Barrow, AK. Nine of those
models had bulk two-moment microphysics and two of them
bin microphysics. Figure 2 compares the three UCLALES–SALSA simula-
tions to the results presented in the Ovchinnikov et al. (2014)
intercomparison paper. In the figure, LES model results from
Ovchinnikov et al. (2014) are separated between bulk and
bin microphysics to highlight the differences between mi-
crophysics schemes. First, Fig. 2a shows LWP for the base-
line simulation without any ice (ICE0). It is evident that our
model agrees well with the other 11 models. The simulated
LWP of UCLALES–SALSA is in the middle of the model
spread. Differences are most likely explained by different
dynamical cores, which is also stated in Ovchinnikov et al. (2014). A more thorough testing of warm-phase cloud mi-
crophysics in UCLALES–SALSA was done in the Tonttila
et al. (2017), and for the remainder of this work we will con- We implemented in UCLALES–SALSA model runs with
the same semi-idealised simulation setup given in Ovchin-
nikov et al. (2014) that attempted to minimise intermodel dif-
ferences by applying identical descriptions for the following
processes: surface properties and fluxes (fluxes set to zero), Atmos. Chem. Phys., 20, 11639–11654, 2020 https://doi.org/10.5194/acp-20-11639-2020 11643 J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA 3.2
Sensitivity on ice concentration However,
these are highly simplified due to the lack of real aerosol-
dependent freezing and related feedback processes. These re-
sults also show the need for more detailed feedbacks since a
constant ice number concentration is not a realistic assump-
tion for real clouds. https://doi.org/10.5194/acp-20-11639-2020 J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA ors, and ice formation is modelled using the implemented
ice nucleation theories, which relate ambient conditions and
droplet properties to their freezing rates. ors, and ice formation is modelled using the implemented
ice nucleation theories, which relate ambient conditions and
droplet properties to their freezing rates. Figure 4a shows that in the prognostic ice simulation LWP
starts to increase after 4.5 h of simulation. This is caused by
a decrease in ice number concentration (Fig. 4c) to such a
low level, which allows more water vapour for condensa-
tion to liquid droplets. The same figure also depicts how the
ice number concentration is set to a target value (simulation
ICE4) and how the concentration is stable until the cloud
dissipates. Figure 4d depicts how droplet number concentra-
tion lowers, especially right after the spin-up period when
ice number concentration is increasing. However, changes in
droplet number concentration are not the driving force be-
hind complete removal of liquid phase. Figure 4e illustrates
how the whole cloud with prognostic droplet freezing de-
scends, and as the cloud in the ICE4 simulation is affected
by entrainment both below and above the cloud, the cloud
gets thinner and dissipates. To see the difference between fixed and prognostic droplet
freezing, we made a prognostic ice simulation that was tar-
geted to have similar IWP during the first 8 h as in the simu-
lation with ice number concentration of 4 L−1 (ICE4) (see
Sect. 3.1 and 3.2). This ICE4 simulation was selected for
comparison because it is close to the tipping point at which
cloud either stabilises or glaciates (see Sect. 3.2). To achieve the target IWP, we adjusted the freezing prop-
erties accordingly of aerosols that can act as an INPs. The to-
tal number concentration and size distribution of the aerosol
remain the same as in the fixed ice number simulations
(Sect. 3.1 and 3.2); thus, they are the same as in Ovchinnikov
et al. (2014). In the absence of more detailed aerosol obser-
vations, INP number concentration and mixing state as well
as contact angle were considered adjustable parameters im-
pacting ice nucleation ability. Here, contact angle represents
the angle between the ice embryo and the ice nucleus in an
aqueous medium. At the beginning of the prognostic ice run, the domain
mean of dust-containing aerosols is approximately 27 L−1. J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA After 32 h of simulation the same mean value is about
13 L−1. Here, the loss of INPs limits the ice number con-
centration. The mixed-phase cloud persists because the ice
number concentration can change. This is so-called self-
adjustment of INPs, which better reproduces the observed
evolution of mixed-phase clouds since they are usually more
resilient in observations than in models (Andronache, 2017;
Morrison et al., 2011a). This is also in line with previous
modelling studies, wherein prognostic INPs will reduce the
number of ice crystals because of precipitation, thus allowing
cloud liquid to be sustained (Fridlind et al., 2012; Solomon
et al., 2015, 2018). The decrease in dust (INPs) mass con-
centration in different hydrometeor types is shown in Fig. 5. Dust is an efficient ice nucleus, so it will soon end up in ice
crystals which are removed from the system by sedimenta-
tion (Fig. 5c). The free troposphere is the only source for
the boundary layer dust, and the relevant mechanisms are
entrainment and large-scale subsidence. Subsidence is de-
scribed with a downward vertical velocity moving mass and
energy. Entrainment in this case describes any other kind of
mass exchange between cloud top and the free troposphere. For instance, subsidence is 0.004 m s−1 at the cloud top and
the aerosol number concentration in dust-containing b bins
above the cloud is about 23 000 m−3, so the dust aerosol
flux from the free troposphere is approximately 90 m−2 s−1. Because radiative cooling is strengthening the supersatura-
tion at the cloud top, the most CCN-active part of these en-
trained dust-containing particles can be activated immedi-
ately as cloud droplets. This can be seen as a higher dust
mass concentration within cloud droplets in the upper layer
of the cloud (Fig. 5b). If the temperature is low enough, these
dust-containing droplets will subsequently freeze during the
following time steps and therefore take part in preserving the
mixed-phase cloud. First, in order to set the INP number concentration, we
incorporated b bins (for more information about bin descrip-
tion, see Sect. 2 and Fig. 1). The proportion x = 150 × 10−6
of the total aerosol number concentration was partitioned in
b bins as INPs. The proportion (1−x) remained in the a bins. The resulting number concentrations of INPs in accumula-
tion and coarse modes were 23 850 and 975 kg−1, respec-
tively. 3.3
Prognostic ice simulation To conclude, the spread between models, especially be-
tween bin and bulk microphysics models, gets wider as
the prescribed ice number concentration gets larger and
closer to the limit when the cloud glaciates completely. In
UCLALES–SALSA this limit of full glaciation is lower than
in other models in Ovchinnikov et al. (2014). This limit is
further examined in Sect. 3.2. One of the unique features of our model is its ability to
keep track of the chemical composition along with a sec-
tional aerosol size distribution in the cloud phase. This
allows us to model freezing processes related to an ice-
nucleating compound like dust. Furthermore, parallel bins
allow for analysing the relative contribution of e.g. dust par-
ticles (INPs) on ice formation. We call this prognostic ice
because here freezing probability is related to dust aerosol,
the mass and number concentrations of which are prognos-
tic variables. We allow interactions between all hydromete- https://doi.org/10.5194/acp-20-11639-2020 Atmos. Chem. Phys., 20, 11639–11654, 2020 11644 J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA Figure 2. Liquid and ice water path time series in UCLALES–SALSA simulations with fixed ice number concentrations of 0, 1 and 4 L−1
(ICE0, ICE1 and ICE4, respectively). Black and grey lines show results in the Ovchinnikov et al. (2014) study. Figure 2 Liquid and ice water path time series in UCLALES SALSA simulations with fixed ice number concentrations of 0 1 and 4 L−1 Figure 2. Liquid and ice water path time series in UCLALES–SALSA simulations with fixed ice number concentrations of 0, 1 and 4 L−1
(ICE0, ICE1 and ICE4, respectively). Black and grey lines show results in the Ovchinnikov et al. (2014) study. Figure 3. Liquid and ice water path time series in UCLALES–SALSA simulations with fixed ice number concentrations of 1, 2, 3, 4, 5 and
6 L−1 (ICE1, ICE2, ICE3, ICE4, ICE5 and ICE6, respectively). Figure 3. Liquid and ice water path time series in UCLALES–SALSA simulations with fixed ice number concentrations of 1, 2, 3, 4, 5 and
6 L−1 (ICE1, ICE2, ICE3, ICE4, ICE5 and ICE6, respectively). Atmos. Chem. Phys., 20, 11639–11654, 2020 https://doi.org/10.5194/acp-20-11639-2020 Atmos. Chem. Phys., 20, 11639–11654, 2020 11645 Atmos. Chem. Phys., 20, 11639–11654, 2020 J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA Second, the INP mixing state was adjusted so that the par-
ticles in the b bins were set to have an insoluble dust core,
50 % of the dry mass and ammonium bisulfate for the other
half. Here, dust acts as INPs. Third, the freezing rate was adjusted by setting the cosine
of the contact angle of dust to mis = 0.57 (Eq. A3 in Ap-
pendix A). It should be noted that the target IWP could have been
reached using different combinations of INP mixing state, x
and mis, but these simulations showed that the results depend
mostly on the resulting ice number concentration rather than
the applied parameterisation. These characteristics of aerosol
are uniform throughout the whole simulation domain. The simulation time for the prognostic ice run was set to
32 h. The water paths of the mixed-phase cloud are quite sta-
ble after that. The simulation time of ICE4, used to compare
with the prognostic run, was not extended any further from
24 h since the cloud dissipates around 12 h of simulation. As in the ICE4 simulation, in the prognostic ice simula-
tion, droplet freezing was set to start after a spin-up of 2 h. Figure 4a and b illustrate that the prognostic ice and ICE4
simulations have similar IWP and LWP during the first 8 h. Hence, the targeting is successful and the initial conditions of
the simulations match each other. After that, the prognostic
ice simulation diverges from the ICE4 simulation. A more detailed examination of droplet activation and ice
formation can be done by studying the time evolution of
the size distribution. Figure 6 shows how particles of dif- https://doi.org/10.5194/acp-20-11639-2020 Atmos. Chem. Phys., 20, 11639–11654, 2020 J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA 11646 Figure 4. Time series of water paths, mean ice number concentration in icy regions, in-cloud cloud droplet number concentration (CDNC),
and the cloud top and base of the 32 h UCLALES–SALSA simulation with prognostic droplet freezing (Prognostic ice) compared with the
24 h UCLALES–SALSA simulation with a fixed ice number concentration of 4 L−1 (ICE4). Figure 4. Time series of water paths, mean ice number concentration in icy regions, in-cloud cloud droplet number concentration (CDNC),
and the cloud top and base of the 32 h UCLALES–SALSA simulation with prognostic droplet freezing (Prognostic ice) compared with the
24 h UCLALES–SALSA simulation with a fixed ice number concentration of 4 L−1 (ICE4). ferent sizes are partitioned between different hydrometeor
types within the cloud layer. Figure 6c and d show how
the larger particles freeze first and their number concentra-
tion decreases quickly as these particles deposit at the sur-
face within falling ice hydrometeors and are removed from
the system. Even though the entrainment from above is pro-
viding more particles, this is not fast enough to maintain
the original concentration. Removal of the smaller INPs is
slower, as those are less likely to activate as cloud droplets,
and the resulting droplets are also less likely to freeze due to
the smaller dust core area. However, with time and because
of continuous mixing of the boundary layer, the smaller INPs
are also eventually able to form cloud droplets within the
strongest updrafts, and formed droplets will freeze within the
cloud. This will lead to stabilisation of the aerosol size distri-
bution. The increase in the total number of particles in bin 1
is a numerical artefact caused by the bin adjustment routine,
which can move particles from one bin to another in order
to keep the dry size within the predefined bin limits. When
a large proportion of particles in bin 2 are activated as cloud droplets, some of the remaining particles are moved to the
smaller bin to avoid numerical problems. However, this nu-
merical artefact does not affect the results. ferent sizes are partitioned between different hydrometeor
types within the cloud layer. Figure 6c and d show how
the larger particles freeze first and their number concentra-
tion decreases quickly as these particles deposit at the sur-
face within falling ice hydrometeors and are removed from
the system. J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA Even though the entrainment from above is pro-
viding more particles, this is not fast enough to maintain
the original concentration. Removal of the smaller INPs is
slower, as those are less likely to activate as cloud droplets,
and the resulting droplets are also less likely to freeze due to
the smaller dust core area. However, with time and because
of continuous mixing of the boundary layer, the smaller INPs
are also eventually able to form cloud droplets within the
strongest updrafts, and formed droplets will freeze within the
cloud. This will lead to stabilisation of the aerosol size distri-
bution. The increase in the total number of particles in bin 1
is a numerical artefact caused by the bin adjustment routine,
which can move particles from one bin to another in order
to keep the dry size within the predefined bin limits. When
a large proportion of particles in bin 2 are activated as cloud Figure 7a and b illustrate the fact that supercooled liquid
droplets are dominant in the upper layers of the mixed-phase
cloud compared to ice crystals. Here the total ice number
concentration stabilises at approximately 0.44 L−1, whereas
it is obvious from Sect. 3.2 that a much higher concentra-
tion is needed to completely glaciate the cloud. Correspond-
ingly, the cloud droplet number concentration stabilises at
approximately 175 cm−3. Remarkably, these values are in
line with aircraft observations (Flight F31) of this ISDAC
case. The observed ice and cloud droplet number concentra-
tions are 0.35 L−1 and 185 cm−3, respectively (McFarquhar
et al., 2011; Savre and Ekman, 2015). The ice number con-
centration is also approximately 2 orders of magnitude less
than the number concentration of efficient INPs above the
cloud layer. From that we can estimate that an above-cloud
INP concentration of the order of 0.1 to 1.0 cm−3 is enough
to glaciate the cloud. https://doi.org/10.5194/acp-20-11639-2020 Atmos. Chem. Phys., 20, 11639–11654, 2020 J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA 11648 Figure 7. Vertical profiles of liquid water, ice and the freezing rate of droplets (nucleation rate) in the UCLALES–SALSA simulation with
prognostic droplet freezing. Figure 7. Vertical profiles of liquid water, ice and the freezing rate of droplets (nucleation rate) in the UCLALES–SALSA simulation with
prognostic droplet freezing. relative proportions of particles in different hydrometeors are
size-dependent and how a sectional description for aerosols
is required to be able to simulate such processes in LES mod-
els. relative proportions of particles in different hydrometeors are
size-dependent and how a sectional description for aerosols
is required to be able to simulate such processes in LES mod-
els. Figure 7c further illustrates an interesting behaviour of ice
particle formation. At the beginning of the simulation ice par-
ticles are formed throughout the cloud, but later the most in-
tensive formation takes place at the top of the cloud where
fresh INPs are entrained into the cloud layer. However, the
maximum supersaturation in these entraining downdrafts is
so low that only the largest particles are able to form cloud
droplets and consequently freeze. The smaller ones penetrate
through the cloud layer as interstitial aerosol particles (i.e. unactivated particle) and are able to form cloud droplets (i.e. activate) and ice particles at the cloud base when they are
recirculated back to the cloud with higher supersaturation. This can be seen in Fig. 8. Figure 8a shows that in size bin 2
cloud droplets and ice particles are more frequent in updrafts
compared to Fig. 8b, which illustrates that aerosols are more
favourable in downdrafts. Additionally, ice particles domi-
nate in bigger sizes as aerosols freeze both in downdrafts
and updrafts (size bin 3 shown in Fig. 8c and d). Simulated
freezing in different vertical velocity conditions in other size
bins does not differ from results shown already in Fig. 6. The
lower peak at the end of the simulation in the vertical pro-
file of freezing rate in Fig. 7c also shows how recirculated
aerosols are frozen in the cloud layer. Such a phenomenon
has been modelled before e.g. in Solomon et al. (2015); how-
ever, here the cloud is simulated with explicit calculation of
in-cloud supersaturation and representation of aerosol size
distribution and chemical composition. If activation is not
modelled with this level of detail, activation and freezing
might happen too early or late and in a wrong part of the
cloud. J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA Overall, Figs. 6, 7c and 8 nicely demonstrate how the J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA 11647 Figure 5. Logarithmic total mass mixing ratios (kg kg−1) of dust in different hydrometeors given as cloud profile time series in the
UCLALES–SALSA simulation from the prognostic ice simulation. Cloud top is not plotted to keep the figure clearer because it is prac-
tically the same as the upper line of RH over ice. Figure 5. Logarithmic total mass mixing ratios (kg kg−1) of dust in different hydrometeors given as cloud profile time series in the
UCLALES–SALSA simulation from the prognostic ice simulation. Cloud top is not plotted to keep the figure clearer because it is prac-
tically the same as the upper line of RH over ice. Figure 6. Relative proportions of hydrometeors at each time step in the cloud layer. A cloud layer is defined when both the cloud liquid water
and ice mixing ratios are over 0.001 (g kg−1). The green line represents the relative change in the total number concentration in each bin. Figure 6. Relative proportions of hydrometeors at each time step in the cloud layer. A cloud layer is defined when both the cloud liquid water
and ice mixing ratios are over 0.001 (g kg−1). The green line represents the relative change in the total number concentration in each bin. Atmos. Chem. Phys., 20, 11639–11654, 2020 Atmos. Chem. Phys., 20, 11639–11654, 2020 https://doi.org/10.5194/acp-20-11639-2020 https://doi.org/10.5194/acp-20-11639-2020 J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA 4
Conclusions In this study we have extended our large-eddy model
UCLALES–SALSA (Tonttila et al., 2017) for ice and mixed-
phase clouds. The model has an exceptionally detailed sec-
tional aerosol description for both aerosol number and chem-
ical composition, which makes this model suitable for exam-
ining aerosol–cloud interactions and dynamics. Specifically,
this allows for the description of an ice-nucleation-active ma-
terial such as mineral dust, which can be used in calculating
ice formation rates from the nucleation theory. As the first step, we compared our model predictions
with those from a mixed-phase cloud model intercompari-
son study (Ovchinnikov et al., 2014) to confirm the accuracy
of the newly implemented ice microphysics. In this simpli-
fied model intercomparison setup, wherein any cloud droplet
will freeze until a specified ice number concentration (from
zero up to 4 particles L−1) is reached, the focus is on cloud
dynamics. In agreement with Ovchinnikov et al. (2014) and
several other studies (e.g. Klein et al., 2009; Morrison et al.,
2011b; Stevens et al., 2018) we conclude that microphysi-
cal details such as the fact that dry particle size is tracked
in UCLALES–SALSA, while most other sectional models
track the ice particle size, have an impact on predictions. Such details become more important close to the tipping https://doi.org/10.5194/acp-20-11639-2020 Atmos. Chem. Phys., 20, 11639–11654, 2020 J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA 11649 Figure 8. Mean profiles of the relative proportions of hydrometeors in different vertical velocity conditions. Velocities smaller than 0.1 m s−1
are neglected. Averaging is done from simulation hours 28 to 32. The green line shows the relative change in the total number of hydrome-
teors. Only bins 2 and 3 are shown since they provide additional information in comparison to Fig. 6. Figure 8. Mean profiles of the relative proportions of hydrometeors in different vertical velocity conditions. Velocities smaller than 0.1 m s−1
are neglected. Averaging is done from simulation hours 28 to 32. The green line shows the relative change in the total number of hydrome-
teors. Only bins 2 and 3 are shown since they provide additional information in comparison to Fig. 6. point at which the further addition of ice particles leads to
the rapid glaciation of the cloud. Thus, the entrainment process is maintaining INP concentra-
tion in the whole boundary layer. https://doi.org/10.5194/acp-20-11639-2020 Atmos. Chem. Phys., 20, 11639–11654, 2020 Appendix A: Immersion freezing The rate J of heterogeneous germ formation by immersion
freezing is a function of temperature T in Kelvin, the radius
of insoluble substrate rN and the equilibrium saturation ratio
Sw at the droplet surface based on Köhler theory; it is deter-
mined as The shape factor f is defined as a function of the ra-
tio x = rN/rcr and m = mis. It is gained from Eq. (2.9) in
Khvorostyanov and Curry (2000), originally from Fletcher
(1962). 2f (m,x) = 1 +
(1 −mx)
φ
3 + x3
2 −3ψ + ψ3
+ 3mx3 (ψ −1),ψ = (x −m)
φ, φ
=
1 −2mx + x21/2
(A8) J (T,rN,Sw) = Chet exp
−1Fact + 1Fcr
kT
s−1
,
Chet = kT
h c1 s4πr2
N,
( J (T,rN,Sw) = Chet exp
−1Fact + 1Fcr
kT
s−1
, Chet = kT
h c1 s4πr2
N,
(A1) (A1) (A8) where k and h are the Boltzmann and Planck constants,
1Fact is the activation energy at the solution–ice interface,
1Fcr is the critical energy of germ formation, Chet is the nor-
malising function, rN is the radius of an insoluble fraction
of an aerosol particle (INP), and c1 s is the concentration of
water molecules adsorbed on 1 cm−2 of a surface (Eq. 2.1 in
Khvorostyanov and Curry, 2004). The parameter values used
are C = 1.7 × 10.99985010 N m−2 and c1 s = 1 × 1019 m−2. Activation energy 1Fact is calculated based on Eq. (15) in The case-dependent parameters ϵ, which is the elastic
strain produced in an ice embryo by the insoluble substrate,
α, which is the relative area of active sites, and mis, which
is the cosine of the contact angle, are defined in our results
(Sect. 3.3) to be ϵ = 0
α = 0
mis = 0.57. (A9) (A9) Activation energy 1Fact is calculated based on Eq. (15) in
Jeffery and Austin (1997): The mis was used as a targeting parameter since the simu-
lation tests were found to be very sensitive for this parameter. Other case-dependent parameters ϵ and α were not altered
and had their default values. J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA where Mw is the molar mass of water (Eq. 2.7 in
Khvorostyanov and Curry, 2000). where Mw is the molar mass of water (Eq. 2.7 in
Khvorostyanov and Curry, 2000). 4
Conclusions This study emphasises the benefits of the detailed aerosol–
cloud–ice module within an LES model. In fact, UCLALES–
SALSA is one of the few cloud-scale models (Fridlind et
al., 2012; Khain et al., 2004; Savre and Ekman, 2015; Fu
and Xue, 2017) with which details about aerosol and cloud
droplet chemical composition can be utilised with a particle-
level theoretical understanding of ice nucleation. The model
will be a useful tool for mixed-phase cloud research, which
has started to attract more widespread interest. In the second part, we constructed a case in which ice
formation is modelled using a heterogeneous ice nucleation
scheme and a prognostic ice-nucleating particle population
containing mineral dust. This so-called prognostic ice sim-
ulation was designed so that it matched the previous fixed
ice number concentration simulation in which the cloud was
close to the tipping point. When the simulation with a fixed
ice concentration showed complete glaciation after about
12 h, the prognostic ice simulation reached an equilibrium
state which lasted up to end of the 32 h simulation. With
this the prognostic simulation showed the importance of the
self-adjustment of ice-nucleation-active particles. This is in
good agreement with previous modelling studies (Fridlind et
al., 2012; Solomon et al., 2015, 2018) and a observational
study in which resilient mixed-phase clouds are seen together
with relatively high ice nuclei concentrations (Filioglou et
al., 2019). Further examination of the prognostic ice simulation re-
vealed that the efficient INPs entrained from the free tropo-
sphere are able to maintain the mixed-phase cloud with an ice
particle number concentration on average 0.1 %–0.2 % of the
INP concentration above the cloud. These entrained particles
do not immediately form ice particles in the cloud top. The
detailed analysis of the model outputs reveals how particle
size and supersaturation-dependent cloud activation eventu-
ally control the formation of ice through immersion freezing. Some entrained INPs penetrate through the cloud as intersti-
tial particles, get mixed within boundary layer air and con-
tribute ice formation later when recycled back to the cloud. Atmos. Chem. Phys., 20, 11639–11654, 2020 https://doi.org/10.5194/acp-20-11639-2020 https://doi.org/10.5194/acp-20-11639-2020 11650 Appendix A: Immersion freezing 1Fact = RT
B
T −T∗
−ln D∗
D0
NA ,
(A2) (A2) where T is temperature in Kelvin, R is the molar gas con-
stant, NA is the Avogadro constant, and parameter values
B = 347, T∗= 177, D∗= 4.17 and D0 = 349 for p = 1 bar
are gained from Table 2 in Khvorostyanov and Curry (2004). J. Ahola et al.: Modelling mixed-phase clouds with UCLALES–SALSA 11651 Surface tension between ice and vapour (Hoose et al.,
2010) is calculated with Surface tension between ice and vapour (Hoose et al.,
2010) is calculated with The ice germ radius is defined as The ice germ radius is defined as
Surface tension between
2010) i
l
l
d
i h rcr =
σis
ρiceLefm ln
T0
T SG
W
,
(B2) (B2) σiv =
(76.1 −0.155Tc) + (28.5 + 0.25Tc)
× 10−3 Jm−2 . (C2) which is the same as in Eq. (A5) with ϵ = 0 (Khvorostyanov
and Curry, 2000). which is the same as in Eq. (A5) with ϵ = 0 (Khvorostyanov
and Curry, 2000). (C2) Hence, we get the critical energy of germ formation
(Khvorostyanov and Sassen, 1998, Eq. 9b): The ice germ radius rcr (Khvorostyanov and Curry, 2000,
Eq. 2.12) is defined as The ice germ radius rcr (Khvorostyanov and Curry, 2000,
Eq. 2.12) is defined as 1Fcr = 4
3πσisr2
cr. (B3) 1Fcr = 4
3πσisr2
cr. rcr =
2σiv
Rv
ρice
Mw T lnSi −Cϵ2
,
(C3) (B3) (C3) Appendix B: Homogeneous freezing Critical energy is based on Eq. (2.10) in Khvorostyanov
and Curry (2000): Homogeneous freezing is possible for any aqueous droplet
with or without insoluble particles. This is applied to the
model according to Khvorostyanov and Sassen (1998). 1Fcr = 4π
3 σisr2
crf (mis,x) −αr2
N (1 −mis),
(A3) (A3) The number of crystals formed by homogeneous nucle-
ation due to the freezing of supercooled pure water or del-
iquescent condensation nuclei is described by Eq. (1) in
Khvorostyanov and Sassen (1998): where where σis = 28 × 10−3 + 0.25Tc × 10−3 Jm−2
(A4) (A4) J = 2Nc
ρwkT
ρiceh
r σis
kT exp
h
−1Fact + 1Fcr
kT
i
,
(B1) is the surface tension between ice and solution and where
Tc is temperature in degrees Celsius (Khvorostyanov and
Sassen, 1998). The ice germ radius is (B1) where k and h are the Boltzmann and Planck constants, ρw is
the density of water, ρice is the density of ice (same as in A)
and Nc = 5.85×1016 m−2 is the number of water molecules
contacting a unit area of ice germ (Khvorostyanov and Curry,
2000). rcr =
σis
ρiceLefm ln
T0
T SG
W
−Cϵ2 ,
(A5) (A5) where ρice = 900 kg m−3 is the density of ice, and T0 =
273.15 K. Case-dependent activation energy 1Fact(T ) = 0.694 ×
10−19 × (1 + 0.027(Tc + 30)) kg m2 s−2 when Tc < −30 ◦C
(Khvorostyanov and Sassen, 1998). Lef
m =
79.7 + 0.708Tc −2.5 × 10−3 × T 2
c
× 4.1868103 Jkg−1
(A The effective latent heat of melting Lef
m is the same as in
Eq. (A6). The dimensionless parameter G is the same as in
Eq. (A7). The surface tension between ice and solution σis is
the same as in Eq. (A4). (A6) is the effective latent heat of melting (Eq. 6 in Khvorostyanov
and Sassen, 1998). The dimensionless parameter is G =
RT
MwLefm
,
(A7) G =
RT
MwLefm
, (A7) m. Phys., 20, 11639–11654, 2020
https://doi.org/10.5194/acp https://doi.org/10.5194/acp-20-11639-2020 Atmos. Chem. Phys., 20, 11639–11654, 2020 Atmos. Chem. Phys., 20, 11639–11654, 2020 https://doi.org/10.5194/acp-20-11639-2020 Atmos. Chem. Phys., 20, 11639–11654, 2020 Appendix C: Deposition freezing where Si is the water vapour saturation ratio over ice, T is
temperature and C is constant at 1.7 × 1010 (N m−2). Deposition freezing is possible for dry insoluble aerosol at
subsaturated conditions (RH < 100 %). This is implemented
following Khvorostyanov and Curry (2000) and additional
parameters from Hoose et al. (2010). From previous values we get the critical energy of germ
formation: 1Fcr = 4
3πσivr2
crf (m,x),
(C4) (C4) The rate of germ formation J (s−1) through deposition
freezing is defined as in Khvorostyanov and Curry (2000,
Eq 2.13) The pre-exponential factor (kinetic coefficient) is
about where the shape factor f (m,x) is defined as in Eq. (A8). where the shape factor f (m,x) is defined as in Eq. (A8). J = 1030 × r2
n exp
−1Fact + 1Fcr
kT
,
(C1) (C1) where rn is the radius of the insoluble portion of the
droplet, k is Boltzmann’s constant and T is temperature. The
pre-exponential factor (kinetic coefficient) is 1026 (cm−2)r2
n
(Fletcher, 1962). Here, the case-dependent activation energy
1Fact is set to zero (Khvorostyanov and Curry, 2000). Atmos. Chem. Phys., 20, 11639–11654, 2020 https://doi.org/10.5194/acp-20-11639-2020 https://doi.org/10.5194/acp-20-11639-2020 Competing interests. The authors declare that there is no conflict of
interest. Competing interests. The authors declare that there is no conflict of
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Nuclear Poincaré cycle synchronizes with the incident de Broglie wave to predict regularity in neutron resonance energies
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Nuclear Poincaré cycle synchronizes with the incident de Broglie
wave to predict regularity in neutron resonance energies Makio Ohkubo1,a
1N.Resonance Lab, 1663-39, Senba-cyo, Mito-shi, Ibaraki-ken 310-0851 Japan 1N.Resonance Lab, 1663-39, Senba-cyo, Mito-shi, Ibaraki-ken 310-0851 Japan Abstract. In observed neutron resonances, long believed to be a form of quantum chaos,
regular family structures are found in the s-wave resonances of many even-even nuclei in
the tens keV to MeV region [M.Ohkubo, Phys. Rev. C 87, 014608(2013)]. Resonance
reactions take place when the incident de Broglie wave synchronizes with the Poincaré
cycle of the compound nucleus, which is composed of several normal modes with peri-
ods that are time quantized by inverse Fermi energy. Based on the breathing model of
the compound nucleus, neutron resonance energies in family structures are written by
simple arithmetic expressions using S n and small integers. Family structures in observed
resonances of 40Ca+n and 37Cl+n are described as simple cases. A model for time quan-
tization is discussed. © 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/). ,
2
EPJ Web of Conferences 12
CNR*15
13003 (2016) ,
2
EPJ Web of Conferences 12
CNR*15
13003 (2016) DOI: 10.1051/epjconf/201612213003 ae-mail: makiohkubo@hi-ho.ne.jp 1 Introduction In neutron-nucleus interactions, a compound nucleus (CN) is formed with excitation energy Ex =
S n + En, where S n is neutron separation energy ∼8 MeV, and En is the neutron kinetic energy in the
center of mass system (CMS). In the compound nucleus, many degrees of freedom will be excited and
mixed to form very complicated states or so-called chaos. Statistical distributions of many observed
neutron resonance data are in good agreement with predictions of the Random Matrix Theory (RMT),
therefore, the neutron resonance region is long believed to be a form of quantum chaos where no
regularity in level spacings/energies is expected. However, as is seen in every field of science, different methods of analysis extract different features
of the system under scrutiny. Many facts contradicting RMT have been reported in observed resonance
energies/spacings over the past five decades that relate the frequent appearance of special spacings by
Dij (spacings between two arbitrary levels) analysis or by Fourier-like analysis [1]. j
In order to understand the mechanism of neutron resonance reactions including special spac-
ings/energies, we developed the “Breathing Model" of the compound nucleus, analogous to classical
resonance phenomena where nuclear Poincaré cycles synchronize with the incident de Broglie wave
at resonances. This model predicts resonance energies by simple arithmetic expressions with good
accuracy. In section 2 of this article, the concept of the Breathing Model is described. In section 3,
theoretical insight into time behaviors of normal modes uses time quantization by inverse Fermi en-
ergy. In section 4, simple cases of two normal modes found in observed resonance data are shown. In
section 5, regularity in resonance energies are described. In section 6, a preliminary time quantization ,
2
EPJ Web of Conferences 12
CNR*15
13003 (2016) DOI: 10.1051/epjconf/201612213003 mechanism of a ‘hot chain’ is described. In section 7, transition from simple CN to complex CN are
described. Discussions and conclusion are found in section 8. 2 Breathing model of the compound nucleus in resonance reactions In the neutron-nucleus reaction, the wave packet of an incident neutron is divided into two compo-
nents: (1) pass-by component without interaction; and (2) penetration component that penetrates the
CN, excites many normal modes, and appears at the CN surface after every recurrence time τrec or
the “Poincaré period" τPP. Interference takes place between the two components, and the observed
cross-sections are the result of the interference between the two wave packet components. We de-
fine the initial “coalescent phase" as the wave crest of the pass-by component, the gather, and the
flare up of penetration components on the CN surface (doorway) during a short time ∼τ0 (described
below). This coalescent phase appears repeatedly with a time period τrec which depends on the en-
semble of normal modes excited at resonance. This behaves as a scattering center for the pass-by
component during the resonance lifetime ∼2πℏ/Γ, where Γ is the resonance width. At resonances,
the incident de Broglie frequency synchronizes with the nuclear Poincaré cycle as illustrated in Fig. 1. If the mechanism above does not exist, an energy independent potential scattering cross-section
(∼10 rmb = 10 × 10−28m2) will be observed. 3 Theoretical insight The penetration component excites the CN to energy S n(∼8 MeV). As the lifetime of the CN formed
by neutron resonance is relatively long, many excited degrees of freedom can be approximated by
an oscillator ensemble rewritten as an ensemble of normal modes. Several combinations of normal
modes are possible. The energy and time period of i-th mode are Ei and τi = 2πℏ/Ei, respectively. For M normal modes,the recurrence periods τrec are integer multiples of τi. (1) τrec = niτi,
ni:integer(i = 1, 2, ..., M),
(1) where the influence of angular uncertainty ∼1 rad is negligibly small for the lifetime of the resonance. From Eq. (1), τi/τj (i, j = 1, 2, ..., M) are integer ratios. Therefore, τi (i = 1, 2, ..., M) are restricted to
be integer multiples of unit time τ0, or time quantization by τ0. For unit time τ0, it is plausible to use
the inverse Fermi energy, τ0 = 2πℏ/G = 1.2 × 10−22s,
(2) (2) where G ∼34.5 MeV is almost the Fermi energy( the maximum energy of the nucleon in nuclear
potential). Hence, the time period of the normal mode is niτ0, and its energy is G/ni where ni is an
integer. Although not all details are known, the Hamiltonian and wave functions of the CN described by
M normal modes are formally written as H = H1 + ... + HM,
(3)
Ψ(x, t) = ψ1(x, t) ⊗... ⊗ψM(x, t). (4) H = H1 + ... + HM,
(3) (3) (4) The time periodic recurrence of ψi(x, t) and Ψ(x, t) are The time periodic recurrence of ψi(x, t) and Ψ(x, t) are The time periodic recurrence of ψi(x, t) and Ψ(x, t) are The time periodic recurrence of ψi(x, t) and Ψ(x, t) are ψi(xi, t) = ψi(xi, t + niτ0),
(5)
Ψ(x, t) = Ψ(x, t + τrec). (6) (5) (6) Ψ(x, t) = Ψ(x, t + τrec). 2 ,
2
EPJ Web of Conferences 12
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EPJ Web of Conferences 12
13003 (2016) DOI: 10.1051/epjconf/201612213003 Figure 1. Time evolution of the compound nucleus at resonance. (a) A toy model is used to show recurrence
of many oscillators rotating with arbitrary frequencies. (b) Penetration component, which excites CN and recurs
(flare up on CN surface) at coalescent phases with the Poincaré periodicity, synchronizing with the pass-by
component (c) at resonance. Figure 1. τw = (k/m)LCM(n1, ..., nM)τ0.
(10) 4 Simple cases Despite expectations of complex features of CN, many simple cases are found in observed resonance
data where only two normal modes are excited. The ratio S n/En are in a simple form S n
En
= (n1 + n2)
k
m
. (12) (12) and S n is and S n is S n =
1
n1
+ 1
n2
G. (13) S n =
1
n1
+ 1
n2
G. (13) (13) In this case, many observed resonances dispose in vicinity of (IVO) In this case, many observed resonances dispose in vicinity of (IVO) In this case, many observed resonances dispose in vicinity of (IVO) En =
m
k
Erec =
m
k
G
n1n2
=
m
k
S n
n1 + n2
,
(14) (14) composing a family structure. 3 Theoretical insight Time evolution of the compound nucleus at resonance. (a) A toy model is used to show recurrence
of many oscillators rotating with arbitrary frequencies. (b) Penetration component, which excites CN and recurs
(flare up on CN surface) at coalescent phases with the Poincaré periodicity, synchronizing with the pass-by
component (c) at resonance. The nuclear Poincaré period τpp or recurrence time τrec is defined as e nuclear Poincaré period τpp or recurrence time τrec is defined as τpp = τrec = LCM(n1, ..., nM)τ0,
(7) (7) where LCM is the least common multiple for a set of integers (n1, ..., nM). The recurrence energy Erec is defined as Erec =
G
LCM(n1, ..., nM). (8) (8) The excitation energy of the CN caused by the penetration component S n is written as The excitation energy of the CN caused by the penetration component S n is written as S n = E1 + ... + EM = G
1
n1
+ ... + 1
nM
= G
M
i=1
1
ni
. (9) (9) Neutron resonances take place when the period of the incident wave (pass-by component) τw(=
2πℏ/En) are in simple integer ratios (k/m) of the nuclear Poincaré period τrec. τw = (k/m)LCM(n1, ..., nM)τ0. (10) (10) 3 ,
2
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13003 (2016) DOI: 10.1051/epjconf/201612213003 CNR*15 That is, the possible resonance energies En in CMS are That is, the possible resonance energies En in CMS are (11) En = (m/k)Erec,
(11) En = (m/k)Erec,
(11) (11) En = (m/k)Erec, composing a family structure, where k, and m are small integers. composing a family structure, where k, and m are small integers. composing a family structure, where k, and m are small integers. 4.1
40Ca+n In s-wave resonances in 40Ca+n [2], we found that S n/En = 17(k/m) for many resonances (40Ca (I))
which provided the clue to this problem [3]. Also, S n is written as S n = 8362 keV = (17/70)G =
(1/7+1/10)G where G = 34434 keV. Among 40 s-wave resonances observed in 0 ⩽Eobs
n
⩽2500 keV
region, 15 resonances are IVO EC
n = (S n/17)(m/k) = 491.9(m/k) keV with (k, m ⩽10), where (m/k)
are 1/3 , 3/7, 1/2, 7/10, 8/9, 1/1, 10/9, 7/6, 5/4, 4/3, 7/4, 2/1, 3/1, 4/1 and 9/2. For 10 resonances, the
deviations δ = Eobs
n
−Ec
n are within a region of 5 keV width. The statistical probability of the appearance of the pseudofamily are calculated assuming a random
distribution of nl = 40 levels in the energy region R = 2330 keV [1]. Candidate cases (m/k) (k, m ⩽
10) are counted on a (k, m) plane to be B = 53. For a level placed at random in the region, the
probability of being on a candidate region is p = (ϵB)/R = 0.114. For 40 levels placed at random, the
expected number of integer ratios is nlp = 4.6. The probability for 10 levels out of 40 being integer
ratios is calculated by the binomial distribution: Pr(10, 40, p) = 40C10p10(1 −p)30 ≈0.0083 = 0.83%,
and the sum Ps = 40
j=10 Pr( j, 40, p) ⩽1.2%. j
In 40Ca+n in the same energy region, another family coexists showing S n/En = 39(k/m). Many
resonances dispose IVO Ec
n = (S n/39)(m/k) = 214(m/k) keV, called 40Ca (II). Similar regular family structures are found in s-wave resonances in target nuclei of 54Cr,(I)(II),
64Ni, 90Zr, and 208Pb. Similar regular family structures are found in s-wave resonances in target nuclei of 54Cr,(I)(II),
64Ni, 90Zr, and 208Pb. For resonances including mixed Jπ, similar family structures are found in target nuclei of 26Mg,
37Cl, 48Ca, 50Ti, and 58Fe. For resonances including mixed Jπ, similar family structures are found in target nuclei of 26Mg,
37Cl, 48Ca, 50Ti, and 58Fe. 4 ,
2
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13003 (2016) DOI: 10.1051/epjconf/201612213003 Figure 2. a) 37Cl+n resonances. Neutron width Γn vs. incident neutron energy up to 700 keV [4]. 4.1
40Ca+n S n =
6107.9 keV can be written as (8/45)G = (1/9 + 1/15)G, where G = 34357 keV. To meet energy region require-
ments, a recurrence energy Erec = (1/225)G = S n/40 = 152.7 keV is used. b) Two normal modes (hot chains) of
periods 9τ0 and 15τ0 couple to the doorway (circle) where coalescent phases appear with a time period of 45τ0
repeatedly. Figure 2. a) 37Cl+n resonances. Neutron width Γn vs. incident neutron energy up to 700 keV [4]. S n =
6107.9 keV can be written as (8/45)G = (1/9 + 1/15)G, where G = 34357 keV. To meet energy region require-
ments, a recurrence energy Erec = (1/225)G = S n/40 = 152.7 keV is used. b) Two normal modes (hot chains) of
periods 9τ0 and 15τ0 couple to the doorway (circle) where coalescent phases appear with a time period of 45τ0
repeatedly. 4.2
37Cl+n As an example, the regular family structures of s-wave resonances in 37Cl+n are described below
where 1+ and 2+ resonances coexist. Original data from Sayer et al. [4] observed up to 1 MeV. The separation energy S n is rewritten as S n = 6107.9 keV = (8/45)G = (1/9 + 1/15)G where
G = 34357 keV. The recurrence energy is Erec = G/45 = S n/8 = 763.5 keV. In observed values
S n/En, a factor 40 appears for many resonances. Then S n = (40/225)G and one fifth harmonics of
Erec = S n/40 = 152.7 keV are used. Many observed resonance energies Eobs
n
dispose IVO Ec
n =
152.7(m/k) keV. Below En ⩽500 keV, 23 resonances are observed. For 10 resonances, m/k are; 3/10, 3/5, 7/8, 8/7, 6/5, 4/3, 7/5, 8/5, 9/5, and 2/1 as illustrated in
Fig.2a. Among these resonances, deviations δ = Eobs
n
−Ec
n are within a width of 0.8 keV for 6
resonances. The probability of the appearance of these dispositions is calculated similar to the 40Ca
(I) described above. The parameters are: nl = 23 in the region R = 500 keV, ϵ = 0.8 keV, B = 55, and
p = ϵB/R = 0.088. Pr = 23C6p6(1 −p)17 = 0.0097 = 0.97 % and Ps are calculated to be 1.3 %. 5 ,
2
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13003 (2016) DOI: 10.1051/epjconf/201612213003 CNR*15 7 From simple to complex The simple cases above are for light or magic nuclei where neutron resonance level densities are
not very high. Few normal modes couple to the doorway as illustrated in Fig. 2(b) and Fig. 3(a). For medium and heavy nuclei where level densities are high, many (M) normal modes couple to the
doorway as illustrated in Fig. 3(b). M are estimated to be M ⩽10 for observed resonances [5]. Several
different resonance families coexist in the same energy region and display complex features. For the
resonances in the eV region, LCM(n1, ..., nM) will be increased to 104 ∼107, and family structure
analysis will be an interesting and difficult problem. 5 Regularity in resonance energies The probability of the appearance of the regular family structures Ps are quite small for several nuclei. In Table II of Ohkubo[1], Ps are listed for seven cases of four target nuclei, 40Ca,(I)(II), 54Cr,(I)(II),
64Ni, and 90Zr, where the average Ps is 1.3 %. In addition, Ps is 1.3 % for 37Cl. We have investigated
neutron resonances of about 40 light and magic nuclides. If we assume random dispositions of levels
for all 40 nuclides, the expectation of it is less than one nuclide. However, it appears in 5 nuclides. The probability U of appearing in 5 nuclides among 40 nuclides with Ps = 1.3% is calculated by the
binomial distribution as U = 40C5(0.013)5(0.987)35 ≈1.5 × 10−4. (15) U = 40C5(0.013)5(0.987)35 ≈1.5 × 10−4. (15) The occurrence of a phenomenon with very small probability means a failure of the random hypothe-
sis, and the existence of regular family structures are verified with the statistical significance level of
∼10−4. Though the random level ensemble substitutes for the RMT ensemble, this conclusion will
not be changed for RMT ensemble. 6 Time quantization of normal modes Time quantization of normal modes is inevitable for resonance reactions because of the finite time
restriction of τrec as described in Eqs. (1)and (2). For the mechanism of time quantization of normal
modes, we consider “hot chains" excited in CN. For example in 40Ca(I), two normal modes of 7τ0 and
10τ0 are excited. These normal modes work as a time-delay-system for the penetration components. For the time-delay-system of 7τ0, we consider a hot chain composed of seven elements. An element of
the chain couples to the doorway, where the penetration component, called fire, enters and exits, and
interference takes place with the pass-by component. The fire transfers to the neighbouring element
in unit time τ0, and the fire circulates in the chain in 7τ0, repeatedly. Similar mechanisms work for the
10τ0 chain. Fires in two chains gather and flare up at the doorway to form a scattering center for the
pass-by component with a period LCM(7, 10) = 70τ0 repeatedly during the lifetime of the resonance. 8 Discussion and Conclusion The classical analogy of resonance phenomena is valid for neutron resonance reactions with some
modification of physical concepts, i.e., the nuclear Poincaré cycle synchronizes with the incident de
Broglie wave. Resonance families derived by arithmetic expressions in Eq. (11), agree well with the
observed resonance energies. Time quantization of normal modes by τ0 is essential to this work and
simplifies the excitation modes in the energy region ∼S n. Since τ0 deviates about ±1% from family
to family depending on the internuclear interactions, the value G = 2πℏ/τ0 ∼34.5 MeV deviates
consequently. 6 ,
2
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13003 (2016) DOI: 10.1051/epjconf/201612213003 Figure 3. (a) Simple case of 40Ca+n compound nucleus. Two normal modes (hot chains) of periods 7τ0 and
10τ0 couple to the doorway (center circle) where coalescent phases appear with time period 70τ0 repeatedly. (b)
Complex compound nucleus. Many (M ⩽10) normal modes couple to the doorway where coalescent phases
appear with time period τrec = LCM(n1, ..., nM)τ0, repeatedly. Figure 3. (a) Simple case of 40Ca+n compound nucleus. Two normal modes (hot chains) of periods 7τ0 and
10τ0 couple to the doorway (center circle) where coalescent phases appear with time period 70τ0 repeatedly. (b)
Complex compound nucleus. Many (M ⩽10) normal modes couple to the doorway where coalescent phases
appear with time period τrec = LCM(n1, ..., nM)τ0, repeatedly. Figure 3. (a) Simple case of 40Ca+n compound nucleus. Two normal modes (hot chains) of periods 7τ0 and
10τ0 couple to the doorway (center circle) where coalescent phases appear with time period 70τ0 repeatedly. (b)
Complex compound nucleus. Many (M ⩽10) normal modes couple to the doorway where coalescent phases
appear with time period τrec = LCM(n1, ..., nM)τ0, repeatedly. Time behaviors of the compound nucleus are extracted by analysing energy structures of neutron
resonance levels. This is mathematically analogous to determining crystal structures from neutron
diffraction patterns using a neutron plane wave exp(i(kx −ωt)), where k is wave vector, x is a space
coordinate, ω is frequency, and t is time. The crystal diffractions are in the (k, x) domain, scatter-
ing from atoms at space-periodic lattices, whereas the neutron resonances are in the (ω, t) domain,
scattering from time-periodic coalescent phases on the CN surface. The traditional idea of quantum chaos in the neutron resonance region is not correct. 8 Discussion and Conclusion For a long
time, it was surmised that regularities in resonance positions/spacings did not exist because of the
success of the Random Matrix Theory, supported by many observed neutron resonance data analysed
by traditional statistical methods. These methods, however, could not detect strong correlations of
family structures as described in this article. To this point, it must be stressed that traditional sta-
tistical analyses have no ability to distinguish between chaos and regularity. Our results offer a new
explanation for a regular system for this region. On the other hand, rules on strengths and Jπ of each resonance, which will depend on spatial com-
ponents of wave functions, are not known at present. However, this approach seems to be promising
to investigate nuclear dynamic structures in discrete energy regions. [2] R.Toepke; KFK-2122, Karlsruhe(1974)
[3] M.Ohkubo; Phys.Rev.C 80, 024607(2009)
[4] R.O.Sayer,K.H.Guber, L.C.Leal and N.M.Larson; Phys.Rev.C 73, 044603(2006)
[5] M.Ohkubo; Phys.Rev.C 53,1325(1996) [3] M.Ohkubo; Phys.Rev.C 80, 024607(2009) References [1] M.Ohkubo; Phys.Rev.C 87, 014608(2013) [1] M.Ohkubo; Phys.Rev.C 87, 014608(2013) 7 7 ,
2
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https://openalex.org/W2245793637
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https://pdf.blucher.com.br/chemicalengineeringproceedings/cobeq2014/1534-18806-163418.pdf
|
Portuguese
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HIDRÓLISE ENZIMÁTICA DE ÓLEO DE SOJA EM REATOR DE LEITO FIXO UTILIZANDO A LIPASE LIPOZYME TL IM
| null | 2,015
|
cc-by
| 2,450
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HIDRÓLISE ENZIMÁTICA DE ÓLEO DE SOJA EM REATOR
DE LEITO FIXO UTILIZANDO A LIPASE LIPOZYME TL IM
E. RAIZER1, T. O. REINEHR1, G. P. de SÁ Jr.1, J. A. AWADALLAK1, C. R. BRITO Jr.1,
C
SILVA2 e E. A. SILVA1 1 Universidade Estadual do Oeste do Paraná, Departamento de Engenharia Química
2 Universidade Estadual de Maringá, Departamento de Engenharia Química
E-mail para contato: eduardoeq@gmail.com RESUMO – Os ácidos graxos possuem diversas aplicações industriais e servem de
substratos para inúmeros produtos, principalmente dentro da indústria alimentícia, onde
um alto grau de pureza se faz necessário e é dificilmente alcançado quando sua produção
envolve catalisadores químicos. Além disso, a reação de hidrólise geralmente necessita de
altas temperaturas e pressões para ocorrer, o que pode ser contornado com o uso de
enzimas (lipases) como catalisador, as quais utilizam condições reacionais mais brandas. Dentro deste contexto, este projeto teve como principal objetivo avaliar o processo de
hidrólise enzimática do óleo de soja em um reator de leito fixo tubular usando como
catalisador a enzima Lipozyme TL IM e avaliar os efeitos da razão mássica água/substrato
e da temperatura no rendimento da reação por meio de um planejamento experimental. As
reações de hidrólise foram realizadas a temperaturas de 20, 25 e 30°C, em concentrações
de 4, 8 e 12% de água em relação à massa total e tempo de residência de uma hora. Nas
melhores condições, foi produzido um óleo com acidez final de 24,46%. Área temática: Engenharia de Reações Químicas e Catálise importante na exploração econômica dessas fontes renováveis. Um significante número de produtos
de alto valor agregado como revestimentos, colas, óleos lubrificantes, xampus e outros produtos de
cuidados pessoais requer o uso de ácidos graxos em sua fabricação (Murty, et al., 2002). importante na exploração econômica dessas fontes renováveis. Um significante número de produtos
de alto valor agregado como revestimentos, colas, óleos lubrificantes, xampus e outros produtos de
cuidados pessoais requer o uso de ácidos graxos em sua fabricação (Murty, et al., 2002). A utilização de catalisadores na hidrólise é muito comum, pondendo ser conduzida através de
catalisadores químicos ou enzimáticos. O uso de processos enzimáticos apresenta certas vantagens,
como a atuação em condições operacionais brandas de temperatura, pH e pressão, velocidades de
reação bastante superiores àquelas obtidas em presença de catalisadores químicos convencionais
(Mendes et al., 2011), assim como uma maior especificidade, gerando menos produtos indesejáveis e
resultando também em uma maior facilidade de separação do meio. No entanto, de forma geral, as
enzimas possuem um alto custo de aquisição, reduzindo assim sua utilização, quando comparadas aos
catalisadores químicos. Nesse contexto, o objetivo deste trabalho foi a produção de ácidos graxos livres com alto grau
de conversão, através da hidrólise do óleo de soja em leito fixo com a utilização da enzima Lipozyme
TL IM. 2.1. Delineamento experimental A
influência da razão água/substrato e da temperatura na formação de ácidos graxos, foi
verificada por um delineamento central composto rotacional com duas variáveis. Como variável
resposta foi considerado o grau de acidez após 1 h de reação. A análise dos dados foi efetuada por
meio do programa Statistica® (versão 10). As hidrólises foram realizadas em um reator encamisado
contendo 20 mL de emulsão numa proporção água:substrato, por um período de 1 h com temperaturas
fixas e agitação magnética (350 rpm). 2. MATERIAIS E MÉTODOS O substrato da reação de hidrólise utilizado neste trabalho foi o óleo de soja. A lipase comercial
Lipozyme TL IM, utilizada como catalisador foi cedida por LFN Latino América. Uma solução de
fenolftaleína (Synth®) foi utilizada como indicador para medidas de acidez do óleo. Etanol absoluto
(Chemco®), Éter etílico (Nuclear®) e NaOH (Synth®) foram utilizados na determinação da acidez. 1. INTRODUÇÃO As atuais tendências nutricionais têm tornado a dieta cotidiana rica em óleos e gorduras, que
aliada à falta de atividades físicas regulares, está associada a um aumento no índice de obesidade da
população, aumentando os riscos de doenças cardiovasculares, diabetes e hipertensão (Francischi et
al., 2000). Entretanto, os óleos e gorduras possuem um papel fundamental no funcionamento do corpo
humano, fornecendo energia, atuando no transporte de vitaminas lipossolúveis e também como fontes
de ácidos graxos essenciais (Castro et al., 2004). Não podendo, dessa forma, ser totalmente
dispensados da alimentação diária (Yang et al., 2004). Um crescente interesse por tecnologias de modificação de óleos e gorduras surgiu nos últimos
anos, impulsionado pela importância dessas substâncias enquanto matérias-primas para as indústrias
químicas, farmacêuticas e alimentícias (Castro et al., 2004). O principal componente dos óleos e gorduras é o Triacilglicerol (TAG), e por meio de sua
hidrólise parcial, obtém-se o diacilglicerol (DAG) e os ácidos graxos. Segundo Awadallak (2012),
apesar de atingir conversões menores, a hidrólise é uma reação direta que utiliza como substratos
óleos e gorduras naturais. A produção de ácidos graxos por esse processo é um componente muito 1 Área temática: Engenharia de Reações Químicas e Catálise 1 importante na exploração econômica dessas fontes renováveis. Um significante número de produtos
de alto valor agregado como revestimentos, colas, óleos lubrificantes, xampus e outros produtos de
cuidados pessoais requer o uso de ácidos graxos em sua fabricação (Murty, et al., 2002). 2.3. Reações de hidrólise As reações de hidrólise enzimática do óleo de soja foram conduzidas em um sistema contínuo
composto por um reator encamisado conectado a um reator de leito fixo tubular encamisado,
preenchido com 1,4 g de enzima e completado com pérolas de vidro, conforme mostra a Figura 1. Figura 1 – Módulo de reação de hidrólise. Figura 1 – Módulo de reação de hidrólise. A mistura foi transportada pelo sistema através de uma bomba peristáltica Cole-Parmer, e a
temperatura foi controlada com auxílio de um banho termostático Marconi. Após a reação, as amostras foram aquecidas durante 24 horas a uma temperatura de 105°C para
assegurar a evaporação da água, e em seguida o teste de acidez foi realizado. 2.2. Preparação das amostras Os experimentos foram preparados dentro de um reator encamisado, em concentrações de 4, 8 e
12% de água deionizada em relação à massa total de substrato (água + óleo), fixada em 20 g. Após a
conexão do reator encamisado ao sistema, as reações foram realizadas a temperaturas de 20, 25 e
30°C. 2 2 Área temática: Engenharia de Reações Químicas e Catálise 2.4. Quantificação da acidez A quantidade de ácidos graxos livres presentes no óleo foi determinada por titulação com
solução de NaOH. Uma solução de 50 mL de etanol absoluto e éter etílico em proporção 1:1 com
cerca de 1 g de óleo e duas gotas de solução de fenolftaleína foi titulada com uma solução de NaOH
0,05 M sob agitação vigorosa até a mudança de coloração (mudança súbita de uma coloração branca
para rosa). A acidez do óleo foi calculada de acordo com a seguinte relação: 𝐴𝑐𝑖𝑑𝑒𝑧𝑚% = 100 𝑉𝑜𝑙 𝑀!"#$ 𝑃𝑀!"#
𝑝𝑎
(01) (01) 3 Área temática: Engenharia de Reações Químicas e Catálise 3 3. RESULTADOS E DISCUSSÃO O estudo das condições controláveis se deu através da metodologia de superfície de resposta,
utilizando um planejamento experimental do tipo delineamento central composto rotacional (DCCR). Para tempos de reação fixos de duas horas, variou-se a razão água/substrato e a temperatura. A Tabela 1 representa as variáveis e níveis para o planejamento experimental. Tabela 1 – Variáveis e níveis para o DCCR
-√2
-1
0
1
√2
Razão água/substrato (%)
2,34
4
8
12
13,66
Temperatura (oC)
17,93
20
25
30
32,07 O projeto do experimento consistiu em um planejamento do tipo delineamento central composto
rotacional para duas variáveis, com quatro repetições no ponto central para a avaliação do erro e dois
pontos axiais no eixo de cada variável, resultando em quinze experimentos. A Tabela 2 apresenta a
estimativa dos efeitos em conjunto com o gráfico de Pareto (Figura 2). Tabela 2 – Estimativa dos efeitos
Variável
Efeito
Erro padrão
p-valor
Coeficiente
Intercepto
16,5642
0,6321
0,000000
16,5642
(1) Temperatura (L)
0,4230
0,7879
0,604400
0,2115
Temperatura (Q)
-3,1520
0,9818
0,010653
-1,5760
(2) Razão Água/Substrato (L)
12,1855
1,1100
0,000002
6,0928
Razão Água/Substrato (Q)
1,1072
1,9659
0,587068
0,5536
Interação (1) com (2)
-0,3977
1,7447 -0,227970
0,8248
Figura 2 – Gráfico de Pareto. Figura 2 – Gráfico de Pareto. 4 4 Área temática: Engenharia de Reações Químicas e Catálise Área temática: Engenharia de Reações Químicas e Catálise A variável Razão água/substrato (linear) e a variável Temperatura (quadrática) apresentaram,
para um nível de significância de 5%, efeitos significativos sobre a conversão em AGL. A variável
Temperatura (quadrática) indica a existência de um ponto máximo de conversão dentro do intervalo
estudado, enquanto a falta de curvatura presente no ajuste quadrático do intervalo testado para a
Razão água/substrato indica que tanto esse intervalo quanto suas proximidades não contém um ponto
ótimo. A Tabela 3 representa a matriz de dados do DCCR para as variáveis estudadas e as respostas
obtidas em termos de porcentagem de acidez experimental e predita. 3. RESULTADOS E DISCUSSÃO Tabela 3 – Condições experimentais e resultados
Amostra
Razão Água/Substrato
(ma/(mo+ma))
Temperatura
(oC)
Acidez
Experimental
(%)
Acidez
Predita
(%)
1
2,34%
25,00
9,01
10,91
2
4,00%
20,00
10,79
10,49
3
4,00%
30,00
12,23
11,23
4
4,00%
25,00
14,44
12,44
5
8,00%
25,00
16,30
16,52
6
8,00%
25,00
16,02
16,52
7
8,00%
32,10
13,82
13,66
8
8,00%
30,00
14,48
15,17
9
8,00%
20,00
14,65
14,72
10
8,00%
25,00
16,42
16,52
11
8,00%
17,90
13,96
13,03
12
12,00%
30,00
18,95
19,68
13
12,00%
20,00
18,16
19,52
14
12,00%
25,00
21,76
21,18
15
13,66%
25,00
24,46
23,28 Tabela 3 – Condições experimentais e resultados Embora o ponto máximo da reação ainda não tenha sido determinado devido ao baixo intervalo
de testes realizados para a razão água/substrato, bons resultados foram alcançados. As respostas
preditas foram obtidas pelo ajuste de um modelo quadrático feito pelo software Statistica®. A acidez
mostrou-se dependente tanto da razão água/substrato quanto da temperatura, segundo o gráfico de
Pareto (Figura 2). Essa relação é descrita através da Equação 2: 𝐴𝑐𝑖𝑑𝑒𝑧𝑚% = −33,00 + 3,25𝑇−0,06𝑇! + 98,53𝑅+ 178,78𝑅! −0,71𝑅𝑇 (02) 𝐴𝑐𝑖𝑑𝑒𝑧𝑚% = −33,00 + 3,25𝑇−0,06𝑇! + 98,53𝑅+ 178,78𝑅! −0,71𝑅𝑇 (02) O coeficiente de correlação ao quadrado R2 é comumente utilizado como parâmetro de
avaliação para a eficiência de modelo modelos matemáticos. Quanto mais próximo da unidade, mais
fiel tende a ser a correlação aos dados experimentais. Neste caso o valor de R2 foi de em 0,371,
indicando um ajuste satisfatório, porém passível de melhora. Através da regressão foi possível traçar o gráfico de superfície para a acidez em gressão foi possível traçar o gráfico de superfície para a acidez em função das 5 Área temática: Engenharia de Reações Químicas e Catálise 5 variáveis de entrada. A Figura 3 mostra a superfície para a acidez final em função da razão
água/substrato e da massa de temperatura. variáveis de entrada. A Figura 3 mostra a superfície para a acidez final em função da razão
água/substrato e da massa de temperatura. variáveis de entrada. A Figura 3 mostra a superfície para a acidez final em função da razão
água/substrato e da massa de temperatura. Através do gráfico é possível perceber que a razão água/substrato está diretamente relacionada
com o desempenho da reação e que o aumento da temperatura está provavelmente associado à
desativação da enzima. Figura 3 – Acidez em função da temperatura e da razão água/substrato. 3. RESULTADOS E DISCUSSÃO Figura 3 – Acidez em função da temperatura e da razão água/substrato. As análises de variância e de resíduos, obtidas através do software Statistica® estão descritas na
Tabela 3 e na Figura 4. As análises de variância e de resíduos, obtidas através do software Statistica® estão descritas na
Tabela 3 e na Figura 4. Tabela 3 – Análise de variância
Fonte de
variação
Soma dos
Quadrados
Graus de
Liberdade
Quadrado
Médio
Fcalc
Ftab
Regressão
208,722
5
41,744
26,816 3,388
Resíduos
14,01
9
1,557
Total
222,732
14 Área temática: Engenharia de Reações Químicas e Catálise Área temática: Engenharia de Reações Químicas e Catálise
6 6 6 Figura 4 – Resíduos. Figura 4 – Resíduos. 4. CONCLUSÕES Para um intervalo de confiança de 95%, as variáveis Temperatura (quadrática) e Razão
água/substrato (linear) apresentaram efeitos significativos sobre a conversão em AGL. A presença de
um ponto máximo de conversão dentro do intervalo de temperatura estudado foi encontrada, enquanto
o intervalo testado para a razão água/substrato evidenciou a falta de curvatura no ajuste quadrático,
mostrando que tanto esse intervalo quanto suas proximidades não possuem ponto ótimo. O coeficiente
de correlação obtido foi de R2=0,9371. Esse resultado em conjunto com os valores relativamente
baixos dos resíduos, bem como sua distribuição aleatória em torno de zero e um valor de Fcalc muito
maior que Ftab são fortes indicações de que o ajuste quadrático é satisfatório e não tendencioso. No
entanto ainda são necessários mais testes em outras faixas de razão água/substrato para determinar
com alto grau de confiança qual o ponto ótimo da reação de hidrólise do óleo de soja em leito fixo. ma:
Massa de água (g) MNaOH: Molaridade das solução de NaOH 7 7 Área temática: Engenharia de Reações Químicas e Catálise mo:
Massa de óleo
(g)
pa:
Massa da amostra de óleo para titulação
(g)
PMAGL: Peso molecular médio dos ácidos graxos presentes no óleo (g/mol)
R:
Razão água/substrato
(Mágua/Mágua+Móleo)
T:
Temperatura
(oC)
Vol:
Volume de solução de NaOH gasto na titulação da solução (L) (g) PMAGL: Peso molecular médio dos ácidos graxos presentes no óleo (g/mol)
R:
Razão água/substrato
(Mágua/Mágua+Móleo)
T:
Temperatura
(oC) (oC) Vol:
Volume de solução de NaOH gasto na titulação da solução (L) 6. REFERÊNCIAS AWADALLAK, J.A. Uso do ultrassom na hidrólise enzimática do óleo de palma: síntese de
diacilglicerol. Dissertação de mestrado em Engenharia Química, Universidade Estadual do
Oeste do Paraná, Toledo, 2012. AWADALLAK, J.A. Uso do ultrassom na hidrólise enzimática do óleo de palma: síntese de
diacilglicerol. Dissertação de mestrado em Engenharia Química, Universidade Estadual do
Oeste do Paraná, Toledo, 2012. CASTRO, H. F.; MENDES A.A.; SANTOS, J.C.; AGUIAR, C.L. Modificações de óleos e
gorduras por biotransformação. Quím. Nova, v. 27, p. 146-156, 2004. FRANCISCHI, R.P.P.; PEREIRA, L.O.; FREITAS, C.S.; KLOPFER, M.; SANTOS, R.C.;
VIEIRA, P.; LANCHA JUNIOR, A.H. Obesity: updated information about its etiology,
morbidity and treatment. Rev. Nutr., v. 13, p. 17-28, 2000. MENDES, A. A.; OLIVEIRA, P. C.; CASTRO, H. F.; GIORDANO, R. L. C. Aplicação de
quitosana como suporte para a imobilização de enzimas de interesse industrial. Quím. Nova, v. 34, p. 831-841, 2011. MURTY, R. V.; BHAT, J.; MUNISWARAN P. K. A. Hydrolysis of Oils by Using Immobilized
Lipase Enzyme: A Review. Biotechnol. Bioproccess Eng. v. 7, p. 57-66, 2002. YANG, T.; ZHANG, H.; MU, H.; SINCLAIR, A.J.; XU, X. Diacylglycerols from butterfat:
Production by glycerolysis and short path distillation and analysis of physical properties. Journal
of the American Oil Chemists’ Society, v. 81, p. 979–987, 2004. 8 8 Área temática: Engenharia de Reações Químicas e Catálise
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The views of parents of children with cancer and pediatric physical therapists on a network for continuity and optimal quality of care for children with cancer: KinderOncoNet
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Supportive care in cancer
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Abstract Purpose Children with cancer require specific therapeutic guidance. Parents prefer physical therapy close to home, while
pediatric physical therapists (PPTs) working in the community may lack specific knowledge. The aim of this study is to
determine the needs of parents of children with cancer and PPTs to inform the design and development of a care network,
named “KinderOncoNet.” Methods We explored the perspectives and needs of parents of children with cancer and PPTs in the community, and we
investigated the added value that KinderOncoNet could offer. We used an iterative process; data collection consisted of (1)
gathering information from parents of children with cancer and PPTs through a survey and (2) co-creation sessions with
stakeholders. Results In total, 98 parents and 177 PPTs participated in the survey. Parents (97%) and PPTs (93%) indicated that the care
network would bring added value. All but one parent stressed the importance of a local PPT being aware of both the condition
and the side and late effects of oncological treatment. Moreover, 40% of PPTs thought they do not have sufficient knowledge
to provide high-quality therapy and that they would embrace opportunities for education. Through the co-creation sessions,
a prototype of the care network was conceptualized. Conclusion KinderOncoNet can contribute to the continuity and quality of physiotherapy care for children with cancer during
and after the oncological treatment. Such a network would allow for sharing knowledge, developing skills, and improving
accessibility and communication in the Netherlands. Keywords Childhood oncology · Care network · Pediatric physiotherapy · Quality of life · Supportive care RESEARCH RESEARCH RESEARCH The views of parents of children with cancer and pediatric physical
therapists on a network for continuity and optimal quality of care
for children with cancer: KinderOncoNet L. B. Kleinlugtenbelt1 · W. J. E. Tissing1,2 · W. J. M. Plieger‑van Solkema3 · P. van der Torre1 · W. J. W. Kollen1 ·
J. W. Gorter4,5 J. W. Gorter4,5 Received: 22 August 2023 / Accepted: 27 November 2023 / Published online: 6 December 2023
© The Author(s) 2023 * L. B. Kleinlugtenbelt
l.b.kleinlugtenbelt-3@prinsesmaximacentrum.nl 1
Princess Máxima Center for Pediatric Oncology,
Heidelberglaan 25, 3584, CS, Utrecht, The Netherlands Supportive Care in Cancer (2024) 32:9
https://doi.org/10.1007/s00520-023-08211-6 Supportive Care in Cancer (2024) 32:9
https://doi.org/10.1007/s00520-023-08211-6 Introduction In the Netherlands, around 600 children are diagnosed with
cancer each year [1]. The treatment of childhood cancer
often comes with many side effects. The increasing survival
due to more intensive treatment protocols has come with an
increased number of side effects. With higher cure rates,
the total number of childhood cancer survivors increases,
resulting in increasing numbers of patients with short-
and long-term medical, physical, and psychosocial needs,
often extending into adulthood [2, 3]. As a consequence of
more side effects and increasing numbers of survivors, [4]
pediatric physiotherapists (PPTs) are seeing more children
and adolescents with cancer, during and after oncological
treatment. 1
Princess Máxima Center for Pediatric Oncology,
Heidelberglaan 25, 3584, CS, Utrecht, The Netherlands 1
Princess Máxima Center for Pediatric Oncology,
Heidelberglaan 25, 3584, CS, Utrecht, The Netherlands 2
Department of pediatric oncology, University of Groningen,
University Medical Center Groningen, Groningen,
The Netherlands 2
Department of pediatric oncology, University of Groningen,
University Medical Center Groningen, Groningen,
The Netherlands 3
Dutch Childhood Cancer Organisation, Utrecht,
The Netherlands 4
Department of Rehabilitation, Physical Therapy Science
and Sports, University Medical Center Utrecht, Utrecht,
The Netherlands 5
UMC Utrecht Brain Center and Center of Excellence
for Rehabilitation Medicine, Utrecht University, Utrecht,
The Netherlands (0121 3456789)
3 9
Page 2 of 9 9
Page 2 of 9 Supportive Care in Cancer (2024) 32:9 Since 2018, the care for children and adolescents with
cancer in the Netherlands is centralized at the Princess Máx-
ima Center in Utrecht. The mission is to cure every child
with cancer with optimal quality of life. Our center aims
to centralize care when needed and to provide care locally
when possible. Parts of the oncological treatment can be
administered in 1 of 15 shared care centers. Our hypothesis
is that this care model could be further optimized by extend-
ing the continuity of physiotherapy care to close to home and
community settings and by sharing our knowledge, exper-
tise, and experience with professionals. means not only prevention of more expensive care in spe-
cialized centers, but also to move care closer to people’s
homes, whenever possible, for better quality and efficiency
[14–16]. Goals of such healthcare networks often have a
quadruple aim, i.e., improving the patient experience of
care, the health of populations, reducing per capita costs,
and improving the work life of those who deliver care [17]. Introduction Looking at existing care networks and best practices in the
Netherlands, such as CP-NET (cerebral palsy), OncoNET
(oncology for adults), CVA-Net (stroke), and Parkinson-
Net (Parkinson disease) [18], or internationally like Acti-
veOncoKids [19, 20], the National Physical Activity and
Childhood Cancer Network in Australia [21], the Italian
rehabilitation group [22], and Pogo [23], we know that
network care can improve the quality of care. In line with
these existing networks, we aim to improve the pediatric
physiotherapy care across settings for children by estab-
lishing a care network, KinderOncoNet (Children’s Oncol-
ogy Network). Currently, pediatric physiotherapy for children with can-
cer is delivered by PPTs in the Sports and Exercise Center
of the Princess Máxima Center while children are admit-
ted or undergo medical treatment or by the local pediatric
physiotherapist when the patients are at home. In 2021, 304
of the 553 (55%) newly diagnosed children were seen by
a pediatric physiotherapist in the Princess Máxima Center,
with 225 of the 553 (41%) of these children being referred
to a local PPT after discharge. The primary objective of this study is to determine the
needs of parents of children with cancer and PPTs across
all settings, to inform the design and development of a care
network. In addition, we asked stakeholders about the added
value that KinderOncoNet could offer. The physiotherapy program in the Princess Máxima
Center is tailored to the needs of each child, adolescent, and
family, depending on the type of cancer, developmental stage
(0–18 years), phase of the oncological trajectory (before,
during, or after treatment), and any complications and/
or late effects of the treatment. Pediatric physical therapy
can be helpful both to reduce the impact of side effects of
the oncological treatment with respect to impairment level
(such as balance impairments due to polyneuropathy) and
to improve activity (e.g., walking) and participation (e.g.,
school, sports), and overall quality of life of the children [5,
6]. The premise is that when children with cancer are better
equipped and supported across settings, it will lead to bet-
ter physical and mental health outcomes, as well as better
functioning in society, and to less costs in the future [7–10]. 1 3 Study setting and design We initiated a project named “KinderOncoNet” in the Prin-
cess Máxima Center for pediatric oncology in Utrecht, the
Netherlands, from March 2021 to March 2022. The study
was designed as an iterative process, to determine the needs
and added value for our care network. The project group
included the following members and stakeholders (total n
= 8): two professionals from the Princess Maxima Center
(a PPT/project leader and a PPT/manager of the department
Sports and Exercise); an employee of the parent/patient
organization: the Dutch Childhood Cancer Organisation
(VKKN); a board member of the Dutch Association for
Pediatric Physical Therapy (NVFK); an employee of Enter
Communication (an ICT company); an employee from the
HU (University of Applied Sciences) Utrecht, the Nether-
lands; lectorate innovations and exercise care; and two stu-
dents from the Bachelor of Physical Therapy Program at the
HU in Utrecht, the Netherlands. Data collection consisted
of two different phases: gathering information from the par-
ents of children with cancer, during and after treatment and
from PPTs through a survey (phase 1), and co-creation ses-
sions with stakeholders, to further expand on the knowledge
obtained in phase 1 (phase 2). g
y
While continuity of care by a PTT at home is critically
important to reduce side and late effects of the oncological
disease [11, 12], we currently lack a network of specialized
PPTs in cancer across settings in the Netherlands. Most of
the local PPTs in the Netherlands only see none to four chil-
dren with childhood oncology in their whole career. With
all the different types of cancer and various needs, a PPT in
the community will thus always lack knowledge and experi-
ence. Therefore, to optimize continuity and quality of care
by physiotherapists close to home, it makes sense that PPTs
need access to up-to-date knowledge and expertise related
to pediatric oncology. Developing a care network could be
a solution to overcome this care gap. Worldwide, there is an understanding that the concept
of network medicine is critical to meet the needs of the
growing population of childhood cancer survivors [13]. Facilitating the right care in the right place is one of the
most important objectives of care networks. This approach 1 3 Supportive Care in Cancer (2024) 32:9 Page 3 of 9
9 9 Selection method The survey for parents was distributed online by the Prin-
cess Máxima Center’s newsletter for parents, the Dutch
Childhood Cancer Organisation, and a Facebook group for
parents. Participation was voluntary. The survey for PPTs
was distributed online by the the Dutch Society for Pediatric
Physiotherapy (NVFK) to affiliated PPTs in the Netherlands
across all settings in the community. The survey asked par-
ticipants to indicate whether they were interested in joining
the consortium. The consortium partners were purposefully
selected by the project group to ensure representative PPTs
across all settings (primary care, shared care center, and
rehabilitation center). The theme of the second in-person co-creation meeting
was “knowledge and expertise.” Based on the questions of
the survey, topics as “what is needed to improve capacity
and competence?,” “how to maintain knowledge and exper-
tise?,” and “which educational needs and what content and
form of educational needs within the care network are neces-
sary?” were further explored and discussed. Also, the con-
ditions to participate in KinderOncoNet were determined. Phase 1: survey Data from the co-creation meetings were collected by
note taking during the sessions and analyzed and coded
through a tree structure of the topics using the software
ATLAS.ti [29]. Needs of parents of children with cancer during and after
treatment, and PPTs, and also beliefs about the added value
to develop a knowledge platform and care network for estab-
lishing KinderOncoNet were determined by a survey. The
survey consisted of closed and open-ended questions and
statements with Likert scale (0, not important, to 10, very
important). The survey for parents consisted of ten questions
(closed and open-ended) about (1) parents’ experiences with
physical therapy care in childhood cancer and (2) parents’
need for developing a care network. The survey for PTTs
consisted of 36 questions about (1) current workplace, (2)
experience with and knowledge of pediatric oncology, and
(3) expectations of a pediatric oncology exercise care net-
work and knowledge platform. Open-ended questions were
used to explore their expectations in developing a network. Statistics Data of the survey (phase 1) were collected by SurveyMon-
key, and descriptive statistics (frequency, distribution) were
generated by Excel [28]. Stakeholder engagement We used a multi-stakeholder approach of parents and PPTs
in phase 1 (survey) and PPTs across all settings (primary
care, shared care center, and rehabilitation center) in phase
2 (co-creation sessions) see Table 1. Ethical approval (see the “Method” section) to further explore what the needs
for PPTs are and to convert this in ideas to create a net-
work, based on an action-oriented research approach [25]. In co-creation sessions, good and productive collaboration
among every participant is very important and is taken into
consideration from the very early steps in conceptualizing
the prototype [26, 27]. All procedures involving human participants were in accord-
ance with the ethical standards of the institutional and/or
national research committee and with the 1964 Helsinki
Declaration and its later amendments or comparable ethi-
cal standards. The study, a quality improvement project, is
not considered subject to the Medical Research Involving
Human Subjects Act (WMO) [24]. This study does not fall
under the scope of the Dutch Medical Research Involving
Human Subjects Act (WMO). It therefore does not require
approval from an accredited medical ethics committee in
the Netherlands. The first co-creation meeting was organized in May
2021. This meeting took place online by Zoom because of
the restrictive measures regarding COVID-19. The second
co-creation meeting was organized in September 2021. This
meeting took place in-person at the Princess Máxima Center
in Utrecht, the Netherlands. The theme of the first’s co-cre-
ation meeting was “find, connect, and trust” and aimed to
identify the needs in terms of collaboration and findability
within the future care network, KinderOncoNet. Topics of
accessibility, privacy, and communication capabilities were
also discussed. Phase 2: co‑creation sessions In total, 98 parents and 177 PPTs participated in the survey. For characteristics of participants of the survey, see Table 1. Based on the results of the surveys, two co-creation sessions
were organized with consortium partners and stakeholders p
p
y
In total, 267 out of 275 (97%) participants (parents
and PPTs) expressed added value in the development of a 1 3 9
Page 4 of 9 Supportive Care in Cancer (2024) 32:9 *Actively involved consortium of PPTs nationwide with knowledge and expertise to carry out the research. The participants were recruited by a call from the NVFK. To select participants for the consortium group,
conditions were set for representing primary, secondary, and tertiary care and shared care center across the
Netherlands
**PPTs pediatric physiotherapists
Participants in survey
Total (N) Range (mean; sd)
Parents
98
During treatment
39
After treatment
59
Treatment of a local PTT during treatment? Yes
79
Yes, but not local
8
No
12
PPTs
177
Gender
Female
158
Male
15
Age (years)
22–70 (42.4; 10.7)
Work experience (years)
0–44 (16.3; 10.2)
Setting
Primary care
154
Secondary care (incl. shared
care and rehabilitation
center)
24
Tertiary care
5
Participants in co-creation sessions
PTTs in the community*
34
PPTs** from primary care settings
19
PPTs from shared care center
6
PTTs nine from rehabilitation center
9 Table 1 Participants in the
survey and co-creation sessions *Actively involved consortium of PPTs nationwide with knowledge and expertise to carry out the research. The participants were recruited by a call from the NVFK. To select participants for the consortium group,
conditions were set for representing primary, secondary, and tertiary care and shared care center across the
Netherlands
**PPTs pediatric physiotherapists **PPTs pediatric physiotherapists percent of the parents are willing to travel 15–30 min from
home to a local PPT. For further outcomes, see Table 2. knowledge platform and care network. In total, 94 out of
98 (96%) of the parents indicated that it is important that
the local PPT is aware of not only the pediatric oncological
condition but also the side effects and late effects of onco-
logical treatment affecting exercise (mean 9.0 on a scale
of 1–10). Parents expected to be referred from the Princess
Máxima Center to a local PTT with expertise and experi-
ence in pediatric oncology (7.7 on a scale of 0–10). Eighty Results from the questionnaire for PPTs are presented in
Table 3. Phase 2: co‑creation sessions Forty percent of the PPTs mentioned they do not
have sufficient knowledge to be able to provide a high-qual-
ity therapy (on a scale of poor, insufficient, sufficient, more
than sufficient, excellent, see Table 3). PPts mentioned the
lack of opportunities for education to gain more knowledge
in the field of pediatric oncology and physiotherapy care. Table 2 Summary of outcome survey parents (n = 98)
Question
Answered Outcome
Experience with pediatric physiotherapy
96
Range (0–10); mean 7.5
When I come to the Princess Máxima Center, I expect to be referred by the Maxima to a pediatric
physiotherapist with expertise in pediatric oncology
95
Range (0–10); mean 7.7
I find it important that the pediatric physiotherapist has experience and up-to-date knowledge in
children with an oncological condition
98
Range (0–10); mean 9.0
For a pediatric physical therapist with pediatric oncology knowledge and experience, I am willing
to invest the following additional travel time
98
No extra travel time; 11 (11.2%)
15 min; 31 (31.6%)
30 min; 47 (48.0%)
Max 60 min; 16 (16.3%)
KinderOncoNet has to be assessable for parents? 98
Yes; 87
No; 11 Table 2 Summary of outcome survey parents (n = 98) Table 2 Summary of outcome survey parents (n = 98)
Question 1 Page 5 of 9
9 Page 5 of 9
9 Supportive Care in Cancer (2024) 32:9 Table 3 Summary of outcome survey PPTs (n = 177)
Question
Answered (n)
Outcome
Percentage
How many children with cancer in treatment during career? 175
0 (n = 15) 1–4 (n = 78) 5–10 (n = 57)
11–20 (n = 14) > 20 (n =
11)
8.6% 44.6%
32.6% 8.0%
6.3%
Willing to connect to KinderOncoNet? 163
Yes (n = 153)
No (n = 10)
93.9%
6.1%
KinderOncoNet is of added value? 164
Yes (n = 153)
No (n = 11)
93.3%
6.7%
KinderOncoNet accessible for parents? Phase 2: co‑creation sessions 168
Yes (n = 107)
No (n = 54)
66.5%
33.5%
General knowledge childhood oncology
168
Poor (n = 4)
Insufficient (n = 61)
Sufficient (n = 87)
More than sufficient (n = 14)
Excellent (n = 2)
2.4%
36.3%
51.8%
8.3%
1.2%
General knowledge hemato-oncology
167
Poor (n = 11)
Insufficient (n = 69)
Sufficient (n = 69)
More than sufficient (n = 17)
Excellent (n = 1)
6.6%
41.3%
41.3%
10.2%
0.6%
General knowledge
168
Poor(n = 13)
Insufficient (n = 72)
Sufficient (n = 68)
More than sufficient (n = 12)
Excellent (n = 3)
7.7%
42.9%
40.5%
7.1%
1.8%
Solid tumors
164
Poor (n = 13)
Insufficient (n = 85)
Sufficient (n = 55)
More than sufficient (n = 9)
Excellent (n = 2)
7.9%
51.8%
33.5%
5.5%
1.2%
General knowledge bone tumors
167
Poor (n = 13)
Insufficient (n = 77)
Sufficient (n = 60)
More than sufficient (n = 14)
Excellent (n = 3)
7.8%
46.1%
35.9%
8.4%
1.8%
General knowledge neuro-oncology
168
Poor (n = 13)
Insufficient (n = 72)
Sufficient (n = 68)
More than sufficient (n = 12)
Excellent (n = 3)
7.7%
42.8%
40.5%
7.1%
1.8%
Sufficient knowledge after Master Pediatric Physical Therapy
165
Poor (n = 13)
Insufficient (n = 66)
Neutral (n = 67)
Sufficient (n = 18)
More than sufficient (n = 1)
7.9%
40.0%
40.6%
10.9%
0.6% General knowledge neuro-oncology Sufficient knowledge after Master Pediatric Physical Therapy Thirty percent of the PPTs indicated that they needed educa-
tion to become more competent in treating a child with the
diagnose childhood cancer, and fifteen percent of the PPTs
indicated that experience is important to become more com-
petent in the field of pediatric oncology. Also, an up-to-date
knowledge platform (8.3%), intervision and peer review in
the professional field (6.3%), and case management (4.1%)
were mentioned to be important. Other topics PPTs provided
in response to the final open-ended question asking about
suggestions for the knowledge hub were (1) knowledge about
specific exercise physiology and treatment in children with childhood cancer (36.5%), (2) pediatric oncology in general
(30.4%), (3) red flags (11.5%), and (4) psychological coun-
seling (7.4%). In addition, the effects of chemotherapy on the
child (6.1%), side effects of medication (5.4%), and informa-
tion about different prognoses (3.4%) were mentioned to a
lesser extent. Phase 2: co‑creation sessions The co-creation sessions were attended by n = 31 (session 1)
and n = 25 (session 2) PPTs of the in total 34 stakeholders in
the project (n = 19 pediatric physiotherapists from primary
care settings, n = 6 from shared care center, and n = 9 from
rehabilitation center) (see Table 1).ii About education and gaining knowledge, it was often
mentioned that both physical and online trainings are desir-
able. The content of these trainings can cover general knowl-
edge about pediatric oncology, related treatments, fatigue
symptoms, and cognitive, traumatic, and psychosocial sup-
port. In addition, it was mentioned that annual trainings
with the possibility of deepening through e-learnings is
needed. During the physical training, there should also an
opportunity to find each other, to connect, and to network
with each other; attendance at physical days may be man-
datory to encourage active membership. Finally, the care
network and knowledge platform should be accessible to
parents, children, and survivors, so they can find a competent
professional close to home and be expanded to other allied
healthcare professionals (dieticians, occupational therapists,
and speech and language therapists) and psychosocial disci-
plines. The result of this study is a prototype of the knowl-
edge platform KinderOncoNet based on the needs of the
participants in this study. The theme of the first’s co-creation meeting was “find,
connect, and trust” and aimed to identify the needs in terms
of collaboration and findability within the future care net-
work, KinderOncoNet. Topics of accessibility, privacy, and
communication capabilities were also discussed. In the
first co-creation session, it was indicated that PPTs outside
the Maxima need good access to professionals within the
Princess Maxima Center for consultation and patient dis-
cussions. Moreover an easy and secure way to share confi-
dential information and files would facilitate collaboration
across settings. Secondly, trust is very important. Thresholds to treat a
child with childhood cancer should be removed. It is there-
fore important that referrals are made to each other with
additional information, knowledge, and clear indications and
that people know the limits of their own competences and
knowledge. The expectations of KinderOncoNet lay mainly in the
creation of an accessible network in which it is easy to com-
municate, where colleagues can easily be found and where
up-to-date knowledge and training are offered. Phase 2: co‑creation sessions Connec-
tion to KinderOncoNet should not take too much time, be
affordable, and should have a form that ensures an active
connection. Phase 2: co‑creation sessions Furthermore, availability of online e-learnings, physi-
cal education, and organization of network meetings were
mentioned. Regarding the accessibility of KinderOncoNet,
the establishment of an active forum for easy contact with
colleagues, acceptable costs, and a not too high investment 1 3 Supportive Care in Cancer (2024) 32:9 9
Page 6 of 9 9
Page the Princess Máxima Center, and treatment is started there;
the transfer to another institution for further treatment is
difficult. The idea from the participants was to initiate this
familiarization and transfer early in the treatment process,
so that child and caregivers are already familiar with profes-
sionals closer to home. So finding a PPT close to home from
the beginning of the therapy would be helpful. Appointing
a case manager could improve the transition and commu-
nication. Thereby, it was found to be important that differ-
ent institutions can reinforce and complement each other in
sharing care, to get the right care in the right place. of time to be part of the network were mentioned. Moreover,
participation in the network should be reimbursed by health
insurance companies, and CME points should be granted
for the education. Strengths and limitations Strengths of our study are that the iterative process in two
phases allowed us to first analyze the data from the survey
of a large number of participants and then categorize the
information into themes as input for the smaller co-creation
sessions. Another strength is that the project study group included
representation of the VKKN and a large number of parents
of children with cancer. The literature demonstrates that
engagement of stakeholders has great benefits, through part-
nership in research, patients, and healthcare professionals
are actively involved throughout the entire research process
[37–40]. The use of co-creation meetings brought the study
closer to the stakeholders and ensured a higher potential for
impact. Secondly, KinderOncoNet might improve knowledge. Results from a study from Gohar [31] show that although
physicians identified musculoskeletal complications in
children with cancer, only a minority of these patients
were referred for PT. This knowledge supports the need
for increasing the awareness of physicians about benefits
of early integration of PT into the therapy plan for a child
and to refer them as soon as possible to a PPT close to
home [31]. Moreover, there is evidence in the importance
of PPT in reducing physical function problems during and
after treatment to keep them as healthy as possible [6, 11,
12, 32]. So when children get more optimal referred to a
PPT close to home, through the collaboration within Kin-
derOncoNet, the quality and continuity of care in physi-
cal function improves. Of note, the number of childhood
cancer survivors is increasing. In 2020, the number of
childhood cancer survivors (CCS) in Europee reached
500,000 [33, 34], of which many experience late effects
[35, 36]. Most young adults reported a need for support,
in particular for information, especially regarding lifestyle
and health risks after childhood cancer. It is important
to empower them to take control over their health [32]. Thus, thirdly, KinderOncoNet has the potential to be the
hub, connecting patients, parents, survivors, and health-
care professionals in the Princess Máxima Center with
professionals closer to home and facilitate the easy access
to knowledge and skills about these rare diseases. By
doing so, KinderOncoNet would indeed be the solution
to find the right healthcare professional close to home. However, some limitations of the study must be acknowl-
edged. This study focused only on physical therapy. Discussion In the report of their taskforce “The Right Care in
the Right Place,” network care is the answer to the exist-
ing friction between a life-transcending demand for care
for (chronic) conditions and the care landscape clustered
in disciplines and care lines [30]. KinderOncoNet could
be the solution for the challenge with the small number of
children diagnose with cancer in need of optimal quality
and continuity of care, the right care at the right place. and Childhood Cancer Network in Australia [21]; the Ital-
ian rehabilitation group [22]; and Pogo [23] that network
care can improve the quality of care [14]. Moreover, our
initiative would be in line with the directions provided by
the Ministry of Health, Welfare and Sport in the Nether-
lands. In the report of their taskforce “The Right Care in
the Right Place,” network care is the answer to the exist-
ing friction between a life-transcending demand for care
for (chronic) conditions and the care landscape clustered
in disciplines and care lines [30]. KinderOncoNet could
be the solution for the challenge with the small number of
children diagnose with cancer in need of optimal quality
and continuity of care, the right care at the right place. Discussion In this study, we determined that almost all parents indi-
cated a need for a local PPT who is knowledgeable about
both the pediatric oncological condition, its side effects, and
late effects of oncological treatment affecting exercise. At
the same time, almost half of the PPTs indicated a gap in
knowledge to be able to provide high-quality therapy and
also a need for education in the field of pediatric oncology
and physiotherapy care. There was a general understanding
that the development of a care network for pediatric oncol-
ogy would improve care, KinderOncoNet that facilitates col-
laboration, communication, and trust in each other. The theme of the second in-person co-creation meeting
was “knowledge and expertise.” Based on the questions of
the survey, the following topics were further explored and
discussed: “what is needed to improve capacity and com-
petence?,” “how to maintain knowledge and expertise?,”
and “which educational needs and what content and form
of educational needs within the care network are necessary?”
Also, the conditions to participate in KinderOncoNet were
determined. During the second co-creation meeting, the
referral from care from the Princess Maxima Center to care
close to home, so the transfer between different institutions,
was mentioned to be a very important subject. All children
diagnosed with childhood cancer start their treatment in the
Princess Maxima Center, and all complex care takes place
in Utrecht. Less intensive care is provided in the shared
care centers closer to home. As diagnosis is established in The findings provide support for the idea to create a
care network, which is in line with existing initiatives and
research. Firstly, we know from care networks such as
Parkinson disease (ParkinsonNet) [18] in the Netherlands
and international initiatives for childhood cancer, like
ActiveOncoKids [19, 20]; the National Physical Activity 1 3 3 Page 7 of 9
9 Supportive Care in Cancer (2024) 32:9 9 and Childhood Cancer Network in Australia [21]; the Ital-
ian rehabilitation group [22]; and Pogo [23] that network
care can improve the quality of care [14]. Moreover, our
initiative would be in line with the directions provided by
the Ministry of Health, Welfare and Sport in the Nether-
lands. • Knowledge platform, with up-to-date information for child/parents, survivors, and allied healthcare professionals
• Realization of a multidisciplinary digital care access map for parents, children, and healthcare professionals, where they can find each other,
and it is visible which quality requirements they meet and to find the nearest allied healthcare professional close to home
• Development of a multidisciplinary “professional in pediatric oncology” training and e-learnings for allied healthcare professionals
• Define responsibilities and care processes agreements within the care network of allied health professionals
• Connect for optimal quality and continuity of care close to home! Table 4 Expectations, wishes, and products to conceptualize and develop KinderOncoNet
Recommendations i
• Connect for optimal quality and continuity of care close to home! Table 4 Expectations, wishes, and products to conceptualize and develop KinderOncoNet
Recommendations
• Knowledge platform, with up-to-date information for child/parents, survivors, and allied healthcare professionals
• Realization of a multidisciplinary digital care access map for parents, children, and healthcare professionals, where they can find each other,
and it is visible which quality requirements they meet and to find the nearest allied healthcare professional close to home
• Development of a multidisciplinary “professional in pediatric oncology” training and e-learnings for allied healthcare professionals
• Define responsibilities and care processes agreements within the care network of allied health professionals
• Connect for optimal quality and continuity of care close to home! Conclusion Scott JM et al (2018) Association of exercise with mortal-
ity in adult survivors of childhood cancer. JAMA Oncol
4(10):1352–1358 Funding The project was funded by the “National Regieorgaan Prak-
tijkgericht Onderzoek SIA” (K.20.01.092) [42]. To disclose addi-
tional grants that funded the first author of this study: ZonMW grant
(10270022110002) - Development and Evaluation of a National Net-
work of Allied Health Professionals working with Children with Can-
cer to improve Participation and Quality of Life (KinderOncoNet) [43]. 9. Gotte M, Taraks S, Boos J (2014) Sports in pediatric oncology:
the role(s) of physical activity for children with cancer. J Pediatr
Hematol Oncol 36(2):85–90 10. Götte M et al (2016) Translation of exercise research into practice
models in children and adolescents with cancer. Oncol Res Treat
39:126f 10. Götte M et al (2016) Translation of exercise research into practice
models in children and adolescents with cancer. Oncol Res Treat
39:126f 11. Morales JS et al (2020) What are the effects of exercise training in
childhood cancer survivors? A systematic review. Cancer Metas-
tasis Rev 39(1):115–125i Data availability The data that support the findings of this study are
available on request from the corresponding author. 12. Stossel S et al (2020) Benefits of exercise training for children
and adolescents undergoing cancer treatment: results from the
randomized controlled MUCKI trial. Front Pediatr 8:243 Strengths and limitations How-
ever, there is also a wide range of other healthcare disci-
plines involved in the care of a child with cancer. A broader
scope of KinderOncoNet could help improve its goal. There-
fore, we plan to expand KinderOncoNet for other health-
care professionals, including dieticians, speech and language
therapists, occupational therapists, and psychologists. We
should also expand the target population to those children
who are receiving long-term follow-up care, so-called child-
hood cancer survivors, and those transitioning from pediatric
care into adult healthcare settings. Another limitation is the
recruitment of participants. The recruitment was voluntary,
for the parents and PPTs. This may have created a stake-
holder group consisting mainly of partners with a high sense
of participation [41]. Based on the results of this study, we
described expectations, wishes, and products to conceptual-
ize and develop KinderOncoNet (see Table 4). In the future,
the prototype will be developed and evaluated in clinical
practice and among other disciplines, such as psychology,
speech therapy, occupational therapy, and dietetics. Once
KinderOncoNet is sufficiently functional and deployable,
further research will be started to show its feasibility and
effectiveness, on the continuity and quality of care for chil-
dren with childhood oncology during and after treatment. We envision a future evaluation of the costs and benefits of 1 3 9
Page 8 of 9 9
Page 8 of 9 Supportive Care in Cancer (2024) 32:9 the national network using the quadruple aim framework to
report on the impact on population health, healthcare experi-
ences, costs, and professional experiences. 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 national network using the quadruple aim framework to
report on the impact on population health, healthcare experi-
ences, costs, and professional experiences. Conclusion 1. SKION "http://www.skion.nl". Accessed 30 Nov 2023 Parents of children with cancer and PPTs clearly indicate
that there is a need to develop a national care network spe-
cialized in pediatric oncology. Through this project, we
now have a conceptual network “KinderOncoNet” that will
facilitate involvement of children/parents/survivors and
PPTs across settings to improve the accessibility, continuity
and quality of care, participation, and quality of life in the
Netherlands. 2. Verdecchia A et al (2007) Recent cancer survival in Europe: a
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activities of daily living, motor performance, and physical activ-
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study. Front Pediatr 10:982996 Acknowledgements We gratefully acknowledge Marjolijn Ketelaar and
Martin Beuzel for sharing their expertise and for their constructive
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cancer. Cochrane Database Syst Rev 3:Cd008796 Author contribution L.B. Kleinlugtenbelt and J.W. Gorter wrote the
main manuscript. L.B. Kleinlugtenbelt prepared the tables and figures. W.J.E. Tissing, W. Plieger- van Solkema (representative of the parent
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does not require approval from an accredited medical ethics committee
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Nov 2023 Competing interests The authors have no relevant financial or non-
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any organization or entity with any financial interest or non-financial
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12(6):573–576fi Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, 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
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The association between clinical laboratory data and chest CT findings explains disease severity in a large Italian cohort of COVID-19 patients
|
BMC infectious diseases
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cc-by
| 7,807
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The association between clinical laboratory
data and chest CT findings explains disease
severity in a large Italian cohort of COVID-
19 patients 1*, Giulia Besutti2,3, Efrem Bonelli1,2, Valentina Iotti2, Marta Ottone4, Laura Albertazzi1,
2 Simone Canovi1*, Giulia Besutti2,3, Efrem Bonelli1,2, Valentina Iotti2, Marta Ottone4, Laura Albertazzi1,
Alessandro Zerbini5, Pierpaolo Pattacini2, Paolo Giorgi Rossi4, Rossana Colla1, Tommaso Fasano1 and on behalf of
the Reggio Emilia COVID-19 Working Group; Canovi et al. BMC Infectious Diseases (2021) 21:157
https://doi.org/10.1186/s12879-021-05855-9 Canovi et al. BMC Infectious Diseases (2021) 21:157
https://doi.org/10.1186/s12879-021-05855-9 Open Access Abstract Background: Laboratory data and computed tomography (CT) have been used during the COVID-19 pandemic,
mainly to determine patient prognosis and guide clinical management. The aim of this study was to evaluate the
association between CT findings and laboratory data in a cohort of COVID-19 patients. Methods: This was an observational cross-sectional study including consecutive patients presenting to the Reggio
Emilia (Italy) province emergency rooms for suspected COVID-19 for one month during the outbreak peak, who
underwent chest CT scan and laboratory testing at presentation and resulted positive for SARS-CoV-2. Results: Included were 866 patients. Total leukocytes, neutrophils, C-reactive protein (CRP), creatinine, AST, ALT and
LDH increase with worsening parenchymal involvement; an increase in platelets was appreciable with the highest
burden of lung involvement. A decrease in lymphocyte counts paralleled worsening parenchymal extension, along
with reduced arterial oxygen partial pressure and saturation. After correcting for parenchymal extension, ground-
glass opacities were associated with reduced platelets and increased procalcitonin, consolidation with increased
CRP and reduced oxygen saturation. Conclusions: Pulmonary lesions induced by SARS-CoV-2 infection were associated with raised inflammatory
response, impaired gas exchange and end-organ damage. These data suggest that lung lesions probably exert a
central role in COVID-19 pathogenesis and clinical presentation. Keywords: COVID-19, Clinical laboratory, Computed tomography * Correspondence: Simone.Canovi@ausl.re.it
1Clinical chemistry and Endocrinology Laboratory, Departement of
Diagnostic Imaging and Laboratory Medicine, AUSL-IRCCS di Reggio Emilia,
viale Risorgimento 80, 42123 Reggio Emilia, Italy
Full list of author information is available at the end of the article Blood tests and RT-PCR Blood tests and RT-PCR
Laboratory results for C-reactive protein (CRP), lactate
dehydrogenase (LDH), white blood cells, lymphocytes,
neutrophils, and platelets were measured on ER admis-
sion in the entire cohort. Results of arterial blood gas
analysis were also measured. For patients presenting to
ER up to March 13, results for total bilirubin, creatinine,
aspartate
transaminase
(AST),
alanine
transaminase
(ALT), albumin, procalcitonin and prothrombin time
(PT) were also collected. Complete blood counts were
obtained
with
Siemens
ADVIA2120i
(Siemens The aim of this study was to evaluate the association
between laboratory data and chest CT findings in a large
Italian cohort of COVID-19 patients. Background territory. The
first
case
of
SARS-CoV-2
infection
occured on February 27, 2020, and up to June 14, 2020,
there were 4950 virologically-confirmed cases. The study
was approved by the Area Vasta Emilia Nord Ethics
Committee on April 7, 2020, with protocol number
2020/0045199. Given the retrospective nature of the
study, patients’ informed consent to participate in the
study was obtained whenever possible. In December, 2019, an outbreak of a novel coronavirus
(SARS-CoV-2) became apparent in China, and has since
become a global concern for human health and a major
challenge for national health services. In Italy, due to the
overwhelming hospital influx of patients by the end of
February 2020, there was serious concern regarding the
national health system’s capacity to cope with severely
infected subjects who required intensive care for the
management of SARS-CoV-2-related pneumonia [1]. Clinical information, laboratory data and CT scans have
been used in the setting of COVID-19 pandemic, periph-
erally as part of the diagnostic work-up, but mostly to
help define patient prognosis and to guide clinical
management. Study design and population In this observational cross-sectional study, we included
all consecutive patients who presented to the provincial
emergency rooms (ERs) between February 27 and March
23 for suspected COVID-19, underwent chest CT scan
and blood tests at ER presentation and tested positive
for SARS-CoV-2 at RT-PCR within 10 days from ER
presentation. A
recent
meta-analysis
systematically
compared
laboratory findings in cohorts of patients grouped by dis-
ease severity or mortality [2]. Clear patterns of inflam-
matory,
hematological,
biochemical
and
immune
biomarker abnormalities were found, warranting inclu-
sion
of
laboratory
parameters
in
risk
stratification
models. The diagnostic protocol for patients presenting to the
ER for suspected COVID-19 in the province of Reggio
Emilia has been previously described [12]. Briefly, it in-
cluded RT-PCR, blood tests, chest X-rays, and chest CT
scan in cases of suggestive X-ray or clinical findings. CT scan provides information on both type and degree
of parenchymal involvement, the first including mostly
ground-glass opacities (GGO) and consolidation, and
the latter by means of estimating, visually or automatic-
ally, the percentage of involved lung parenchyma. A po-
tential prognostic role of chest CT findings, especially
the extent of parenchymal involvement, has been pro-
posed [3–6]. © The Author(s). 2021 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. Canovi et al. BMC Infectious Diseases (2021) 21:157 Page 2 of 9 Data collection Data were retrieved from the COVID-19 Surveillance
Registry coordinated by the National Institute of Health
and implemented in each Local Health Authority. This
registry collects information about date of symptom
onset, diagnosis, hospitalization and death or recovery of
patients testing positive for SARS-CoV-2 RNA by RT-
PCR. Information is directly collected from the patient
himself/herself through daily telephone contact when
cared for in an outpatient setting and from electronical
medical
records
when
hospitalized. Data
from
the
COVID-19 Surveillance Registry were linked with the
provincial Radiology Information System to search for
CT scans performed at the moment of or after the onset
of COVID symptoms. For all included patients, hospital
discharge databases were linked to the COVID-19 Regis-
try to identify hospital admissions in the 10 years pre-
ceding COVID-19 hospitalization in order to calculate
the Charlson index for each patient [13]. Few studies have also combined clinical, laboratory,
and CT findings [7, 8]. Some of these suggest that the
performance of the prognostic model is better when
adding CT features, while the others show that CT find-
ings had insufficient prognostic power to be used in
combination models. In this setting, it would be useful to understand how
CT changes relate to laboratory data describing different
clinical
and
pathophysiological
derangements
in
COVID-19. Indeed, studies reporting the association be-
tween lung imaging and clinical laboratory data in
COVID-19 patients are scarce and heterogeneous in
terms of study population, laboratory tests and imaging
interpretation. While consistent associations are mostly
reported for some laboratory data [9–11] conflicting re-
sults have been described for others [5]. CT structured reporting and retrospective analysis p
g
p
y
In the period between March 13 and March 23, during
routine CT reporting, each radiologist completed both
the usual radiology report as well as a structured report,
including the presence/absence of GGO and consolida-
tions, and the extension of pulmonary lesions using a
visual scoring system (< 20%, 20–39%, 40–59%, and ≥
60% of parenchymal involvement) (Fig. 1) [12]. Chest
CTs which were performed in this time frame were not
retrospectively reviewed. CT scans performed in the time period between
February 27 and March 13 were retrospectively reviewed
by an experienced radiologist. Besides the three parame-
ters described above (GGO, consolidations, and visual
scoring), data about the presence of crazy-paving pat-
tern, pleural effusion, and mediastinal or hilar lymph
node enlargement (short axis > 1 cm) were also recorded. The interrater agreement in CT disease extension evalu-
ation has already been reported to be excellent [14]. Statistical analyses Laboratory results are reported as medians (interquartile
range, IQR). Distribution of CT findings across multiple
qualitative and quantitative (lung extension) classes are
reported; the association between qualitative CT find-
ings, clinical and demographic variables and degree of
parenchymal involvement was evaluated through Fisher’s
exact test. The associations between CT findings and
laboratory results were evaluated with multiple linear
regression models adjusted for confounders sex and age. P values are reported as continuous measures and no
preset significance threshold was used. Analyses were
performed using software packages R 3.1.0 (R Founda-
tion for Statistical Computing, Vienna, Austria) and
MedCalc 18.2.1 (MedCalc Software, Ostend, Belgium). To diagnose SARS-CoV-2 infection, a commercial
one-step reverse transcriptase-polymerase chain reac-
tion (RT-PCR) (GeneFinder™COVID -19 PLUS Real
Real Amp Kit) was used and RT-PCR assay was per-
formed on an Applied Biosystems 7500 Sequence
Detection System (Applied Biosystems, Foster City,
CA, United States). Methods
Setting The Reggio Emilia province (532,000 inhabitants), lo-
cated in Northern Italy, counts six hospitals in its Canovi et al. BMC Infectious Diseases (2021) 21:157 Page 3 of 9 GE Medical Systems. Other details on CT acquisition
technique have been previously reported [12]. Healthineers, Erlangen, Germany) on BD Vacutainer K2-
EDTA-anticoagulated whole blood (Becton Dickinson,
Franklin Lakes, NJ, US); PT was measured with Siemens
Thromborel S on a Sysmex CS-5100 automated coagul-
ometer (Sysmex Corporation, Kobe, Japan) in plasma
samples obtained after centrifugation at 1500 g for 15
min of whole blood collected in 1.8 mL BD Vacutainer
tubes with 3.2% sodium citrate 0,109 M; arterial blood
gases were analyzed with ABL800 flex (Radiometer,
Copenaghen, Denmark) on heparinized blood collected
in BD syringes for arterial blood collection; procalcitonin
concentrations were measured with LIAISON BRAHMS
PCT II GEN on a LIAISON XL (DiaSorin, Saluggia VC,
Italy) in plasma samples collected in lithium heparin BD
Vacutainers. All other biochemical tests were measured
with Siemens automated methods (wide-range CRP,
AST, ALT, TBIL_2, ALB, CREA_2, LDPL) on ADVIA-
1800 chemistry analyzers in lithium heparin plasma sam-
ples: immunoturibidmetric wide-range CRP, enzymatic
methods for AST, ALT and LDH (forward reaction), col-
orimetric methods for creatinine (kinetic Jaffe reaction),
bilirubin (vanadate oxidation method) and albumin (bro-
mocresol green endpoint). Internal quality control and
external quality assessment were implemented for all
these measurands during the study period. Results involvement, going from 15.7 to 37.9% and from 5.0 to
43.3%, respectively. Clinical outcome was associated with
the degree of parenchymal involvement evaluated by CT
analysis: indeed, hospitalization rates and proportions of
early (within 30 days) deaths raised from 24.8 and 5.0%
in patients with parenchymal involvement < 20 to 79.1%
and 43.3% in cases with ≥60% compromised lungs,
respectively. Overall, 837 (96.7%) patients had GGO and
547 (63.2%) had consolidation. Retrospective review of
CT revealed 126 (60.3%) cases with crazy-paving pattern;
enlarged lymph nodes and pleural effusion were present
in 38 (12.0%) and 27 (8.5%) patients, respectively. Each
individual qualitative finding was associated with the
degree of lung involvement: GGO were observed in
92.3% of patients with parenchymal extension < 20% and
in 100% of patients with extension ≥60%; likewise, the
proportion of patients with consolidation increased from
55.2 to 73.1% (Table 1). Included in the study were 866 patients (318 from Feb-
ruary 27 to March 13 and 548 from March 14 to March
23). Median age was 60 years (range: 18–96); there were
527 (60.9%) males and 339 (39.1) females. Charlson co-
morbidity index ranged from 0 to 10, with 151 (18.4%)
patients having an index greater than or equal to 1. Pa-
tients were clinically evaluated after a median of 7 days
(IQR: 4–9 days) since symptom onset. Table 1 summarizes demographic and clinical vari-
ables and CT findings, grouped according to the exten-
sion
of
underlying
parenchymal
involvement. The
proportion of patients over 60 years of age increased
with increasing parenchymal extension evaluated by CT
imaging, going from 39.2% in individuals with extension
< 20 to 62.7% in cases with extension ≥60%. The same
was true for the percentage of male patients, which
increased from 54.6 to 73.1% in cases with extension <
20% or ≥60%, respectively. At the same time, the pro-
portion of individuals with a Charlson index greater than
or equal to 1 and the proportion of deaths within 30
days of admission increased with increasing parenchymal The association between parenchymal extension and
laboratory results, corrected for age and sex, is reported
in Table 2. Proportions of demographic/clinical variables and CT findings across CT extension classes [n (% of class column)]. Data for ground glass opacities and
consolidation are obtained from the entire cohort (n = 866); raw numbers and frequencies for the other CT categories are calculated from patients observed until
March 13 (n = 318) CT acquisition technique CT scans were performed without contrast media injec-
tion, with one of the following scanners: 128-slice Soma-
tom
Definition
Edge,
Siemens
Healthcare;
64-slice
Ingenuity, Philips Healthcare; 16-slice GE Brightspeed, Fig. 1 Coronal chest CT images representing the visual scoring system used to evaluate the degree of parenchymal involvement: < 20% (a), 20–
39% (b), 40–59% (c), > = 60% (d). Parenchymal qualitative CT findings evaluated in this study were: ground-glass opacities (asterisks in panels a
and d), consolidation (arrows in panel b), crazy-paving pattern (arrowheads in panel c) Fig. 1 Coronal chest CT images representing the visual scoring system used to evaluate the degree of parenchymal involvement: < 20% (a), 20–
39% (b), 40–59% (c), > = 60% (d). Parenchymal qualitative CT findings evaluated in this study were: ground-glass opacities (asterisks in panels a
and d), consolidation (arrows in panel b), crazy-paving pattern (arrowheads in panel c) Page 4 of 9 Canovi et al. BMC Infectious Diseases (2021) 21:157 Canovi et al. BMC Infectious Diseases (2021) 21:157 Page 4 of 9 Page 4 of 9 Results Circulating concentrations of total leuko-
cytes, neutrophils, C-reactive protein, creatinine, AST, Table 1 Summary of demographic, clinical variables and CT findings
Parenchymal extension
< 20%
20–39%
40–59%
≥60%
Fisher’s exact test p-value
Demographic and clinical variables
Age
≥60 y (n = 432)
133 (39.2)
175 (51.5)
82 (68.3)
42 (62.7)
< 0.0001
< 60 y (n = 434)
206 (60.8)
165 (48.5)
38 (31.7)
25 (37.3)
Sex
Male (n = 527)
185 (54.6)
205 (60.3)
88 (73.3)
49 (73.1)
0.0001
Female (n = 339)
154 (45.4)
135 (39.7)
32 (26.7)
18 (26.9)
Charlson comorbidity index
≥1 (n = 151)
50 (15.7)
48 (15.0)
28 (24.8)
25 (37.9)
< 0.0001
0 (n = 667)
268 (84.3)
273 (85.0)
85 (75.2)
41 (62.1)
Hospitalization
N (n = 503)
255 (75.2)
197 (57.9)
37 (30.8)
14 (20.9)
< 0.0001
Y (n = 363)
84 (24.8)
143 (42.1)
83 (69.2)
53 (79.1)
Death within 30 days
N (n = 773)
322 (95.0)
317 (93.2)
96 (80.0)
38 (56.7)
< 0.0001
Y (n = 93)
17 (5.0)
23 (6.8)
24 (20.0)
29 (43.3)
CT findings
Ground-glass opacities
N (n = 29)
26 (7.7)
2 (0.6)
1 (0.8)
0 (0.0)
0.0051
Y (n = 837)
313 (92.3)
338 (99.4)
119 (99.2)
67 (100)
Consolidation
N (n = 319)
152 (44.8)
118 (34.7)
31 (25.8)
18 (26.9)
0.0002
Y (n = 547)
187 (55.2)
222 (65.3)
89 (74.2)
49 (73.1)
Crazy-paving pattern
N (n = 126)
66 (60.6)
38 (33.3)
15 (25.0)
7 (20.6)
0.0042
Y (n = 191)
43 (39.4)
76 (66.7)
45 (75.0)
27 (79.4)
Enlarged lymph nodes
N (n = 279)
104 (95.4)
102 (89.5)
49 (81.7)
24 (70.6)
0.0005
Y (n = 38)
5 (4.6)
12 (10.5)
11 (18.3)
10 (29.4)
Pleural effusion
N (n = 290)
106 (97.2)
104 (91.2)
52 (86.7)
28 (82.4)
0.0101
Y (n = 27)
3 (2.8)
10 (8.8)
8 (13.3)
6 (17.6)
Proportions of demographic/clinical variables and CT findings across CT extension classes [n (% of class column)]. Data for ground glass opacities and
consolidation are obtained from the entire cohort (n = 866); raw numbers and frequencies for the other CT categories are calculated from patients observed until Table 1 Summary of demographic, clinical variables and CT findings Proportions of demographic/clinical variables and CT findings across CT extension classes [n (% of class column)]. Data for ground glass opacities and
consolidation are obtained from the entire cohort (n = 866); raw numbers and frequencies for the other CT categories are calculated from patients observed until
March 13 (n = 318) CT findings Canovi et al. BMC Infectious Diseases (2021) 21:157 Page 5 of 9 Table 2 Association between parenchymal CT extension and laboratory data
CT lesion extension
< 20% (n = 339)
20–40% (n = 340)
40–60% (n = 120)
> 60% (n = 67)
N
Median (IQR)
N
Median (IQR)
N
Median (IQR)
N
Median (IQR)
P
White blood cells (10^9/L) 304 4.79 (3.84–5.91)
315 5.11 (4.02–6.50)
113 5.60 (4.33–7.85)
66 6.22 (4.66–7.97)
0.0005
Neutrophils (10^9/L)
265 3.02 (2.32–4.04)
291 3.64 (2.66–4.76)
101 4.13 (3.05–6.16)
62 4.79 (3.49–6.49)
< 0.0001
Lymphocytes (10^9/L)
264 1.14 (0.86–1.57)
290 1.05 (0.77–1.39)
100 0.89 (0.68–1.29)
62 0.85 (0.63–1.05)
0.0004
Platelets (10^9/L)
301 180.00 (146.51–
221.25)
313 176.00 (147.29–
218.75)
113 180.00 (140.19–
218.25)
65 214.00 (159.00–
269.25)
0.0086
Prothrombin time (ratio)
29
1.00 (0.96–1.05)
55
1.10 (1.02–1.15)
37
1.08 (1.02–1.19)
25 1.10 (1.05–1.14)
0.1367
C-reactive protein (mg/L)
299 15.00 (7.02–29.73)
315 43.02 (24.03–85.38)
112 84.60 (46.68–135.66)
64 122.50 (49.55–
206.40)
< 0.0001
Procalcitonin (μg/L)
59
0.090 (0.05–0.12)
75
0.13 (0.09–0.20)
46
0.18 (0.11–0.41)
27 0.26 (0.11–0.60)
0.6363
Creatinine (μmol/L)
90
75.60 (67.20–91.96)
109 84.88 (67.91–95.67)
59
88.42 (74.36–114.95)
33 92.84 (79.14–128.21)
0.0162
AST (U/L)
68
28.50 (23.20–36.00)
90
38.50 (29.00–49.00)
58
43.00 (34.00–62.28)
29 45.00 (40.00–52.95)
< 0.0001
ALT (U/L)
71
25.00 (19.25–39.50)
93
30.73 (19.00–43.50)
57
32.00 (23.54–41.11)
29 30.00 (23.22–39.50)
0.0091
Total bilirubin (μmol/L)
63
8.55 (6.84–11.97)
86
10.26 (8.48–13.68)
51
10.26 (6.84–13.68)
30 11.12 (8.55–17.10)
0.9055
Albumin (g/L)
14
40.90 (38.30–42.20)
30
38.60 (36.30–41.20)
25
37.50 (35.55–39.52)
13 38.30 (32.63–40.10)
0.0077
LDH (U/L)
249 408.50 (358.15–
487.00)
273 510.53 (422.77–
599.75)
88
639.50 (474.85–
783.50)
49 707.00 (496.81–
896.50)
< 0.0001
PCO2 (mmHg)
73
35.70 (32.58–38.35)
86
34.40 (31.00–37.20)
53
33.30 (30.88–36.45)
31 36.30 (32.15–42.08)
0.6574
PO2 (mmHg)
73
79.50 (69.33–87.05)
85
71.00 (64.20–78.40)
53
65.50 (61.68–74.42)
31 60.80 (54.63–72.65)
0.0014
SO2 (%)
284 96.50 (95.00–97.80)
286 95.20 (93.80–96.50)
103 93.70 (91.80–95.78)
62 91.80 (88.30–94.40)
< 0.0001
P: p-value for coefficient of CT variable in a multiple linear regression controlled for confounders age and sex Table 2 Association between parenchymal CT extension and laboratory data ALT and LDH showed a progressive increase with wors-
ening parenchymal involvement (Fig. 1); an increase in
platelets, on the other hand, was appreciable only in as-
sociation with the highest burden of lung involvement. CT findings A small decrease in circulating albumin concentrations
and lymphocyte counts paralleled worsening parenchy-
mal extension, along with reduced arterial oxygen partial
pressure and saturation. Finally, total bilirubin, procalci-
tonin and prothrombin time showed only very modest
increments with the extension of lung involvement by
SARS-CoV-2 infection. creatinine concentrations. The presence of enlarged
lymph nodes was associated with increased neutrophil
counts, whereas pleural effusion with increased LDH
and creatinine concentrations, reduced albumin and re-
duced oxygen saturation levels (Additional file 1). ALT and LDH showed a progressive increase with wors-
ening parenchymal involvement (Fig. 1); an increase in
platelets, on the other hand, was appreciable only in as-
sociation with the highest burden of lung involvement. A small decrease in circulating albumin concentrations
and lymphocyte counts paralleled worsening parenchy-
mal extension, along with reduced arterial oxygen partial
pressure and saturation. Finally, total bilirubin, procalci-
tonin and prothrombin time showed only very modest
increments with the extension of lung involvement by
SARS-CoV-2 infection. Discussion We report the results of a cross-sectional study evaluat-
ing the association between CT imaging and laboratory
data in a large cohort of SARS-CoV-2-infected patients
observed in northern Italy during the peak of COVID-19
outbreak, taking into account quantitative and qualita-
tive radiologic findings and their relation with changes
in laboratory results reflecting inflammatory response,
gas exchange and end-organ damage. The main findings
of the association between laboratory data and the ex-
tension of pulmonary lesions are reported in Fig. 2. The
clinical spectrum induced by SARS-CoV-2 is broad, go-
ing from subclinical infections to severe cases of pneu-
monia with respiratory failure, sepsis and multiple organ
failure [15, 16]. In this setting, radiological and labora-
tory tests have established clinical roles, mainly for the
prognostication of COVID-19 patients. However, the Since a correlation was found between the extent of
parenchymal involvement and the presence of individual
CT findings, we were interested in evaluating whether
the association between laboratory results and GGO,
consolidation,
crazy-paving
pattern,
enlarged
lymph
nodes and pleural effusion was independent from the
degree of parenchymal involvement. Consequently, the
analysis was corrected for age, sex and extension of lung
lesions (Table 3). After correction, GGO were associated
with reduced platelet counts and increased procalcitonin
concentrations; consolidation with increased CRP and
reduced oxygen saturation levels; crazy-paving pattern
with reduced oxygen pressure and an increasing trend in Canovi et al. Discussion BMC Infectious Diseases (2021) 21:157 Page 6 of 9 Table 3 Association between CT findings and laboratory data (corrected for parenchymal extension)
Ground-glass opacities
Consolidation
Crazy paving
N (n = 29)
Y (=837)
P*/P**
N (n = 319)
Y (n = 547)
P*/P**
N (n = 126)
Y (n = 191)
P*/P**
White blood
cells (10^9/L)
5.77 (3.84–7.10)
(n = 26)
5.10 (4.02–6.51)
(n = 772)
0.5890/
0.1833
5.00 (3.98–6.34)
(n = 285)
5.15 (4.02–6.60)
(n = 513)
0.1979/
0.5441
5.11 (3.99–6.57)
(n = 110)
5.29 (4.16–6.63)
(n = 173)
0.5099/
0.7682
Neutrophils
(10^9/L)
3.52 (2.71–4.67)
(n = 24)
3.56 (2.58–4.72)
(n = 695)
0.7394/
0.4439
3.42 (2.60–4.63)
(n = 252)
3.61 (2.56–4.80)
(n = 467)
0.1437/
0.5918
3.57 (2.66–4.71)
(n = 89)
4.06 (2.71–5.33)
(n = 154)
0.4268/
0.8354
Lymphocytes
(10^9/L)
1.47 (0.81–1.93)
(n = 24)
1.04 (0.77–1.40)
(n = 692)
0.0887/
0.3354
1.08 (0.82–1.46)
(n = 252)
1.02 (0.75–1.39)
(n = 464)
0.2403/
0.5464
1.00 (0.72–1.31)
(n = 89)
0.94 (0.69–1.34)
(n = 152)
0.7478/
0.2758
Platelets
(10^9/L)
205.00
(159.50–267.75)
(n = 25)
180.00 (145.12–
222.19) (n =
767)
0.0422/
0.0201
174.72 (149.00–
221.75) (n =
283)
184.00 (144.00–
223.61) (n =
509)
0.7304/
0.9761
178.00 (142.75–
212.25) (n =
109)
174.00 (139.50–
222.19) (n =
175)
0.1109/
0.3102
Prothrombin
time (ratio)
0.95 (0.95–0.95)
(n = 1)
1.08 (1.00–1.14)
(n = 145)
0.3795/
0.4951
1.09 (1.03–1.12)
(n = 32)
1.07 (1.00–1.16)
(n = 114)
0.5081/
0.4801
1.04 (0.98–1.12)
(n = 49)
1.09 (1.02–1.17)
(n = 96)
0.8513/
0.5647
C-reactive
protein (mg/
L)
7.05 (1.30–
29.90) (n = 26)
34.45 (14.90–
82.80) (n = 764)
0.0197/
0.7841
23.80 (9.92–
53.42) (n = 281)
43.60 (17.25–
96.04) (n = 509)
<
0.0001/
< 0.0001
37.50 (14.10–
80.96) (n = 107)
74.70 (29.23–
126.95) (n =
171)
0.0065/
0.6653
Procalcitonin
(μg/L)
0.09 (0.05–0.15)
(n = 12)
0.12 (0.08–0.25)
(n = 195)
0.0416/
0.0245
0.10 (0.05–0.19)
(n = 56)
0.13 (0.09–0.28)
(n = 151)
0.7360/
0.6694
0.11 (0.07–0.25)
(n = 66)
0.14 (0.09–0.25)
(n = 125)
0.8514/
0.9498
Creatinine
(μmol/L)
75.16 (60.39–
92.40) (n = 13)
83.82 (69.41–
98.94) (n = 278)
0.6436/
0.8846
84.88 (65.61–
95.76) (n = 79)
84.88 (71.53–
99.03) (n = 212)
0.3396/
0.4231
82.23 (68.08–
96.82) (n = 104)
84.00 (71.09–
102.57) (n =
172)
0.1572/
0.0468
AST (U/L)
28.00 (21.50–
35.25) (n = 11)
38.76 (28.00–
50.00) (n = 234)
0.0709/
0.5098
38.00 (27.00–
49.00) (n = 73)
38.00 (28.40–
50.00) (n = 172)
0.6819/
0.9841
31.00 (24.00–
44.25) (n = 85)
40.34 (30.96–
52.00) (n = 146)
0.0061/
0.0698
ALT (U/L)
25.00 (19.00–
36.50) (n = 11)
29.00 (20.00–
41.34) (n = 239)
0.2297/
0.5579
29.83 (22.00–
44.00) (n = 74)
28.00 (19.00–
40.00) (n = 176)
0.4871/
0.3426
27.00 (19.00–
38.00) (n = 87)
29.00 (21.07–
43.00) (n = 148)
0.0422/
0.1239
Total
bilirubin
(μmol/L)
9.41 (8.55–
13.68) (n = 10)
10.26 (7.39–
13.68) (n = 220)
0.9687/
0.8017
8.55 (6.84–
11.97) (n = 69)
10.26 (8.55–
13.77) (n = 161)
0.1239/
0.1136
9.41 (7.46–
11.97) (n = 81)
10.26 (8.11–
13.68) (n = 135)
0.2978/
0.2033
Albumin (g/L)
(n = 0)
38.35 (35.90–
40.70) (n = 82)
ND
38.15 (36.75–
41.45) (n = 16)
38.50 (35.70–
40.40) (n = 66)
0.3277/
0.3778
38.90 (36.20–
40.55) (n = 32)
38.00 (35.55–
40.55) (n = 49)
0.7033/
0.3991
LDH (U/L)
376.00
(354.25–471.50)
(n = 23)
490.00 (390.65–
619.75) (n =
635)
0.0413/
0.4449
459.00 (379.00–
565.00) (n =
245)
502.00 (394.78–
644.00) (n =
413)
0.0039/
0.0821
469.00 (379.50–
596.00) (n = 68)
572.00 (449.71–
724.00) (n =
116)
0.0063/
0.6203
PCO2
(mmHg)
40.05 (30.90–
45.95) (n = 8)
34.60 (31.50–
37.58) (n = 235)
0.0580/
0.0781
35.80 (31.70–
38.65) (n = 72)
34.30 (31.10–
37.50) (n = 171)
0.6043/
0.6623
34.25 (30.90–
37.70) (n = 82)
34.90 (31.50–
37.58) (n = 147)
0.7452/
0.8746
PO2 (mmHg)
76.70 (72.35–
87.15) (n = 8)
70.65 (63.50–
80.60) (n = 234)
0.6333/
0.8540
73.15 (65.60–
83.70) (n = 72)
69.50 (62.40–
80.00) (n = 170)
0.3262/
0.5264
74.35 (65.30–
84.80) (n = 82)
68.05 (62.20–
77.30) (n = 146)
0.0034/
0.0396
sO2 (%)
96.90 (94.55–
98.30) (n = 23)
95.40 (93.50–
96.80) (n = 712)
0.1516/
0.6693
95.80 (94.03–
97.10) (n = 275)
95.20 (93.15–
96.70) (n = 460)
0.0005/
0.0154
95.55 (93.40–
96.90) (n = 90)
93.85 (91.70–
95.70) (n = 150)
0.0203/
0.3603
Data are reported as median (interquartile range). Discussion P: p-value for coefficient of CT variable in a multiple linear regression controlled for confounders age and sex
(P*) or age, sex and CT extension (P**). P-values for predictor CT extension in these models were < 0.05 for total leukocytes, neutrophils, platelets, CRP, creatinine,
AST, ALT, LDH, albumin and oxygen partial pressure and saturation Data are reported as median (interquartile range). P: p-value for coefficient of CT variable in a multiple linear regression controlled for confounders age and sex
(P*) or age, sex and CT extension (P**). P-values for predictor CT extension in these models were < 0.05 for total leukocytes, neutrophils, platelets, CRP, creatinine,
AST, ALT, LDH, albumin and oxygen partial pressure and saturation relationship between imaging findings and laboratory
results is poorly studied. remains elusive to this day. The lung itself could be
the
primary
site
of
production
of
these
pro-
inflammatory mediators [18]; on the other hand, the
cytokine storm could also contribute to pulmonary in-
jury and worsening respiratory function. Like other
viral diseases, the increase in procalcitonin concentra-
tions was only marginal, probably due to the inhibitory
effect of cytokines such as interferon gamma [19]. Fi-
nally, we found another aspect of dysregulated im-
mune
response
associated
with
worsening
lung
involvement: a progressive reduction in circulating
lymphocytes, the degree of which has been reported to
be correlated with clinical severity of COVID-19 [20]. First of all, we observed that the extent of lung par-
enchymal involvement was associated with raised in-
flammatory
response,
as
suggested
by
increased
circulating white blood cells, neutrophil counts and
CRP concentrations. It has indeed been demonstrated
that levels of pro-inflammatory cytokines such as IL-6
(the main stimulus for CRP production) are increased
during SARS-CoV-2 infection, probably playing an im-
munopathologic role in cases of overproduction (so-
called “cytokine storm”) [17]. However, the exact link
between pulmonary lesions and inflammatory reaction Canovi et al. BMC Infectious Diseases (2021) 21:157 Page 7 of 9 Fig. 2 The scheme summarizes the significant variations in laboratory data related to the extension of penumonia evaluated on CT scans e summarizes the significant variations in laboratory data related to the extension of penumonia evaluated on CT scans Fig. 2 The scheme summarizes the significant variations in laboratory data related to the extension of penumonia evalu filling of airspaces or interstitial thickening. Discussion In our
cohort, GGOs were present in almost 97% of included
patients: the remaining had mostly negative CT scans,
showing no prominent signs of pneumonia. In this con-
text, the finding suggests that patients with COVID-19
pneumonia have lower platelet counts than patients
without obvious pulmonary lesions. However, higher
degrees of pulmonary involvement were associated with
increased platelets, a finding compatible with the under-
lying pro-inflammatory state. At the same time, the
increasing trend in prothrombin time with worsening
parenchymal involvement was negligible. These findings
are inconclusive and should be interpreted with caution. In particular, our data lack D-dimer concentrations,
which are significantly more sensitive than platelets
and PT in evaluating the presence of COVID-19 coag-
ulopathy [28]. As to qualitative CT findings, consolidation was inde-
pendently associated with CRP levels, confirming the
findings of previous studies [21, 22], while procalcito-
nin levels were slightly higher in patients with GGO. Finally, we found an independent association between
enlarged lymph nodes and increased neutrophil count. As to qualitative CT findings, consolidation was inde-
pendently associated with CRP levels, confirming the
findings of previous studies [21, 22], while procalcito-
nin levels were slightly higher in patients with GGO. Finally, we found an independent association between
enlarged lymph nodes and increased neutrophil count. The extension of parenchymal lesions was associated
with impaired respiratory gas exchange and hypoxemia. This is not surprising since affected lungs show diffuse
alveolar damage and interstitial edema [23]. Moreover,
vascular abnormalities [24] and perfusion anomalies [25]
probably concur to impair oxygen exchange. The pres-
ence of consolidation at CT, defined as an increase in
parenchymal density obscuring vessels and airways, cor-
responds to alveolar filling by pathological fluids and
cells; in COVID-19 patients this frequently presents with
aspects of organizing pneumonia [26]. Hence, the inde-
pendent association between consolidation and impaired
gas exchange (reduced oxygen saturation level) is to be
expected. Similarly, as the crazy-paving pattern consists
in the superimposition of interlobular septal thickening
on GGO, resulting from alveolar edema and interstitial
inflammation [26], its independent relationship with
reduced oxygen pressure is probably linked to alveolar-
capillary interface dysfunction. Finally, compromised
cardio-respiratory mechanics could further aggravate
oxygen exchange in the presence of pleural effusion. Finally, laboratory tests are clinically useful in recog-
nizing end-organ damage, possibly culminating in multi-
organ failure in the most severe cases. Funding
Thi This research did not receive any specific grant from funding agencies in the
public, commercial, or not-for-profit sectors. Authors’ contributions
SC C
l
D SC: Conceptualization, Data curation, Formal analysis, Methodology, Writing -
original draft, Writing - review and editing. GB: Conceptualization, Data
curation, Methodology, Visualization, Writing - original draft, Writing - review
and editing. EB: Conceptualization, Data curation, Formal analysis, Writing -
review and editing. VI: Data curation, Writing - review and editing. MO: Data
curation, Formal analysis, Methodology, Writing - review and editing. LA:
Writing - review and editing. AZ: Writing - review and editing. PP: Writing -
review and editing. PGR: Data curation, Formal analysis, Methodology,
Writing - review and editing. RC: Writing - review and editing. TF:
Conceptualization, Data curation, Methodology, Visualization, Writing -
original draft, Writing - review and editing. All authors read and approved
the final manuscript. Conceptualization, Data curation, Methodology, Visualization, Writing -
original draft, Writing - review and editing. All authors read and approved
the final manuscript. Ethics approval and consent to participate Research involving human subjects complied with all relevant national
regulations, institutional policies and is in accordance with the tenets of the
Helsinki Declaration (as revised in 2013), and has been approved by the Area
Vasta Emilia Nord Ethics Committee on April 7, 2020 (protocol number 2020/
0045199). Acknowledgments Acknowledgments
We thank Jacqueline Costa for the English language editing and all
personnel of the Clinical chemistry and Endocrinology Laboratory and
Radiology Unit of AUSL-IRCCS, Reggio Emilia. Reggio Emilia COVID-19 Working Group: Massimo Costantini, Roberto Grilli,
Massimiliano Marino, Giulio Formoso, Debora Formisano, Paolo Giorgi Rossi,
Emanuela Bedeschi, Cinzia Perilli, Elisabetta La Rosa, Eufemia Bisaccia, Ivano
Venturi, Massimo Vicentini, Cinzia Campari, Francesco Gioia, Serena Broccoli,
Marta Ottone, Pierpaolo Pattacini, Giulia Besutti, Valentina Iotti, Lucia Spag-
giari, Pamela Mancuso, Andrea Nitrosi, Marco Foracchia, Rossana Colla, Ales-
sandro Zerbini, Marco Massari, Anna Maria Ferrari, Mirco Pinotti, Nicola
Facciolongo, Ivana Lattuada, Laura Trabucco, Stefano De Pietri, Giorgio Fran-
cesco Danelli, Laura Albertazzi, Enrica Bellesia, Simone Canovi, Mattia Corra-
dini, Tommaso Fasano, Elena Magnani, Annalisa Pilia, Alessandra Polese, Silvia
Storchi Incerti, Piera Zaldini, Efrem Bonelli, Bonanno Orsola, Matteo Revelli,
Carlo Salvarani, Carmine Pinto, Francesco Venturelli. We thank Jacqueline Costa for the English language editing and all
personnel of the Clinical chemistry and Endocrinology Laboratory and
Radiology Unit of AUSL-IRCCS, Reggio Emilia. The main limitations of this study are that for some
laboratory tests we did not have a large sample for ana-
lysis (e.g. prothrombin time, total bilirubin) and we
lacked other potentially important tests such as D-
dimer. This is due to the fact that we gathered real-life
data regarding a novel disease during its initial outbreak,
when available evidence was very scarce in guiding clini-
cians in appropriately requesting laboratory tests. None-
theless, we were still able to evaluate many tests
describing different possible clinical and pathophysio-
logical derangements. Also, CT data collection was influ-
enced by the specific period of the epidemic. Since a
structured report was implemented into practice two
weeks after the initial outbreak, CT scans performed in
the initial time period were retrospectively reviewed. However, the reproducibility of CT visual scoring was
high [8, 14], and the different data collection is unlikely
to have influenced the study results. For the same rea-
sons, we lacked information on other CT signs including
findings of organizing pneumonia or interstitial lung fi-
brosis [30]. Availability of data and materials The datasets generated and analysed during the current study are not
publicly available due to privacy resctrictions, but are available from the
corresponding author on reasonable request. Consent for publication
Not applicable. Consent for publication
Not applicable. Competing interests
The authors declare that they have no competing interests. Discussion In this regard, we
observed an increase in circulating levels of intracellular
enzymes with worsening pulmonary involvement: in par-
ticular, LDH had the most sensitive increase, with AST
and especially ALT showing only modest changes. Since
these biomarkers are distributed in various tissues and
organs, it is difficult to interpret their elevation and
probably reflect multi-organ injury (e.g. lungs, liver, kid-
neys, skeletal muscle etc.). We also found a modest
increase in creatinine concentrations with worsening
lung involvement, while we observed only a small
increasing trend in total bilirubin concentrations. COVID-19-related
coagulopathy
shares
similarities
with diffuse intravascular coagulation, being associated
with increased fibrinolytic activity (and raised D-dimer
concentrations), slightly prolonged prothrombin time
and reduced platelet counts [27]. We found that patients
with GGO showed a modest decrease in platelet counts. GGO is the main finding in viral pneumonia and con-
sists in a hazy increase in attenuation with preservation
of bronchial and vascular structures, representing partial Our data are concordant with the most frequent and
consistent changes previously reported in association
with worsening lung involvement, concerning increased
CRP and LDH concentrations and reduced lymphocyte
counts and oxygen level [29]. In addition, we were able Canovi et al. BMC Infectious Diseases (2021) 21:157 Canovi et al. BMC Infectious Diseases (2021) 21:157 Page 8 of 9 Abbreviations ALT: Alanine transaminase; AST: Aspartate transaminase; COVID-
19: Coronavirus disease – 19; CRP: C-reactive protein; CT: Computed
tomography; ER: Emergency room; GGO: Ground-glass opacities;
IL: Interleukin; LDH: Lactate dehydrogenase; PT: Prothrombin time; RT-
PCR: Reverse transcriptase-polymerase chain reaction; SARS-CoV-2: Severe
Acute Respiratory Syndrome – Coronavirus – 2 Competing interests Competing interests
The authors declare that they have no competing interests. Conclusions In conclusion, pulmonary lesions induced by SARS-
CoV-2 infection and evaluated by CT imaging are asso-
ciated with raised inflammatory response, impaired gas
exchange and end-organ damage, as evidenced by clin-
ical laboratory data [31]. These data suggest that lung le-
sions
probably
exert
a
central
role
in
COVID-19
pathogenesis and clinical presentation. More studies are
needed to explore the link between lung lesions and the
COVID-19 related coagulopathy. Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12879-021-05855-9. to include laboratory tests less frequently reported, such
as total leukocytes, neutrophil counts and aminotransfer-
ases, which further highlighted the relationships existing
between pulmonary lesions and inflammatory reaction
or organ damage. While the association between CT
findings and hemostatic changes has not been thor-
oughly investigated before, our data are inconclusive and
more studies are definitely needed. Finally, unlike previ-
ous works [21], we distinguished between the associa-
tions
with
qualitative
CT
findings
(e.g. GGO,
consolidation etc.) and those related to the extension of
the underlying lesions. In fact, since in a preliminary
analysis the qualitative CT findings were found to be
related with disease extension, we corrected the associ-
ation between laboratory data and imaging results for
the degree of parenchymal involvement, which was
found to be related with the most prominent changes in
laboratory results. In so doing we found interesting
results that have not been previously reported, e.g. con-
solidation was not associated with a significant increase
in circulating LDH levels but related to impaired oxy-
genation and increased CRP concentrations. Supplementary Information
The online version contains supplementary material available at https://doi. org/10.1186/s12879-021-05855-9. to include laboratory tests less frequently reported, such
as total leukocytes, neutrophil counts and aminotransfer-
ases, which further highlighted the relationships existing
between pulmonary lesions and inflammatory reaction
or organ damage. While the association between CT
findings and hemostatic changes has not been thor-
oughly investigated before, our data are inconclusive and
more studies are definitely needed. Finally, unlike previ-
ous works [21], we distinguished between the associa-
tions
with
qualitative
CT
findings
(e.g. GGO,
consolidation etc.) and those related to the extension of
the underlying lesions. In fact, since in a preliminary
analysis the qualitative CT findings were found to be
related with disease extension, we corrected the associ-
ation between laboratory data and imaging results for
the degree of parenchymal involvement, which was
found to be related with the most prominent changes in
laboratory results. In so doing we found interesting
results that have not been previously reported, e.g. con-
solidation was not associated with a significant increase
in circulating LDH levels but related to impaired oxy-
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1 Author details
1Clinical chemistry and Endocrinology Laboratory, Departement of
Diagnostic Imaging and Laboratory Medicine, AUSL-IRCCS di Reggio Emilia, Page 9 of 9 Page 9 of 9 Page 9 of 9 Canovi et al. BMC Infectious Diseases (2021) 21:157 Canovi et al. BMC Infectious Diseases (2021) 21:157 Canovi et al. BMC Infectious Diseases (2021) 21:157 Canovi et al. BMC Infectious Diseases (2021) 21:157 viale Risorgimento 80, 42123 Reggio Emilia, Italy. 2Radiology Unit,
Departement of Diagnostic Imaging and Laboratory Medicine, AUSL-IRCCS di
Reggio Emilia, viale Risorgimento, 80, 42123 Reggio Emilia, Italy. 3Clinical and
Experimental Medicine PhD program, University of Modena and Reggio
Emilia, Modena, Italy. 4Epidemiology Unit, AUSL-IRCCS di Reggio Emilia, viale
Risorgimento, 80, 42123 Reggio Emilia, Italy. 5Laboratory of autoimmunity,
allergology and innovative biotechnologies, Departement of Diagnostic
Imaging and Laboratory Medicine, AUSL-IRCCS di Reggio Emilia, viale
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ہندوستان میں برطانوی دور اقتدار میں مسلم متکلمین کی مشنریز سے مزاحمت
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دنہواتسن
می
رباطن ی
دور ادتقار یم
ملسم یملکتم ک
مشن ی سے
زمامح
Muslim Theologians Resistance to Missionaries During the
British Rule in India
Muhammad Anis-ur-Rahman
Doctoral Candidate, Hitec University, Taxila
Hafiz Zafar Mehmood
Doctoral Candidate, AIOU, Islamabad
Dr. Muhammad Altaf
Lecturer, Minhaj University, Lahore دنہواتسن
می
رباطن ی
دور ادتقار یم
ملسم یملکتم ک
مشن ی سے
زمامح
Muslim Theologians Resistance to Missionaries During the
British Rule in India
Muhammad Anis-ur-Rahman
Doctoral Candidate, Hitec University, Taxila
Hafiz Zafar Mehmood
Doctoral Candidate, AIOU, Islamabad
Dr. Muhammad Altaf
Lecturer, Minhaj University, Lahore Al-Qamar, Volume 6, Issue 1 (January-March 2023) Al-Qamar, Volume 6, Issue 1 (January-March 2023) Abstract Abstract
Evidence of the existence of Christianity in India can be found from
the second century AD. When the industrial revolution came in
European countries, European countries established colonies in other
countries of the world and in this context, many European countries
turned to India,but Britain was successful in them. The British
government through the East India Company like When he started
gaining power, he made a plan to convert the people of India into
Christianity. For this purpose, priests from European countries were
called to preach Christianity to the Muslims here. The religious
literature of India was made suspicious and various missionary
organizations were given full support by the government to force the
Indian people to accept Christianity through threats or greed. All the
doubts raised by him were countered in the form of books and
debates, so in this article the life conditions and services of these
scholars will be presented. Abstract Key words: Missionaries, Muslim Theologians, Resistance, British
Rule, India 23 23 دنہواتسن
می
رباطن ی
دور ادتقار یم
ملسم یملکتم ک
مشن ی
سے
زمامح 24
تمہی
برصغیر
یم اسیعتیئ اک آاغز دورسی دصی وسیعی ےس رشوع وہا
۔ رصم ےک رہش
ادنکسرہی ےس
دنہواتسن ےک اترجوں ےک اسھت
ھچک اپدری آ رکآابد وہےئسج ےس دنہواتسن یم اسیعتیئ ک اینبد اقمئ وہیئ
۔ وچیھت دصی وسیعی
یم یھب
اشم ےک
اسیعیئ
دنہو اتسن آرکدنمسرےک اسیلح العق
یم آابد وہ
ئے۔
رقبی ےک ادوار یم بج
وی ریپ اوقام ےن
داین ےک درگیاممکل
وک ہضبق
رک ےک اس یم اکولوینں وک آابد رکےن اک وصنمہب انبای ۔ وت
دنہواتسن یم رفاسن، رپاگتل او
ر
اڈنیلگن ےن
اسیعویئں ک
نشم ی ومیظنتں
ےک اپدری اور نیغلبم
کو
دنہواتسن
اجیھب ۔انچہچن ایس دوران اڈنیلگن
ےکودف ےن لغم ابداشہ
اربک ےس اجتریت ااجزت انہم
احلص رک
ےک ا
ٹسی اڈن
ای ینپمک
ک اینب د ریھک۔ اس ینپمک یم فلتخم اپدری وموجد ےھت،
جن
وک لغم
ابداشہ
(اربک م1605ء)
،اہجیگن(م1627ء)،اشاہجہ (م ں1666
ء )ےک درابروں یم ارث روسخ احلص اھت۔سج رپ ان
اپدرویں ےن
وعام اور لغم زہش
ادوں یم اسیعتیئ ک غیلبت اک
آاغز یک ۔نکیل بج اٹسی اڈنای ینپمک وک1857ء ک گنج آزادی ےک دعب وپر ے
دنہواتسن رپ ہبلغ احلص وہ
ا، تو
رباطنی وکحتم ےن فلتخم ہمہ
تہج وصنم
وبں رپ لمع ریپا وہرک املسم
نں
اور االسم ک خیب ینک اک
زعم یک۔سج یم ان
اسیعیئ اپدرویں ےن االس
م ک اخمتفل یم، ،اضمنیم ،انمرظوں اتکوبں اک وطبر رپوڈنگیپا
اہسرا ایلسج
می
رقآن دیجم ،وضحرﷺ ک ذات اور املسمنں ےک اقعدئ وابعدات وک اشنہن انبای
اجات ۔اھت
اس دوران املسمنں ک دینی اقب اور ظفحت
ےک ےیل ربریغص ےک دنچ یملکتم اسےنم اےی۔وہنجں ےن وکحتم وتق ےک وکربج ابالےئ اطق رھک رک ان اسیعیئ اپدرویں
اورنشمویں اک رمداہن وار اقمہلب
یک۔ نج ک
یملع دخامت وک اس آرلکیٹ یم ومسےن ک وکشش ک یئگ ےہ۔
دنہواتسن یم اسیع تیئ ک غیلبت اک
اباقدعہ آاغزاور نشمی یمیظنت
دنہواتسن یم اسیعتیئ ک اباقدعہ غیلبت اک آاغز1813
یمء اس
،وتق وہا
بج اڈنیلگن ےک دار اوعلام ( اپرٹنمیل )ےس ہی لب اپس
وہا ہک دنہ واتسن ےک وعام وک اسیعیئ انبےن ےک ےیل
، رپزعم
ارفاد اور اپ دری
وطبر نشم دنہواتسن
جا
ںیئ ےگاور ان ک وخب وحہلص
ازفایئ ک اجےئ یگ
۔اس لب ےک اپس وہےت یہ ویرپ اور ارمہکی ےس اپدرویں ےک فلتخم
ووفد ،نشم ی
یمیظنت اور اںینمجن غیلبت
ےک ےیل دنہواتسن آان رشوع وہںیئ ۔ اکی ادنازےےکاطمقب1900ء یم ان
( امہمت
ش شز
م )
ک دعتاد ایبسیل کت چنہپ یئگ
یھت نشمرہ۔ ےک تحت فلتخم ادارے
اکم رکرےہ ےھت۔ہی ادارے میتی اخےن،اتپسہل ،وکسل انبےتسج یم اسیعتیئ وک ہیفخ
تبلیغ
ک اجیت ۔ اس ےکالعوہ
دنہوؤں ےک دتّل اخدنانں وک امل ودوتل دے رک اسیع یئ انبای اجات اھت۔نشم ےک ارفاد اور انمجن
اہنتی
اچالک اور وہایشری ےس فلتخم واس
لئ اور ذراعئ اک اامعتسل رکےت ۔1
نشمویں اک رطہقی غیلبت
نشمویں ےن دنہوؤں اور املسمنں ےک ےیل اگل اگل اپ
دری رقمر ےیک ےھت۔ان اپدرویں وکاخمنیبط ک ذینہ اس تخ اور ذمبہ
ےک انمتبس ےس
رتتیب دی اجیت یھت اور اس ذمبہ ےک
زمکور ولہپؤں اک ااحہط یک اجات اتہک ان ک غیلبت
زایدہ اتمرث وہ۔اس ےک
اسھت اسھت ان ک اعموتن یم ول
لک اسیع
یئ یھب رےھک اجےت ےھت۔ان اسیعیئ اپدرویں دنہواتس ن ےک اقمیم املسمنں اور
دنہوؤں یم رفنت دیپا رک ےن ےک ےیل یھب یئک ادقاام
ت ےیک انچہچن یئک ارگنی ومرنیخ ےن ایسی اتکںیب رحتری ک نج یم املسمن
رمکحانں وک
ظا
مل اور اجرب اظرہ یک ایگ۔ہک ان املسمن رمکحانں ےن دنہ وؤں اک لتق اعم یک
اور ان ےک دنمروں وک اصقنن اچنہپ ای۔
نشمویں ک اکایمب
اگراسن
داتیس ےن ریپس ک اکی ویوینریٹس یم ابلط ء وک اس وحاےل ےس رچکیل دای ےہ۔سج وکاس ےن
ارگن ی آرسفوں ےک رفامہ
رکدہ روپسٹ
ےس ایتر یک سج یم
ےتھکل ںیہ ہک دنہواتسن یم میلعت ایہتف ولگ اسیع ب ب ےک
تیئ وبقل رکےت ںیہ انچہچن دمراس ےک ب
اہھت رپ5200
ارفاد ےن اورانوپگر یم اکی رجنم نشمی ےک اہھت رپ14
زہار ولوگں ےن اسیعتیئ وبقل ک۔دیلہ
یم دو املسمن تمہی
برصغیر
یم اسیعتیئ اک آاغز دورسی دصی وسیعی ےس رشوع وہا
۔ رصم ےک رہش
ادنکسرہی ےس
دنہواتسن ےک اترجوں ےک اسھت
ھچک اپدری آ رکآابد وہےئسج ےس دنہواتسن یم اسیعتیئ ک اینبد اقمئ وہیئ
۔ وچیھت دصی وسیعی
یم یھب
اشم ےک
اسیعیئ
دنہو اتسن آرکدنمسرےک اسیلح العق
یم آابد وہ
ئے۔
رقبی ےک ادوار یم بج
وی ریپ اوقام ےن
داین ےک درگیاممکل
وک ہضبق
رک ےک اس یم اکولوینں وک آابد رکےن اک وصنمہب انبای ۔ وت
دنہواتسن یم رفاسن، رپاگتل او
ر
اڈنیلگن ےن
اسیعویئں ک
نشم ی ومیظنتں
ےک اپدری اور نیغلبم
کو
دنہواتسن
اجیھب ۔انچہچن ایس دوران اڈنیلگن
ےکودف ےن لغم ابداشہ
اربک ےس اجتریت ااجزت انہم
احلص رک
ےک ا
ٹسی اڈن
ای ینپمک
ک اینب د ریھک۔ اس ینپمک یم فلتخم اپدری وموجد ےھت،
جن
وک لغم
ابداشہ
(اربک م1605ء)
،اہجیگن(م1627ء)،اشاہجہ (م ں1666
ء )ےک درابروں یم ارث روسخ احلص اھت۔سج رپ ان
اپدرویں ےن
وعام اور لغم زہش
ادوں یم اسیعتیئ ک غیلبت اک
آاغز یک ۔نکیل بج اٹسی اڈنای ینپمک وک1857ء ک گنج آزادی ےک دعب وپر ے
دنہواتسن رپ ہبلغ احلص وہ
ا، تو
رباطنی وکحتم ےن فلتخم ہمہ
تہج وصنم
وبں رپ لمع ریپا وہرک املسم
نں
اور االسم ک خیب ینک اک
زعم یک۔سج یم ان
اسیعیئ اپدرویں ےن االس
م ک اخمتفل یم، ،اضمنیم ،انمرظوں اتکوبں اک وطبر رپوڈنگیپا
اہسرا ایلسج
می
رقآن دیجم ،وضحرﷺ ک ذات اور املسمنں ےک اقعدئ وابعدات وک اشنہن انبای
اجات ۔اھت
اس دوران املسمنں ک دینی اقب اور ظفحت
ےک ےیل ربریغص ےک دنچ یملکتم اسےنم اےی۔وہنجں ےن وکحتم وتق ےک وکربج ابالےئ اطق رھک رک ان اسیعیئ اپدرویں
اورنشمویں اک رمداہن وار اقمہلب
یک۔ نج ک
یملع دخامت وک اس آرلکیٹ یم ومسےن ک وکشش ک یئگ ےہ۔
دنہواتسنیماسیع
تیئکغیلبتاک
بڈنیلگےوعاٹنمیےہلاتویدناتیاسیتیاادغیلباااباقدعہآاغزاورنشمییمیظنت 24 24 ا رمقل، دلج6
، امشر ہ1
، (ونجر ی–
امر چ 3
202
)
اعمل ومولی دفصر یلع،ومولی امعدادلنی ےن یھب
عیسا
تیئ اایتخرک۔اس وتق دنہواتسن یم آھٹ الھک وتھککل اسیع یئ ےہ۔زین
اس ےک اسھت یئک ناوبں ےن اےنپ رخےچ ےس رگاجرھگ انبےن رشوع ےیک ںیہ سج یم ہکلم وھباپل
یھب اشلم ےہ۔2
ذہلا اس ےس
اظرہ وہات
ےہ ہک اسیعتیئ وکحتم ےک اسہی یم رپوان ڑچھ ریہ یھت نکیل دنچ
یملکتم ےن آ
ٹھ
رک اس وطافن ےک اےگ دنب
ابدنھ
رک اتم ہملسم ک افحتظ ک نج ک لیصفت ذلی یم ذرک ک اجیت ےہ۔
وجاد اساب ط (م1834
)ء
وجاد اک قلعت رتک ےکاسدات اخدنان ےس اھت۔آپ ےک وادل اک انم ا ربامیہ اسابط ےہ۔اربامہ اسابط تنطلس امثعہین ےک
دہعدارےھت۔وجا د ک دیپاشئ1774ء وک رت
ک ےک رہش امرہی یم وہیئ ۔ادتبایئ میلعت رھگ ےس احلص ک۔5
اسل ک رمعیم وادل اک
ااقتنل وہایگ ۔وادل ےک وافت ےک دعب وادلہ ےن
آپ ک میلعت اک وصخیص ایخل راھک۔21
ک اسل
رمع یم
وادلہ اک یھب ااقتنل وہ ایگ
۔سج ےک دعب وجاد
ےن ایستح وک وطبر ہشیپ اایتخر رکایل کریغص ۔ رب
ایستح رکےت وہےئ بج آپ ےن داھکی ہک اہیں
االسم
ےکالخف اسیعویئں ک نشمی یمیظنت اور اپدری رحتم
ک ںیہ ہکبج املسمن
وخاب تلفغ اک اکشر ںیہ،وت
آپ ےن دنہواتسن یم
راہشئ اایتخر رکےن
اک ہیہت رک ایل
۔ اتہک ان نشمی ومیظنتں اور اپدرویں اک اقمہلب یک اجےئ
۔
اسیعتیئ ےک الخف اکم رکےن ےک
ےئل وجاد ےن نشم ےک ارفاد
ےسرقبی
وہےن ےک ےیلاور ان ک اچولں ےس واقف حا لص
ی ت رکےنےک ےیل اظبرہ اسیعتیئ وبقل
رکیل ۔ نشم ےک ارفاد ےن آپ ےک ذےم ابلبئ
کے
رعب رتہمج اکاٹکس دای ۔سج رپآپ ےننشم ےکاسھت10
اسل
زگارے۔ اس دوران آپ ان ےکبتک ےس اپدرویں ک طلغ اقعدئاور االسم ک اقحتین اک وماد عمج رکےت رےہ ۔سج ےک دعب دو
رتہبنی اتکںیب آپ ےن
رد اسیع تیئ
یم رحتری ںیک وج دنہواتسن یم رد اسیعتیئ ےک ےیل اینبد ںینب ۔1
۔ اربلاهی نش
اسلابطي ه
۔2
۔ ارصلارصااسلابطي ه
۔ آپ ک اتکب ربانیہ ےسرمح اہلل ریکانی ےن
بھی
وخب اافتسدہ یک ۔3
آپ ک درگی
اصتفین ہی ںیہ۔1
۔
رتہمج الیجن رعب زابن یم2
۔رتہمج الیجن افریس زابن یم ۔
نش ااسلابطي ه
الحبیش :اس اتکب یم وجادےن
الیجن ےک افریس رتےمج ےک وح
اےل ےس ھچک
امہ واضںیتح شیپ ںیک ۔
ومالان آل نسح وماہین (م
1872
)ء
ومالان آل نسح اک ہلسلس بسن ہی ےہ۔دیس آل نسح نب
الغم دیعس نب وہیج ادلنی نب دمحم فینح نب الغم یلع ےہ۔ اور اےگ اجرک ہی
ہلسلس بسن نیسحؓ ےک اسھت لم اجات ےہ۔ومالان ک والدت1787
ءوک علض اانؤ
کے
اگؤں وماہن
یم وہیئ ۔ادتبایئ میلعت آپ
ےنؤنھکل یم احلص ک۔ میلعت لمکم وہےن رپ اہٰل آاب
د یم رحمر رقمر وہےئ
سج یم رتیق وہےن رپ آپ اک ابتدہل وپحتفرہ
وہا۔وپحتفرہ یم آپ اک اسانم اسیعیئ اپد رویں ےک اسھت وہا،سج رپ آپ ےناسیعویئں اسیکھت انمرظے ےک وحاےل وخب ایتری
ک اور رھپ اپ درویں
کو
یئک وماعق رپ تسکش دی ۔سج رپوکحتم ےن
آپ رپ دقمامت انبےئ
۔آپ
ےن ن رکی ےس اٰیفعتس دای
اور اہجن آابد رہش وک اانپ نکسم انبای
۔ واہں یھب رد اسیعتیئ رپ اکم رکےت رےہ نکیل
ھچک رعہص دعب دیحر آاب د ےک ناب ک وخاشہ رپ
دیحر آابدآ
ےئ اور رھپ اہی
ں
انمظ ااعلہیل رقمر وہےئ۔آپ ک وافت75
اسل ک رمع یم1872ء وک وہیئ۔ ومالان وماہ ین وک انمرظہ
یم وخ
ب ہکلم احلص اھت۔ نکیل آپ زابین انمرظہ رپ رحتریی انمرظہ وک رتحیج دےتی ےھت۔4 آپ ک اصتفین ہی ۔ںیہ1
۔ :ااسفتسر
(/اپدری ڈنپرKarl Gottieb Pfander
)ےن
زیمان اقحل ےکانم ےس االسم ےک رد
یم اتکب یھکل سج ےک وجاب یم
ومالان وماہین ااسفتسر
یھکل اور
االسم اک
دافع رکےت وہےئاسیعیئ اپدرویں ےس ااھٹرہ وساالت ےیک۔ اسي یبش ا ر :ومالان وماہین ےک
اتکب ااسفتسر ےک رد یم اپدری
ڈنپر ےن لح االاکشل ےک انم ےس اتکب یھکل سج رپ دوابرہ ومال انوماہین ےن لح االاکشل ےک
وجاب یم اسي یبش ا ر ھکل
یل۔2
۔
اتکب رموغب در امذخوجاابت ہب صن ری3
:۔ومجمع اونلرنی اس اتکب یم اولتیہ اور راستل
رپ ثحب ک یئگ ےہ۔4
۔ راسہل ودحوتوجد 5۔ ااختنب رتہمج
اراشدات وسیع ی ۔ ا رمقل، دلج6
، امشر ہ1
، (ونجر ی–
امر چ 3
202
)
ھیےیساھیتاندتتیایت اعمل ومولی دفصر یلع،ومولی امعدادلنی ےن یھب
عیسا
تیئ اایتخرک۔اس وتق دنہواتسن یم آھٹ الھک وتھککل اسیع یئ ےہ۔زین
اس ےک اسھت یئک ناوبں ےن اےنپ رخےچ ےس رگاجرھگ انبےن رشوع ےیک ںیہ سج یم ہکلم وھباپل
یھب اشلم ےہ۔2
ذہلا اس ےس
اظرہ وہات
ےہ ہک اسیعتیئ وکحتم ےک اسہی یم رپوان ڑچھ ریہ یھت نکیل دنچ
یملکتم ےن آ
ٹھ
رک اس وطافن ےک اےگ دنب
ابدنھ
رک اتم ہملسم ک افحتظ ک نج ک لیصفت ذلی یم ذرک ک اجیت ےہ۔
وجاداساب (م ط1834
) وجاد اساب ط (م1834
)ء
وجاد اک قلعت رتک ےکاسدات اخدنان ےس اھت۔آپ ےک وادل اک انم ا ربامیہ اسابط ےہ۔اربامہ اسابط تنطلس امثعہین ےک
دہعدارےھت۔وجا د ک دیپاشئ1774ء وک رت
ک ےک رہش امرہی یم وہیئ ۔ادتبایئ میلعت رھگ ےس احلص ک۔5
اسل ک رمعیم وادل اک
ااقتنل وہایگ ۔وادل ےک وافت ےک دعب وادلہ ےن
آپ ک میلعت اک وصخیص ایخل راھک۔21
ک اسل
رمع یم
وادلہ اک یھب ااقتنل وہ ایگ
۔سج ےک دعب وجاد
ےن ایستح وک وطبر ہشیپ اایتخر رکایل کریغص ۔ رب
ایستح رکےت وہےئ بج آپ ےن داھکی ہک اہیں
االسم
ےکالخف اسیعویئں ک نشمی یمیظنت اور اپدری رحتم
ک ںیہ ہکبج املسمن
وخاب تلفغ اک اکشر ںیہ،وت
آپ ےن دنہواتسن یم
راہشئ اایتخر رکےن
اک ہیہت رک ایل
۔ اتہک ان نشمی ومیظنتں اور اپدرویں اک اقمہلب یک اجےئ
۔
اسیعتیئ ےک الخف اکم رکےن ےک
ےئل وجاد ےن نشم ےک ارفاد
ےسرقبی
وہےن ےک ےیلاور ان ک اچولں ےس واقف حا لص
ی ت رکےنےک ےیل اظبرہ اسیعتیئ وبقل
رکیل ۔ نشم ےک ارفاد ےن آپ ےک ذےم ابلبئ
کے
رعب رتہمج اکاٹکس دای ۔سج رپآپ ےننشم ےکاسھت10
اسل
زگارے۔ اس دوران آپ ان ےکبتک ےس اپدرویں ک طلغ اقعدئاور االسم ک اقحتین اک وماد عمج رکےت رےہ ۔سج ےک دعب دو
رتہبنی اتکںیب آپ ےن
رد اسیع تیئ
یم رحتری ںیک وج دنہواتسن یم رد اسیعتیئ ےک ےیل اینبد ںینب ۔1
۔ اربلاهی نش
اسلابطي ه
۔2
۔ ارصلارصااسلابطي ه
۔ آپ ک اتکب ربانیہ ےسرمح اہلل ریکانی ےن
بھی
وخب اافتسدہ یک ۔3
آپ ک درگی
اصتفین ہی ںیہ۔1
۔
رتہمج الیجن رعب زابن یم2
۔رتہمج الیجن افریس زابن یم ۔
نش ااسلابطي ه
الحبیش :اس اتکب یم وجادےن
الیجن ےک افریس رتےمج ےک وح
اےل ےس ھچک
امہ واضںیتح شیپ ںیک ۔
ومالانآلنسحوماہین (م1872
)ء ومالان آل نسح وماہین (م
1872
)ء
ومالان آل نسح اک ہلسلس بسن ہی ےہ۔دیس آل نسح نب
الغم دیعس نب وہیج ادلنی نب دمحم فینح نب الغم یلع ےہ۔ اور اےگ اجرک ہی
ہلسلس بسن نیسحؓ ےک اسھت لم اجات ےہ۔ومالان ک والدت1787
ءوک علض اانؤ
کے
اگؤں وماہن
یم وہیئ ۔ادتبایئ میلعت آپ
ےنؤنھکل یم احلص ک۔ میلعت لمکم وہےن رپ اہٰل آاب
د یم رحمر رقمر وہےئ
سج یم رتیق وہےن رپ آپ اک ابتدہل وپحتفرہ
وہا۔وپحتفرہ یم آپ اک اسانم اسیعیئ اپد رویں ےک اسھت وہا،سج رپ آپ ےناسیعویئں اسیکھت انمرظے ےک وحاےل وخب ایتری
ک اور رھپ اپ درویں
کو
یئک وماعق رپ تسکش دی ۔سج رپوکحتم ےن
آپ رپ دقمامت انبےئ
۔آپ
ےن ن رکی ےس اٰیفعتس دای
اور اہجن آابد رہش وک اانپ نکسم انبای
۔ واہں یھب رد اسیعتیئ رپ اکم رکےت رےہ نکیل
ھچک رعہص دعب دیحر آاب د ےک ناب ک وخاشہ رپ
دیحر آابدآ
ےئ اور رھپ اہی
ں
انمظ ااعلہیل رقمر وہےئ۔آپ ک وافت75
اسل ک رمع یم1872ء وک وہیئ۔ ومالان وماہ ین وک انمرظہ
یم وخ
ب ہکلم احلص اھت۔ نکیل آپ زابین انمرظہ رپ رحتریی انمرظہ وک رتحیج دےتی ےھت۔4 آپ ک اصتفین ہی ۔ںیہ1
۔ :ااسفتسر
(/اپدری ڈنپرKarl Gottieb Pfander
)ےن
زیمان اقحل ےکانم ےس االسم ےک رد
یم اتکب یھکل سج ےک وجاب یم
ومالان وماہین ااسفتسر
یھکل اور
االسم اک
دافع رکےت وہےئاسیعیئ اپدرویں ےس ااھٹرہ وساالت ےیک۔ اسي یبش ا ر :ومالان وماہین ےک
اتکب ااسفتسر ےک رد یم اپدری
ڈنپر ےن لح االاکشل ےک انم ےس اتکب یھکل سج رپ دوابرہ ومال انوماہین ےن لح االاکشل ےک
وجاب یم اسي یبش ا ر ھکل
یل۔2
۔
اتکب رموغب در امذخوجاابت ہب صن ری3
:۔ومجمع اونلرنی اس اتکب یم اولتیہ اور راستل
رپ ثحب ک یئگ ےہ۔4
۔ راسہل ودحوتوجد 5۔ ااختنب رتہمج
اراشدات وسیع ی ۔ 25 دنہواتسن
می
رباطن ی
دور ادتقار یم
ملسم یملکتم ک
مشن ی
سے
زمامح دنہواتسن
می
رباطن ی
دور ادتقار یم
ملسم یملکتم ک
مشن ی
سے
زمامح دنہواتسن
می
رباطن ی
دور ادتقار یم
ملسم یملکتم ک
مشن ی
سے
زمامح ( ومالان اقمس اننوتی و1880
)ء
اقمس اننوتی اک ہلسلس بسن دمحم اقمس نب ادس یلع
نب الغم اشہ نب دمحم شخب ےہ۔ہی ہلسلس بسن آےگ اجرک
اوبرکب دصقیؓ
ےس لم
اجاتےہ۔اقمس اننوتی ک دیپاشئ اننہت،اہسرن وپر یم1833ء وہیئ۔ادتبایئ میلعت اےنپ اگؤں ان یم نہت
احلص ک
۔ سج یم
آپ ےن رقآن دیجم اور وخیطخش یھکیس ۔اس ےک دعب
افریس ےک صنب ےک ےیل دویدنب رشتفی ےل ےئگ ۔بتک ونف ن ک لیمکت ےک
ےیل آپ ےن1844ء وک
ان ولمم ومال
ک یلع اننوتی (م1851
ء)ےک اپس رعکب اکجل دیلہ یم داہلخ ایل ۔ بتک ونفن ک لیمکت ےک
دعب اذخ دح ثی ےک ےیل اشہ
دبع اینغل (م1878ء)ےکہقلح درس یم اشلم وہےئ۔دینی میلعت لمکم وہےن رپ
ااسحن و
ولسک ےک
ےیل احیج ادماد اہلل اک ااختنب یک۔5
میلعت لمکم وہےن رپ عبطم ادمحی ےک اسھت وطب ر ححصم کلسنم وہےئ۔ اہجں رپ آپ ےن یئک
اتکوبں رپ اقیلعتت
لکھی ۔1857
ء یم بج گنج آزادی اک آاغز وہاوت آپ
اھتہن وھبن
اور اشیلم ےک اقمم رپ ارگنی وفج
ےسوخب
ڑلےئ ۔گنج ےک دعب دو اسل کت رووپش رےہ اور رھپ جح ےک ےیل رشتفی ےل ےئگ۔ جح رکےک
1861
ء وک دنہواتسن واسپ
ولٹ اےئ اور ریمھٹ یم اکی عبطم ےسکلسنم وہےئ۔ ریمھٹ ک ایقم دور ان آپ ےن
دویدنب ک اقمم رپ داراولعلم انبےن ک وجتی
دی اور اس داراولعلم ےک وش
ر ی ےک رنک اور ااتسدیھب ےنب۔ارپلی1880
ء وک آپ وک اسسن ک فیلکت رشوع وہیئ رمض ڑبےنھ
پر15
ارپلی1880
ء وکآپ اک ااقتنل وہایگ۔6
آپ ےک ااسذتہ یم ومالان ولمم ک یلع (م1851
ء)،ومالان اشہ دبع اینغل
(م1878ء)اشلم ںیہ۔1
۔
ااصتنر االالسم :اس اتکب یم ومالان اقمس ےن
اسیعویئں ےک اقعدئ
پر
یلقع اور یلقن داللئ ےس
رد ک
ےہ۔ آپ
ےناصتفین ےک اسھت اسھت اسیعیئ اپدروی
ں ےس انمرظے یھب ےیک وج دنمرہج ذلی ۔ںیہ1
۔اچدن وپر :اس
انمرظے یم
اسیعویئں ک رطف ےس اپدری نسل
ےھت ۔ومالان اقمس ےن اقحتین االسم رپ اےسی داللئ دےی رک اپدری ولغم ب
ا۔ وہ2۔اشاہجہن وپر : اشاہجہن وپر یم ارگنی وکحتم ےک رطف ےس
اکی ابمہثح اک اامتہم یک ایگ
جسکا مقصد یہ
اھت ہک بس ولگ
اسیعیئ اپدرویں ےس اتمرث وہ اجںیئ ےگ اوراسیعتیئ
وبقل رکےگنیل۔اس انم رظے یم اسیعویئں ک رطف ےس اپدری یج یٹ
ااکسرٹ اور اپدری نسل ےھت ،
املسمنں
ک
رطف ےس ومالان اقمس ےھت اور دنہو ؤں
ک
رطف ےس ڈنپت داینن رسوسیت
تھے
۔اس
انمرظ ے یم ومالان اقمس ےن االسم ک اقحتین رپ اےسی داللئ دےی ہک اسرے اپدری اور ڈنپت الوجاب وہ ےئ۔
( ومالان رمح اہلل ریکانی م1890
)ء ااوسبلسلہالہمووناےیمربدنلہبیبنلہجیب ( ومالان اقمس اننوتی و1880
)ء لسلہاوامادنماحمدسب کیرلہمااو
م
آپ اک ہلسلس بسن ومالان
رمح اہلل نب ومولی
لیلخ نب میکح بیجن اہلل نب بیبح اہلل نب دبع ارلمیح ےہ ۔آپ اک
ہلسلس بسن
امثعنؓ ےس اج تلم
ےہ۔7ومالان رمح اہلل ک دیپاشئ علض رفظم رگن ،ریکاہن یم وہیئ۔آپ رضحت امثعنؓ ےک اخدن ان ےس
ےھت۔آپ ےن ریتہ ربس
ک رمع کت رقآن دیجم اور افریس اور رع
ب ک ادتب
ایئ اتکوب ں ک میلعت رھگ ےس احلص ک ۔ادتبایئ میلعت
ےک دعب دیلہ ےک دمرہس ایحت یم دالخ وہےئ
۔ میلعت لمکم وہےن رپبط ک اہمرت یھب احلص ک اور اےنپ اگؤں ریکاہن یم
دترسی رش وع ک نکیل ہب دلجاپ درویں اور نشمویں ےک رطخے وک اھبپن ایل سج رپ دترسی وھچ
ڑ رک آپ رد اسیعتیئ ےک
رطف وتمہج وہ ےئ۔8
1857ء ےک گنج آزادی ےک دعب یم وکحتم رباطہین آپ ےک درےپ وہیئ ۔سج رپ آپ ےن دنہواتسن
ےس ہکم رکمہم رجہت رکیل ۔ہکم رکمہم اجرک آپ ےن دمرہس وصہیتل ک اینبد ریھک ۔ہکم رکمہم
می
ایقم ےک دوران رتک ےکہفیلخ ےن
آپ ک وخب زعت
ازفایئ ک
اور ہغمت دیجم ہی ےس نزا۔اس دوران ہفیلخ ےن اپدری ڈنپر اسیکھت انمرظے ک رودات رپ اکی
ےنھکل اتکب ک دروخاتس ک سج رپ آپ ےن ااہظر اقحل فینصت ک۔ آپ ک وافت22
راضمن1890ء وک75
ک اسل رمع یم ہکم
رکمہم یم وہیئ۔آپ ۃنج اٰیلعمل یم رضحت دخہجی ؓ ےک
ربق ےک رقبی
دموفن ںیہ ۔9
آپ ےک ااسذتہ یم ومالان دمحم ایحت ،ومالان
دبع ارلنمح یتشچ (م1824
،)ء یتفم دعس اہلل رما
د آابدی (م1877
ء)، ومالاناامم شخب ابہصیئ (م1858
ء)، ومالان ادمح یلع
، دبؤیل
میکح دمحم (
ب
ب
م1825ء) اشلم ںیہ ۔ آپ ےکوہشمر اشرگدوں یم
ومالان ادمح ادلنی وکچا، یل ومالان دبعاعیمسل 26 ا رمقل، دلج6
، امشر ہ1
، (ونجر ی–
امر چ 3
202
)
اقیدحقرااوسرریںخیرو رمقل، دلج6
، امشر ہ1
، (ونجر ی
امر چ 3
202
)
راوپمری (م
1900ء)، ومالان نر ادمح ارمرستی ،ومالان رشف اقحل دصیقی ،اشہ اوباریخل ، ںیہ۔ آپ ک اصتفین دنمرہج ذلی
:ہیں
1
۔ااجعز وسیعی :اس اتکب یم آپ ےن ابلبئ ےک نتم
ےک اضتدات وک لیصفت ےس ایبن یک ےہ2
۔
انسح ادحلثی یف ااطبل
ي لی ث
الی :اس اتک
ب یم صن ریٰ ک دیقعہ ثیلثت رپاققحمہن ثحب ےہ۔اس اتکب وک ادارہ االسایمت الوہر ےن اشعئ یک
ہے۔3
احبل
وارحتلفی
ش
ارشلفی یف اابثت الیشسخ :
اس اتکب یم آپ ےک انمرظہ آرگہ ک لیصفت ےہ4
۔ ازاله اوکشلک : رمزا رخفادلنی
وج لغم ابداشہ اہبدر اشہ رفظ(م1862ء )ےک ےٹیبےھت ان
وک اپدرویں ےن االسم رپ ارتعااضت اکاکی راسہل اجیھب۔ رمزا
رخفادلنی ےنہی راسہل رمح اہلل ریک
ا
نی وک دایاوران ےک وجاابت ےنھکل ک دروخاتس ک سج رپ
ریکا نی ےنہی اتکب رحتری
ک۔آلکج ہی وم
الان قیتع ادمح وتسبی ک قیقحت ےس ہبتکم دہعمارشلہعی ےن4 دلجوں یم اشعئ ک ےہ۔5
۔
ربوق العم :اس اتکب
یم ریکا نی ےنالہ اتکب ےک دینی ادب ےس وضحر ﷺک اشبرت وک اثتب یک ےہ۔6
۔ازاۃل االواہم :ہی اتکب ومالان ریکانی
ےن افریس زابن یم یھکل یھت اورہی آپ ک یلہپ اتک
ب یھت
۔ہی اتکب آپ ےن ڈنپر ےک اتک ب زیمان اقحل ےک رد یم یھکل
۔دنہواتسن یم
اس اتکب اکاردو رتہمج داعف االاقسم ےک انم ےسےہ۔ہکبج اپاتسکن یم ڈارٹک دمحم اامسلیع اعریف ےن
اس اک
اردو
رتجبمہ کي ا ےہ ۔وج ہبتکم دار اولعلم رکایچ ےن480
احفصت ےک اسھت اشعئ یک ےہ۔7
۔ ق
ت لی ثب
اطمل ع :ہی اتکب ومالان ریکانی
ےناپدری اھتمس ک اتکب
قیقحت دنی قح ےک رد یم یھکل ۔ ۔8
۔ااہظر اقحل
: رتک ےک ہفیلخ ےک دروخاتس رپومالان ریک
ا نی ےن
ہی اتکب یھکل
ےہ۔ اس یم اپدری ڈنپر ےک اسھت وہےن واےل انمرظے ک لیصفت اور اس ےک ارتعااضت ےک وجاابت یلیصفت
وطر رپ ذرک ںیہ
۔9
۔ اانملرظ ةالهيشد ٰاربکلی: ہی اتکب رعب ز
ابن یم ےہ۔اس یم
آپ ےک انمرظوں
ک لیصفت
ےہ۔10
۔ دعمل اوعاجج ازیملان :اس اتکب یم اپدری ڈنپر ک اتکب
زیمان اقحل
ک رتددی ےہ۔
رمح اہلل ریکانی ےک
انمرظے
1
۔ ڈارٹکوزری اخن اوررمح اہلل ریکانی ےن آرگہ یم
اپدری رفچن اور اپدری یٹک ےسانمرظہ یکاور ان وک ولغمب یک
۔10
2۔وم
الان
ریکانی اک وہشمرانمرظہ اپدری ڈنپرےس وہا۔ڈنپرےن اجعم دجسم دیلہ یم اج رک املسمنں وکیلع
ا لا
العن جنلیچ یک ۔
سج رپ رمح اہلل ریکانی ےن22
امرچ1854ء وک اکی اپدری ڈنپر ےکانم طخ اھکل
ا ور اس
یم جنلیچ وک وبقل یک۔سج ےک دعب
10
ارپلی1854
وک ہی انمرظہ رش وع وہا۔انمرظے اک وموضع اولتیہ حیسم وثیلثت، خسن،وبنت دمحم ،یﷺ رحت فی،رقآن
دیجم اک الکم اہلل وہان
رقم ر وہا۔ انمرظہ یئک دنں کت اجری راہ اور اس دوران اپدری ڈنپ ر اوراپدری رفچن یئک
رمہبت اس ابت
کے
اقلئ وہےئ ہک اس ہلئسم یم اسیعیئ اپدری یطلغ رپ ںیہ ۔11انمرظے ےس ےلہپ رفنیقی ےک درایمن یم یئک وطخط اک ابتدہل یھب
وہا ۔ وج افریس یم رمالست ذمیبہ ےک ونعان ےس
اشعئ وہیئ ںیہ ۔ رعب یم اس انمرظے ےک رو داد دبعااقلدر لبنح ےن
اانملرظہ اربکلی ےک انم ےس
اشعئ ک ےہ۔
رسدیس ادمح اخن (م1898
)ء
رسدیس ادمحاخن ک دیپاشئ دیلہ رہش یم1817ء وک وہیئ۔ رھگ رپ رقآن دیجم ک میلعت احلص ک افریس،رعب اورراییض ک میلعت
بتکم ےس اح
ک۔ لص1838
ءوک21
اسل ک رمع یم دعاتل یم انبئ یشنم ےک نرکی رپ
رھبیت وہےئسج یم وتق اسیکھت
رت
یق رکےتےئگ۔ اہبدر اشہ رفظےک رطف ےس1842
ء یم آپ وکوجاد ادلوۃلاور دیس
ادمح اعرف گنج اک اطخب یھب الم۔1855
ء
وک ونجبر یم آپ اک ابتدہل وہ ایگ۔گنج آزادی ےک دوران آپ
نے
یئک ارگنیوں ک افحتظ ک۔سج رپوکحتم رباطہین ےن آپ
وک یئک ااعنامت ےس نازا۔1858
ء وک آپ اک رم
اد آابد ابتدہل وہااہجں رپ آپ وک دصر ادصلور رپ رتیق دی یئگ۔ رماد آاب د آرک آپ
ےن اکی دمرہس اک ایقم یک۔رماد آابد یم نرکی ےس دکبسوش وہ ےن رپ آپ ےن یلع ڑگھ یم
قیا
م اک آاغز یک اور اس اسیک ھت یلع راوپمری (م
1900ء)، ومالان نر ادمح ارمرستی ،ومالان رشف اقحل دصیقی ،اشہ اوباریخل ، ںیہ۔ آپ ک اصتفین دنمرہج ذلی
:ہیں
1
۔ااجعز وسیعی :اس اتکب یم آپ ےن ابلبئ ےک نتم
ےک اضتدات وک لیصفت ےس ایبن یک ےہ2
۔
انسح ادحلثی یف ااطبل
ي لی ث
الی :اس اتک
ب یم صن ریٰ ک دیقعہ ثیلثت رپاققحمہن ثحب ےہ۔اس اتکب وک ادارہ االسایمت الوہر ےن اشعئ یک
ہے۔3
احبل
وارحتلفی
ش
ارشلفی یف اابثت الیشسخ :
اس اتکب یم آپ ےک انمرظہ آرگہ ک لیصفت ےہ4
۔ ازاله اوکشلک : رمزا رخفادلنی
وج لغم ابداشہ اہبدر اشہ رفظ(م1862ء )ےک ےٹیبےھت ان
وک اپدرویں ےن االسم رپ ارتعااضت اکاکی راسہل اجیھب۔ رمزا
رخفادلنی ےنہی راسہل رمح اہلل ریک
ا
نی وک دایاوران ےک وجاابت ےنھکل ک دروخاتس ک سج رپ
ریکا نی ےنہی اتکب رحتری
ک۔آلکج ہی وم
الان قیتع ادمح وتسبی ک قیقحت ےس ہبتکم دہعمارشلہعی ےن4 دلجوں یم اشعئ ک ےہ۔5
۔
ربوق العم :اس اتکب
یم ریکا نی ےنالہ اتکب ےک دینی ادب ےس وضحر ﷺک اشبرت وک اثتب یک ےہ۔6
۔ازاۃل االواہم :ہی اتکب ومالان ریکانی
ےن افریس زابن یم یھکل یھت اورہی آپ ک یلہپ اتک
ب یھت
۔ہی اتکب آپ ےن ڈنپر ےک اتک ب زیمان اقحل ےک رد یم یھکل
۔دنہواتسن یم
اس اتکب اکاردو رتہمج داعف االاقسم ےک انم ےسےہ۔ہکبج اپاتسکن یم ڈارٹک دمحم اامسلیع اعریف ےن
اس اک
اردو
رتجبمہ کي ا ےہ ۔وج ہبتکم دار اولعلم رکایچ ےن480
احفصت ےک اسھت اشعئ یک ےہ۔7
۔ ق
ت لی ثب
اطمل ع :ہی اتکب ومالان ریکانی
ےناپدری اھتمس ک اتکب
قیقحت دنی قح ےک رد یم یھکل ۔ ۔8
۔ااہظر اقحل
: رتک ےک ہفیلخ ےک دروخاتس رپومالان ریک
ا نی ےن
ہی اتکب یھکل
ےہ۔ اس یم اپدری ڈنپر ےک اسھت وہےن واےل انمرظے ک لیصفت اور اس ےک ارتعااضت ےک وجاابت یلیصفت
وطر رپ ذرک ںیہ
۔9
۔ اانملرظ ةالهيشد ٰاربکلی: ہی اتکب رعب ز
ابن یم ےہ۔اس یم
آپ ےک انمرظوں
ک لیصفت
ےہ۔10
۔ دعمل اوعاجج ازیملان :اس اتکب یم اپدری ڈنپر ک اتکب
زیمان اقحل
ک رتددی ےہ۔
رمح اہلل ریکانی ےک
انمرظے
1
۔ ڈارٹکوزری اخن اوررمح اہلل ریکانی ےن آرگہ یم
اپدری رفچن اور اپدری یٹک ےسانمرظہ یکاور ان وک ولغمب یک
۔10
2۔وم
الان
ریکانی اک وہشمرانمرظہ اپدری ڈنپرےس وہا۔ڈنپرےن اجعم دجسم دیلہ یم اج رک املسمنں وکیلع
ا لا
العن جنلیچ یک ۔
سج رپ رمح اہلل ریکانی ےن22
امرچ1854ء وک اکی اپدری ڈنپر ےکانم طخ اھکل
ا ور اس
یم جنلیچ وک وبقل یک۔سج ےک دعب
10
ارپلی1854
وک ہی انمرظہ رش وع وہا۔انمرظے اک وموضع اولتیہ حیسم وثیلثت، خسن،وبنت دمحم ،یﷺ رحت فی،رقآن
دیجم اک الکم اہلل وہان
رقم ر وہا۔ انمرظہ یئک دنں کت اجری راہ اور اس دوران اپدری ڈنپ ر اوراپدری رفچن یئک
رمہبت اس ابت
کے
اقلئ وہےئ ہک اس ہلئسم یم اسیعیئ اپدری یطلغ رپ ںیہ ۔11انمرظے ےس ےلہپ رفنیقی ےک درایمن یم یئک وطخط اک ابتدہل یھب
وہا ۔ وج افریس یم رمالست ذمیبہ ےک ونعان ےس
اشعئ وہیئ ںیہ ۔ رعب یم اس انمرظے ےک رو داد دبعااقلدر لبنح ےن
اانملرظہ اربکلی ےک انم ےس
اشعئ ک ےہ۔
رسدیس ادمح اخن (م1898
)ء
رسدیس ادمحاخن ک دیپاشئ دیلہ رہش یم1817ء وک وہیئ۔ رھگ رپ رقآن دیجم ک میلعت احلص ک افریس،رعب اورراییض ک میلعت
بتکم ےس اح
ک۔ لص1838
ءوک21
اسل ک رمع یم دعاتل یم انبئ یشنم ےک نرکی رپ
رھبیت وہےئسج یم وتق اسیکھت
رت
یق رکےتےئگ۔ اہبدر اشہ رفظےک رطف ےس1842
ء یم آپ وکوجاد ادلوۃلاور دیس
ادمح اعرف گنج اک اطخب یھب الم۔1855
ء
وک ونجبر یم آپ اک ابتدہل وہ ایگ۔گنج آزادی ےک دوران آپ
نے
یئک ارگنیوں ک افحتظ ک۔سج رپوکحتم رباطہین ےن آپ
وک یئک ااعنامت ےس نازا۔1858
ء وک آپ اک رم
اد آابد ابتدہل وہااہجں رپ آپ وک دصر ادصلور رپ رتیق دی یئگ۔ رماد آاب د آرک آپ
ےن اکی دمرہس اک ایقم یک۔رماد آابد یم نرکی ےس دکبسوش وہ ےن رپ آپ ےن یلع ڑگھ یم
قیا
م اک آاغز یک اور اس اسیک ھت یلع رسدیس ادمح اخن (م1898
)ء
رسدیس ادمحاخن ک دیپاشئ دیلہ رہش یم1817ء وک وہیئ۔ رھگ رپ رقآن دیجم ک میلعت احلص ک افریس،رعب اورراییض ک میلعت
بتکم ےس اح
ک۔ لص1838
ءوک21
اسل ک رمع یم دعاتل یم انبئ یشنم ےک نرکی رپ
رھبیت وہےئسج یم وتق اسیکھت
رت
یق رکےتےئگ۔ اہبدر اشہ رفظےک رطف ےس1842
ء یم آپ وکوجاد ادلوۃلاور دیس
ادمح اعرف گنج اک اطخب یھب الم۔1855
ء
وک ونجبر یم آپ اک ابتدہل وہ ایگ۔گنج آزادی ےک دوران آپ
نے
یئک ارگنیوں ک افحتظ ک۔سج رپوکحتم رباطہین ےن آپ
وک یئک ااعنامت ےس نازا۔1858
ء وک آپ اک رم
اد آابد ابتدہل وہااہجں رپ آپ وک دصر ادصلور رپ رتیق دی یئگ۔ رماد آاب د آرک آپ
ےن اکی دمرہس اک ایقم یک۔رماد آابد یم نرکی ےس دکبسوش وہ ےن رپ آپ ےن یلع ڑگھ یم
قیا
م اک آاغز یک اور اس اسیک ھت یلع 27 27 دنہواتسن
می
رباطن ی
دور ادتقار یم
ملسم یملکتم ک
مشن ی
سے
ارفیاہےسوےریےراہیارزانازمامح دنہواتسن
می
رباطن ی
دور ادتقار یم
ملسم یملکتم ک
مشن ی
سے
زمامح
ڑگھ وک اینپ رحتکی اک رمزک انبای۔12
1870
ء یم آپ ےن رباطہین
اک رفس یک۔اہجں ےس واسپ وہےنرپ آپ ےنآرک یلع ڑگھ یم
اکجل ،راسہل ہت بی االالخق اور ممد ن اوسینشی اک ارجاء یک۔ہیقب اسری زدنیگ آپ اکجل ےک دخامت یم دن رات رصموف
۔ نس ےہر1898ء ےک رشوع یم امیبر رےنہ
ےگل۔آہتسہ آہتسہ رمض ےن دشت اایتخر ک اور27
امرچ وک ااقتنل رکےئگ۔آپ
ک دتنیف دجسم دمرۃس اولعلم ےک اپس ک یئگ۔13آپ ک اصتفین ک لیصفت
ہی ےہ:
1
۔ ابطخ ت ادمحہی : اسیعیئ اپدری ومیل ویمر ےنوضحرﷺ ک ریست رپ دی الفئ آف دمحم ےک انم ےس اتکب یھکل۔اس اتکب
یم ومیل ویمر ےن وضحرﷺ ک ریست رپیئک ارتعااضت ےیک۔رسدیسادمح اح ن ےن اس ےکردیم ابطخت ادمحہی
ےک انم
ےساتکب یھکل اور ریست روسل ﷺ ےک وح
اےل ذموکرہ اتکب ےک امتم اہبشت ےک دملل وجا ب دےی۔ ہی اتکب392
احفصت ےک اسھت دوتس اوسییس اٹی
الوہ اشےنر
عئ یک ےہ۔2
۔
راسہل ااکحم الہ اتکب :اس فینصت یم آپ ےن اسیعویئں
ےکاسھت رشکی وہرک اھکان
اھکےن ک لیصفت ایبن ک ےہ ہک اسیعویئں اسیکھت اھک
ان اھکان بت درتس وہ
اگبج ہی ملع وہ ےک و ہ زیچ
رحام ںیہن ےہ۔3
۔نیبت االکلم
:اس اتکب یم الہ اتکب اور املسمنں یم ام رفلق اہب
اور امہب االافتق اسملئ وک ایبن یک ےہ اور
وج زیچںی امہب ارفلق یھت اس ک فلتخم وتاہیج
ت ایبن ک ےہ۔4
۔ الجء اولقلب ذبرک المحیبو ب :اس اتکب یم وضحرﷺک
زجعمات ا
ور ریست ےک نج ولہپؤں رپ اسیعیئ
اپدرویں ےن ااکشالت املسمنں ےک دول ں یم ڈاےل ےھت اس وک ایبن یک
۔5
۔ لف
صنری ک قیقحت : ارگنیوکحتم اقمئ وہےن دعب املسمن ان ےک ےیل فل صنری اک اامعتسل یک رکےت ےھت سج رپ ارگنی وکحتم
ےک اکردنے ہصغ وہےت اور املسمنں وک
اںیئ زس دےتی سج رپ رسدیس ےن ہی اتکب یھکل اور فل صنری ک قیقحت شیپ ک اتہک
وکحتم اور وعا م دونں اک
اغمہطل متخ وہاجےئ۔6
۔ ہتبی االالخق : رس دیس ےن یلع ڑگھ اکجل ےک ےل اس راسہل اک ارجاء یک
جس
یم وخد اوردرگی یئک نیققحم فلتخم اضمنیم اشعئ رکےت
ےھت۔7
۔ اابسب اغبوت
دنہ : گنج آزادی ےک دعب رسدیس ےن ارگنی
وکحتم ک ان ووجاہت اک ایبن اس اتکب یم یک ہک
سج انبء رپ
دنہواتسین
وعام وبجمر وہےئگ ےھت ہک وکحتم وتق ےک الخف
اغبوت رکے۔8
۔
ہفحت نسح
:اس اتکب یم
ؓرضحت اوبرکب رپےیک ےئگ ارتعااضت ےک وجاابت ےہ۔9ریسفت ارقلآن
:اس انم
ےسآپ
ےنرقآن دیجم
ک ریسفت یھکل ےہ۔ آپ ےک درگیاصتفین یم ہی اشلم ےہ۔ ۃملک اقحل ،
راہ تنس در رددب ع ،
لیہست یف
رج الی ق ي ل،آاثر اانصلددی ،
اترخی رسیشک ونجبر ،
ۃلسلس اولملک۔ وااانتوابیراو دنہواتسن
می
رباطن ی
دور ادتقار یم
ملسم یملکتم ک
مشن ی
سے
زمامح ارفیاہےسوےرےیےراہیارزانا ومالانانعتی روسل ابعیس رچایوک ( یٹ م1902
)ء
ومالان انعتی روسل
اک ہلسلس بسن انعتی روسل نب یلع اربک نب
الغم ومحمد نب دبعامصل ےہ وج آےگ اجرک ؓدبعاہلل نب ابعس
سے
لم اجات ےہ ۔14
آپ ک والدت1828ءوک رچایوکٹ
می
۔ وہیئ
رصف وحن اور ونفن ک دریس بتک آپ ےن
آےنپ
وادل اور چچ
سے
ڑپھ
۔
ملع دحثی
ےک ےیل
رایتس وٹنشکح لے ےئگ
۔ افر غ وہےن رپ رچای وکٹ واسپ وہےئ۔اس
دوران ربعاین زابن ےک وصح ل
ےکےیل ہتکلک اک رفس
اایتخر یک ۔ہتکلک یم
ربعاین زابن ک میلعت دےنی ےک ےیل وکیئ وہیدی
ایت ر ںیہن
وہاسج رپ آپ ےن
وبجمراً اکی
اسیعیئ اپدری ےس ارگنیی اورویانین زابںین ھکیس یل
جس
ک اینبد رپ ربعاین زابن ےنھکیسےک وماعق ےنلم
رشوع
وہن ی يبل رخ
ہب قشم
ےک دعب ربعاین زابن رپ لمکم ریصبت احلص ک
۔
ہتکلک ےس اغزی وپر
رشتفی ےل
اور ےئگ اغزی وپر یم
آپ ےن و اک
تل وک وطبر
ہشیپ اایتخر یک
۔
آپ اک ااقتنل وشال
ےک ےنیہم یم1902ء
وک ہعمج ک بش اوہ ۔15
آپ ےک ااسذتہ یم ومالان ادمح یلع
(م
1880ء)
،اقیض یلع اربک (وادل)،ومالان لضف روسل دبویاین(م1872ء)اشلم ںیہ۔ آپ ےک اشرگدوں یم ومالان افروق رچای
وکیٹ(م1911
،)ء
رس دیس
ا دمح اخن(م1898ء ) اشلم ںیہ۔۱ ۔ ٰرشبی : اس اتکب یم
فنصم ےن
وترات اور الیجن ےس
وضحرﷺ ےک ابرے یم
اشبروتں
ک لیصفت،ابلبئ یم اہلیب اور اقلیب اک واہعق،
فل افرقل
ی ط ک قیقحتؑ ،اثیمق اایبنء رکام
وتراات
ور رقآن دیجم
ےک انترظ یم،زجعمہ قش ارمقل رپ
وارد اہبشت اور ان ےک وجاابت، وہید ےک ااہتجدات اور رحت افیت ،
واہعق 28 ا رمقل، دلج6
، امشر ہ1
، (ونجر ی–
امر چ 3
202
)
تیمصنیفہےحثرلاارفحا ا رمقل، دلج6
، امشر ہ1
، (ونجر ی–
امر چ 3
202
)
تیمصنیفہےحثرلاارفحا رعماج اک یلقع ویلقن وبثت،زوبر
فلیم میتی اک االطق رپ ثحب ک ےہ۔ ہی فینصت434
احفصت رپ لمتشم ےہ ۔
آپ ک یئک
غیر
وبطمع بتک یھب ےہ نج یم
درہج ذلی اک ذترکہ تلم ےہ:
1
۔ :ااجعزارقلآن
ومالان رچای وکیٹ ےن اس اتک ب
یم درگی بتک امسو
ی اوررقآن دیجم اک اقتلب،اور رقآن دیجم رپ وہےنواےل
ارتعاضت ےک وجاابت
اور رقآن دیجم ےکووجہ
ااجعز
کو
ایبن ک ےہ۔2
۔ :اونلر اس اتکب یم آپ ےن
ملع انم
رظہ رپ
اکی لمکم
ثحب ک ےہ ۔16
آپ ےک درگی اصتفین ہی ےہ۔ اقم الت دضعہی ،
زابن ربعاین ےکوقادع ،
اہشدت انہم ۔17پآ ےن
رفتمق
راسول(ں ہتبی االالخق،اابخر ربلل،اابخر اولتق
اور راسہل زامہن)
یم ومفلتخم وضاعت رپ
اضمنیم رحتری ےیک سج ک لیصفت
ذلی یم ےہ۔1
۔ دجول نینس وطافین
:اس ومضمن یم
آپ ےن
ابلبئ ےک ابب السنیط رپ ثحب ک
ہے۔2
۔ وترات والیجن ےس
:وضحرﷺ ک نشیپ وگیئ ہی ومضمن آپ ےن
انب رس ےکاکی اپدری ےک رتددی یم یھکل ےہ ۔اس ےن املسمنں یم اس وقل ک
رپاچر ک
کہ
وترات اور الیجن یم وضحرﷺ ک وحاےل ےس وکیئ
اشبرت
ںیہن ےہ ۔اس العوہ ،
تق یر ،
وجاب انب ردش ،
داۃب
االرض ،
اویلم یف اوتلر ا ت
ےک ونعان ےس یھب اضمنیم ےھکل۔ واااردنول اتوی
میوکھےینضاھیےنوے
( ومالان انرص ادلنی دولہی م1903
)ء
آپ ک دیپاشئ1821ء وک انوپگر رہش یم وہیئ
۔
آپ اک انم انرص ادلنی ےہ۔ نکیل آپ ا ینپ تینک اوب اوصنملر ےس یھب وہشمرےھت۔
آپ اک
شیبس ہ رز
تش
ونقج ےکدیس اخدنان ےس
تھا۔
1857ء کے
گنج آزادی ےکدعب دیلہ رجہت ک اورہیقب زدنیگ دیلہ یم زگارےن
ک وہج ےسدولہی
بھی
الہکےئ
۔ آپ ےن آ
دتبایئ میلعت انوپگر ےک املعء ےس احلص ک
۔ دینی میلعت لمکم رکےن ےکدعب رد اسیعتیئ
اک ذوق وہےن ک وہج ےس
الیجن ک میلعت
احلص ک۔6 اسل فلتخم اپدرویں
ےک تبحص یم رےہ ۔اس دوران آپ ان ک امتم
اسزوشں
ےس وافق وہ
ئے865
1 ء
وک آپ ےن اہٰل آابدیم انمرظہ رکےک رد اسیعتیئ اک آاغز یک۔18
آپ ےن رد اسیعتیئ
می
وک املعء
انمرظے اک رطہقی ھکس
ےن ےک ےیل
داراال امتم
ےک انم ےس دمرےس ک اینب د ریھک
۔ اس ادارے
کے
افرغ الیصحتل املعء
ےن رد اسیعتیئ
پر
ہب اکم یک۔دیلہ
کے
املسمنں ےن
اکی میظنت انمجن االسہیم ےک انم ےس اقمئ ک ۔ سج یم آپ ےک ذہم
ڈوییٹ اگلیئ یئگ ہک وج
املسمن نشمویں ےک اج ل یم آ رک اسیعیئ وہ اجےت
ںیہ ان وک
دوابرہ املسمن
رکاان ےہ۔ یمسج
ہب ےس
ول گ آپ ک تنحم ک وہج ےس اسیع یئ وہےن
ےک دعب دوابرہ املسمن وہےئ
۔
آپ ک وافت1903ء
وک وہیئ ۔19
رد اسیعتیئ رپ
آپ ےن دنمرہج ذلی اقلب دقر بتک
لکھیں: قیضاہےندراااوندمااتاتا کتبدلا
ےدنہج لی
پکھیں
1
۔ قع نبه
ااضلیل:
ومالان انرصادلنی ےن ہی
اتکب اپدری امعد ادلنی (م1900ء)
ک اتکب دہاتی ایملسمل
ےک رد
یم یھکل
۔2۔
نحل داودی
: اپدری امعد ادلنی(م
1900ء)اورونھکلےکاکی الہ عیشت
ےک اعمل درایمن اکی رحتریی انمرظہ وہا
۔
سج وک
امعد ادلنی ےن مغن
وبنطری ےک انم
ےس اشعئ یک سج
یم االسم رپ یئک ارتعااضت ےیک ےھت اس ےک
وجاب یم ومالان انرص
ادل ی يشلحنش دا
ودی ےک انم ےس ہی اتکب یھکل
۔3
۔ :ندی اجودی اس اتکب یم االسم رپ
لقع ک رو ےس صنری ک وارد ارتعااضت
ےک وجاابت دےی ےئگ ںیہ
۔4
:رحز اجن
اپدری دبعاہلل امھت ک اتکب اتیلص رقآن
ےک رد
یم ہی اتکب یھکل یئگ
ےہ۔5
۔ :زیمان ازیملان
اپدری
ڈنف (م ر1865
)ء ک اتکب زیم ان اقحل ےک
رد یم ہی اتکب یھکل۔6
۔
ااخفم ااصخلم :اس اتکب
یم اپدری رارجز
ک اتکب شیتفت االالسم اک رد یک ایگ ےہ۔7
۔ :ازعاز رقآن فینصت ہی
انرص ادلنی دولہی شاپدری رام
(م دنچر1880
)ء ب ک اتک
ااجعز رقآن ےک رد یم یھکل ےہ ۔8
۔ ا اصیتسل:
اس اتکب یم فنصم ےن اپدری رام دنچر (م
1880
)ء ک اتکب حیسم ادلاجل
اک احمہمک یک ےہ۔9
۔
ایبتن :
اس اتکب یم
اپدروی
ں ک رطف ےس
آےئابرہ
وساالت ےک
وجاابت ںیہ
۔10
۔ حیحصت ااتلولی :اپدری امعدادلنی ک ریسفت اکمافشت رپ دیقنت ورصبت
ہ ےہ۔11
۔ ااعنم اعم :اس اتکب یم اسیعیئ
اپدری ربج یلع ک اتکب آنیئ
االسم
اک رد
ےہ۔12
۔ ابصمح االربار :اپدری
(م رڈنف1865
)ء ک اتکب اتفمح االرسار ےک رد 29 ا رمقل، دلج6
، امشر ہ1
، (ونجر ی–
امر چ 3
202
)
لطایتااحمدےویوسیحیی ا رمقل، دلج6
، امشر ہ1
، (ونجر ی–
امر چ 3
202
)
ہے۔3۔
مت اسیعیئ ویکں وہےئ
: اسیعیئ اپدری اطلس
ن دمحماپل ک اتکب‘‘یم یحیسم ویکں وہا’’ ےک
رد
یم یھکل سج یم اپدری ےنآایت
ابمرہک
اور ااح
دثی
رپ ارتعااضت
کیے تھے۔4۔
وتدیح،ثیلثت اور راہ اجنت
:اس اتکب یم
عیسا ئیت
ےک اقعدئ و اسملئ اک زجتہی ےہاور
االسم ےک قح وہےن رپ یلقن اوریلقع داللئ ںیہ ۔37
5۔
اقتلب الثہث
:اس
تصنیف
یم ارمرستی ےن رقآن دیجم ،وترات اور الیجن اک اقتیلب
اطمہعل یک ےہ
۔اس اتکب
وموض ےک
اعت ہی
ںیہ۔ دوعی ااہلم،دوعی وتدیح، دالن لصا عن،افصت دخاودنی
اور ااکحم
رشتعی 6۔ االسم اور
تیحیسم :اس اتکب یم اسیعتیئ ےک امتم ولہپوّں اک اجزئہ
شیپ یک
ےہ ۔زیناس اتکب یم اپدری ربتک اہلل ک نیت
راسولں
اک وجاب یھب
ہے۔7۔
امنزارہعب ۔اس
فینصت یم املسمن ،اسیعیئ ،،دنہو
اور
آرہی ےک امنز اک اقتلب ےہ
۔8۔
االسم اور ربشٹ الء :اس یم االسیم وقانین
او
ر ربشٹ الءاک
ومازہن
شیپ یک ۔ےہ9۔
نرااشفں یم تملظ :اس
اتکب یم اسیعویئں ےک نر ااشفں اابخر
ک اضمنیم ک رگتف ےہ۔
آپ
اسھٹ ےن
کت انمرظے اسیعتیئ،
آرہی امسج
اور رمزاتیئ ےک الخف
ک ےہ نج یم وہشمر
انمرظہ،وخرہج انمرظہ
ہنیگن ،انمرظہ،ونجبر
انمرظہ
ون لم علض رفظم رگن
ےہ۔آپ ےن الوہر یم
اسیعویئں ےک اسھت ‘‘اولتیہ حیسم’’رپ اہل آابد یم ‘‘وتدیح اور ثیلثت رپ’’رجگاناہل
یم‘‘ وتدیح ’’ رپ انمرظے
ےیک ںیہ۔
دبع ازعلی ونھکلی
اسیعیئ مش يشکے ارفاد ےن غیلبت اسیعتیئ ہک دوران یتخس ےس رتددی رکےت ےھت۔ ہک ابلبئ
یم وضحرﷺ ےک وحاےل ےس اشبرت
نہیں
ےہ۔ اس ےک ردیم دبعازعلی ؤنھکلی ےن
اشبرت دمحمی
ےک انم ےس اکی زہار احفصت ک اتکب1309
/ ھ1891ءیم
یھکل۔اس فنصم ےک احتل ےک ابرے یم ولعمامت ںیہن لم ےکس۔38
ومالان ابع س اجم
وجیئ
آپ اک انم ابعس یلع نب لضف اہلل افرویق ومجی ےہ۔آپ ےک احال ت ہن لم ےکس نکیل آپ ےن1832
ء یم اسیعیئ اپردری وامیل
ویمرےس انمرظہ یک ۔آپ ک دو اتکںیب ںیہ
۔ وصله الضشعشت م
یلع ادعاءانب
رممی
:ہی اتکب یلیصفت وطر رپ رحتری ک یئگ یھت۲۔
الخصه
وصله الضشعشت م
:ہی اتکب املبق اتک ب اک الخہص ےہ108
احفصت ےک اسھت1842
یم ہبتکم نینس ےس
عبط دشہ ےہ۔39
الخص
بحث
دنہواتسن یم بج ےس رباطہین ےک ارگنیوں وک ہبلغ انلم رشوع وہاوت اوہنں ےن دنہواتسن ےک وعام وک اےنپ
مہ ایخل انبےن ےک ےیل رضوری
اھجمس ہک ان وک اسیعتیئ یم اشلم یک اجےئ
سج ےک ےیل اوہنں ےن ویرپ اور ارمہکی ےس ڑبےڑبے اپدرویں اور اسیعتیئ ک غیلبت ےک
لیے
نشمی ومیظنت ں
ےک ارفاد وک دنہواتسن
انجیھب رشوع یک اور وکحتم ےک رطف ےس لمکم وپسرٹ رفامہ ک اجیت یھت۔ ا ن
اپدرویں ےن
دنہواتسن ےک املسمن وعام وک رمگاہ رکےن ےکےیل رہ رحہب اامعتس ل یک نکیل اس رپمسکیس ک احتل یم یھب املسمنں یم ےس دنچ رمد اجمدہ
املعء ان اپدرویں اور
نشمویں ےک اسےنم ہسیس الپیئ
دویار ک رطح اثتب دقیم ےس ڑھکے رےہ اور ان اک اقمہلب رہ دیمان یم یک
۔ ذہلا
اس آرلکیٹ یم ان املعء ک رصتخم احالت ز
دنیگ اوررد اسیعتیئ ےک وحا ےل ےس ان ےک دخامت اک ااحہط یک ایگ۔
References
d
h
d ʻ
h
h ا رمقل، دلج6
، امشر ہ1
، (ونجر ی–
امر چ 3
202
)
ہے۔3۔
مت اسیعیئ ویکں وہےئ
: اسیعیئ اپدری اطلس
ن دمحماپل ک اتکب‘‘یم یحیسم ویکں وہا’’ ےک
رد
یم یھکل سج یم اپدری ےنآایت
ابمرہک
اور ااح
دثی
رپ ارتعااضت
کیے تھے۔4۔
وتدیح،ثیلثت اور راہ اجنت
:اس اتکب یم
عیسا ئیت
ےک اقعدئ و اسملئ اک زجتہی ےہاور
االسم ےک قح وہےن رپ یلقن اوریلقع داللئ ںیہ ۔37
5۔
اقتلب الثہث
:اس
تصنیف
یم ارمرستی ےن رقآن دیجم ،وترات اور الیجن اک اقتیلب
اطمہعل یک ےہ
۔اس اتکب
وموض ےک
اعت ہی
ںیہ۔ دوعی ااہلم،دوعی وتدیح، دالن لصا عن،افصت دخاودنی
اور ااکحم
رشتعی 6۔ االسم اور
تیحیسم :اس اتکب یم اسیعتیئ ےک امتم ولہپوّں اک اجزئہ
شیپ یک
ےہ ۔زیناس اتکب یم اپدری ربتک اہلل ک نیت
راسولں
اک وجاب یھب
ہے۔7۔
امنزارہعب ۔اس
فینصت یم املسمن ،اسیعیئ ،،دنہو
اور
آرہی ےک امنز اک اقتلب ےہ
۔8۔
االسم اور ربشٹ الء :اس یم االسیم وقانین
او
ر ربشٹ الءاک
ومازہن
شیپ یک ۔ےہ9۔
نرااشفں یم تملظ :اس
اتکب یم اسیعویئں ےک نر ااشفں اابخر
ک اضمنیم ک رگتف ےہ۔
آپ
اسھٹ ےن
کت انمرظے اسیعتیئ،
آرہی امسج
اور رمزاتیئ ےک الخف
ک ےہ نج یم وہشمر
انمرظہ،وخرہج انمرظہ
ہنیگن ،انمرظہ،ونجبر
انمرظہ
ون لم علض رفظم رگن
ےہ۔آپ ےن الوہر یم
اسیعویئں ےک اسھت ‘‘اولتیہ حیسم’’رپ اہل آابد یم ‘‘وتدیح اور ثیلثت رپ’’رجگاناہل
یم‘‘ وتدیح ’’ رپ انمرظے
ےیک ںیہ۔
دبع ازعلی ونھکلی
اسیعیئ مش يشکے ارفاد ےن غیلبت اسیعتیئ ہک دوران یتخس ےس رتددی رکےت ےھت۔ ہک ابلبئ
یم وضحرﷺ ےک وحاےل ےس اشبرت
نہیں
ےہ۔ اس ےک ردیم دبعازعلی ؤنھکلی ےن
اشبرت دمحمی
ےک انم ےس اکی زہار احفصت ک اتکب1309
/ ھ1891ءیم
یھکل۔اس فنصم ےک احتل ےک ابرے یم ولعمامت ںیہن لم ےکس۔38
ومالان ابع س اجم
وجیئ
آپ اک انم ابعس یلع نب لضف اہلل افرویق ومجی ےہ۔آپ ےک احال ت ہن لم ےکس نکیل آپ ےن1832
ء یم اسیعیئ اپردری وامیل
ویمرےس انمرظہ یک ۔آپ ک دو اتکںیب ںیہ
۔ وصله الضشعشت م
یلع ادعاءانب
رممی
:ہی اتکب یلیصفت وطر رپ رحتری ک یئگ یھت۲۔
الخصه
وصله الضشعشت م
:ہی اتکب املبق اتک ب اک الخہص ےہ108
احفصت ےک اسھت1842
یم ہبتکم نینس ےس
عبط دشہ ےہ۔39
الخص
بحث
دنہواتسن یم بج ےس رباطہین ےک ارگنیوں وک ہبلغ انلم رشوع وہاوت اوہنں ےن دنہواتسن ےک وعام وک اےنپ
مہ ایخل انبےن ےک ےیل رضوری
اھجمس ہک ان وک اسیعتیئ یم اشلم یک اجےئ
سج ےک ےیل اوہنں ےن ویرپ اور ارمہکی ےس ڑبےڑبے اپدرویں اور اسیعتیئ ک غیلبت ےک
لیے
نشمی ومیظنت ں
ےک ارفاد وک دنہواتسن
انجیھب رشوع یک اور وکحتم ےک رطف ےس لمکم وپسرٹ رفامہ ک اجیت یھت۔ ا ن
اپدرویں ےن
دنہواتسن ےک املسمن وعام وک رمگاہ رکےن ےکےیل رہ رحہب اامعتس ل یک نکیل اس رپمسکیس ک احتل یم یھب املسمنں یم ےس دنچ رمد اجمدہ
املعء ان اپدرویں اور
نشمویں ےک اسےنم ہسیس الپیئ
دویار ک رطح اثتب دقیم ےس ڑھکے رےہ اور ان اک اقمہلب رہ دیمان یم یک
۔ ذہلا
اس آرلکیٹ یم ان املعء ک رصتخم احالت ز
دنیگ اوررد اسیعتیئ ےک وحا ےل ےس ان ےک دخامت اک ااحہط یک ایگ۔
References عباااو
ساجم
ئیو
آپ اک انم ابعس یلع نب لضف اہلل افرویق ومجی ےہ۔آپ ےک احال ت ہن لم ےکس نکیل آپ ےن1832
ء یم اسیعیئ اپردری وامیل
ویمرےس انمرظہ یک ۔آپ ک دو اتکںیب ںیہ
۔ وصله الضشعشت م
یلع ادعاءانب
رممی
:ہی اتکب یلیصفت وطر رپ رحتری ک یئگ یھت۲۔
الخصه
وصله الضشعشت م
:ہی اتکب املبق اتک ب اک الخہص ےہ108
احفصت ےک اسھت1842
یم ہبتکم نینس ےس
عبط دشہ ےہ۔39
الخص
بحث لاحث
دنہواتسن یم بج ےس رباطہین ےک ارگنیوں وک ہبلغ انلم رشوع وہاوت اوہنں ےن دنہواتسن ےک وعام وک اےنپ
مہ ایخل انبےن ےک ےیل رضوری
اھجمس ہک ان وک اسیعتیئ یم اشلم یک اجےئ
سج ےک ےیل اوہنں ےن ویرپ اور ارمہکی ےس ڑبےڑبے اپدرویں اور اسیعتیئ ک غیلبت ےک
لیے
نشمی ومیظنت ں
ےک ارفاد وک دنہواتسن
انجیھب رشوع یک اور وکحتم ےک رطف ےس لمکم وپسرٹ رفامہ ک اجیت یھت۔ ا ن
اپدرویں ےن
دنہواتسن ےک املسمن وعام وک رمگاہ رکےن ےکےیل رہ رحہب اامعتس ل یک نکیل اس رپمسکیس ک احتل یم یھب املسمنں یم ےس دنچ رمد اجمدہ
املعء ان اپدرویں اور
نشمویں ےک اسےنم ہسیس الپیئ
دویار ک رطح اثتب دقیم ےس ڑھکے رےہ اور ان اک اقمہلب رہ دیمان یم یک
۔ ذہلا
اس آرلکیٹ یم ان املعء ک رصتخم احالت ز
دنیگ اوررد اسیعتیئ ےک وحا ےل ےس ان ےک دخامت اک ااحہط یک ایگ۔ (
y
)
6Thānvī, Research Review of Religious and Academic Services of Moulānā Qāsim, 34-51. دنہواتسن
می
رباطن ی
دور ادتقار یم
ملسم یملکتم ک
مشن ی
سے
زمامح ے دنہواتسن
می
رباطن ی
دور ادتقار یم
ملسم یملکتم ک
مشن ی
سے
زمامح ے 30
دنہواتسن
می
رباطن ی
دور ادتقار یم
ملسم یملکتم ک
مشن ی
سے
زمامح
یم ہی اتکب یھکل ےہ ۔20
( ومالان دبعاقحل اقحین م1917
)ء
دبعاقحل اقحین ک دیپ
اشئ اگوں کمت ھ لہ ، علض اابنہل، اجنپب
می
1851ء
وک وہیئ ۔ادتبایئ میلعت اگوں
رپ احلص ک
۔اس
ےک دعب
ونفن ک دریس اتکںیب
اکوپنر اجرک ڑپھ
۔ملع دحثی ےک ےیل رمادآابد رشتفی ےل ےئگ
۔ دیلہ اج رک
دیس ذنری نیسح
(م1902ء)ک رشف ذملت یھب احلص ک۔
تعلیم
لمکم رکےنرپ
دیلہ یم
دمرہس حتف وپرہی ےس وطبر
ااتسد وہےئ کلسنم ۔اہجں رپ
آےنپ
یئک اسل کت درس دای ۔بج وکحتم
دیحر آابد ےک رطف ےس آپ اک و
ہفیظ رقمر وہ ا۔وت آپ ےن فینصت واتفیل
کے
ےیلآےنپ آپ وک لمکم افرغ یک۔ا ہتبل
دمرہس اعہیل ہتکلک
ےک ارصار رپ آپ وھتڑے رعےص ےک ےیل ااتسد
رقمر وہےئ ۔ومالان
دبعاقحل
وک وکحتم ےک رطف ےس
شمس
ااملعلء اک اطخب الم اھت۔ آپ ک وافت1917ء
وک وہیئ۔21
آپ ےک ااسذتہ یم ذنری
نیسح دولہی(م1902ء)
،ومالان فطل اہلل(م1919ء
) اور
دبعاقحل اکوپنری
(م1885
ء) اشلم
ںیہ۔1
۔ اایبلن یف ولعم
ارقلآن: ہی اتکب ریسفت اقحین اکدقممہ ہ ے
۔
سج یم
آپ ےن الکیم ابمثح ےسیجووجد ابری اعت
یل ، وتدیح،وبنت
ویح،ت زجعما
،ااہلم ،
ادتسراج،اراہاصت، اور اعمل وکلمت رپ ثحب ک ےہ۔2
۔
ریسفتحتف اانملن :ہی ریسفت اقحین ےک
انم ےس
وہشمر
۔ےہ اس
ی فشی رز
می
اسیعویئں ک ارتعااضت اور رس دیس(م1898ء)
ک ریسفت یم وارد
اتوالیت اور الکیم ابمثح رپ
بحث
وموجد
ےہ۔3
۔ رحت فی ارقلآ ن :ہی اتکب اپدری رام دنچر
(م1880
)ء ےک اتکب ےک رد یم یھکل یئگ ےہ۔ آپ ےک درگی اصتفین ہی
ےہ اقعدئ االالسم، اہشب اثبق،ااقحق قح ۔
ومالاندیس دمحم یلع ومیگن ( ی م1927
)ء
آپ اک ہلسلس بسن دیسدمحم یلع نب
دیس دبع ایلعل نب دیس وغث ےہ۔وج آےگ اجرک خیش
دبعااقلدر الیجین ےس اج رک لم اجات ےہ۔22
محمد
یلع بج
دو اسل ےک رمع ےک وہےئوت ان
وادل وافت
رکےئگسج رپ
دادا ےن رپورش رشوع ک نکیل
ابرہ اسل ک
رمع یم دادا یھب
ااقتنل رک ۔ےئگ
جس
ےک دعب
اکیف الکشمت اک اسانم وہا نکیل
تمہ ںیہن اہری اور اس ےک یھب میلعت اجری ریھک انچہچن
چچ ےس رقآن
مجید
ڑپھ
۔ افریس
،رصف و حن
اورقطنم ک اتکںیب اجعم ضیف
سے پڑ ھیں ۔
اس ےک دعب وم الان فطل اہلل یلع ڑگھ
(م1919
)ےک ء اپس یلع ڑگھ اجرک دمرہس اجعم دجسمیلع
ڑگھ یم دالخ وہےئ۔اذخ دحثی ےک ےیل ومالان لیلخ ادمح
اہسروپنری ک ذملت اایتخر رک ےک احصح ہتس ،وماط اامم امکل وم اور ط اامم دمحم
ا
ن ےس ڑپھ۔23دمرہس ضیف اعم اکوپنر
کے جلسہ
داتسر دنبی1892ء رپ املع ء ک اکی اشمورت وہیئ
۔ سج یم اکی انمجن دنوۃ ااملعلءےک
انم ےس
ایقم اک ہلصیف وہا۔
جسکے
انمظ
اول دمحم یلع ومیگنی وک رقمر یک ایگ
۔
آپ ےن اس انمجن ےک تحت اکی دمرےس
اک ااتتفح یک
۔سج
ےن
دعب یم اجعم دنوۃ ااملعلء
اک لکش اایتخر یک
۔1903
وکء آپ ےن اسی عفی د
ی اور آ ابیئ
اگؤں
ومیگن
رشتفی ےل ےئگ
۔ومیگن یم آپ ےناخاقنہ اور دمرہس
رامحہین ک اینبد ریھک
۔
آپ لضف ارلنمحٰ جنگ رماد آابدی( م1895ء)
ےس تعیب ےھت۔24
آپ ک وافت ربمتس1927
ء وک وہیئ
۔آپ وک اےنپ رجح
ے ےک نحص یم دنف رک دای
ایگ ۔25
26
آپ ےک
وہشمر
ی انعتی ادمح اکوکری
ااسذتہ می ںمفت (م1863ء) ،
ومالاندیسنیسح اشہ ،ومالان فطل اہلل یلع ڑگھ
(م1915
)،لیلخ ء ادمح اہسروپنری(م1927ء
) اشلم ںیہ۔ آپ ےک رد
اسیعتیئ دنمرہج ذلی اصتفین ںیہ:
1
۔ ۃ رمآ
ایقیل :ہی اتک ب آپ ےن
اپدری امع
د ادلنی
(م1900
)ء ک
اتکب هد ان ه ایملسمل ےک رد یم یھکل ۔اپدری امعد ادلنی
ےن رمح اہلل ریکانی ک اتکب ااجعز وسیعی ےک ارتعااضت وجاابت دےنی ک ان اکم وکشش ک یھت
۔زین اس اتکب یم الیجن
ےک ر ادن رحتفی وک
اپدرویں ک بتک ےس اثتب یک ےہ۔2۔
آنیئ االسم : ہی اتکب اپدر
ی یشنم دفصر یلع (م1899
)ء
ک اتکب اینز
دنماہن ےک رد یم یھکل سج یم یشنم دفص ر اصبح ےنرشتعی ےک دو ںیمسق انبیئ یھت ریمس اوراالخیق رشتعی وموسی رشتعی یتیسایںیفصالہند
1
۔ ۃ رمآ
ایقیل :ہی اتک ب آپ ےن
اپدری امع
د ادلنی
(م1900
)ء ک
اتکب هد ان ه ایملسمل ےک رد یم یھکل ۔اپدری امعد ادلنی
ےن رمح اہلل ریکانی ک اتکب ااجعز وسیعی ےک ارتعااضت وجاابت دےنی ک ان اکم وکشش ک یھت
۔زین اس اتکب یم الیجن
ےک ر ادن رحتفی وک
اپدرویں ک بتک ےس اثتب یک ےہ۔2۔
آنیئ االسم : ہی اتکب اپدر
ی یشنم دفصر یلع (م1899
)ء
ک اتکب اینز
دنماہن ےک رد یم یھکل سج یم یشنم دفص ر اصبح ےنرشتعی ےک دو ںیمسق انبیئ یھت ریمس اوراالخیق رشتعی وموسی رشتعی 30 ا رمقل، دلج6
، امشر ہ1
، (ونجر ی–
امر چ 3
202
)
رلاعنیعترمیاعدےےر 31
ا رمقل، دلج6
، امشر ہ1
، (ونجر ی–
امر چ 3
202
)
وک ریمس رقار دای اور اسیعتیئ وک االخیق رقار رک ےک اس ےک دعب دوابرہ ریمس رشتعی (ینعی االسم رماد ےہ) ک اعمد وک
ریغ رضوری
رقار دای
۔ اس اتک ب یم اس رفموےض اک دملل رد یک۔3
۔
دعف الي ليبی شا ت : اپدری امعد ادلنی ( م1919
)ء
ک اتکب اقیلعتت
کے
رد یم یھکل
ےہ۔ا س یم اابثت وبنت دمحمی
اور
اانلیج ک رحتفی وک اثتب یک ےہ۔4
۔ رتاہن اجحزی
:ہی اتکب اپدری امعد
ادلنی
(م1900
)ء
اکرحتریی انمرظہ ےہ
۔وج اپدری ذموکر ےن الہ عیشت اعمل
ےک اسھت
وطخط ےک لکش یم ک یھت اور اس وک مغن
وبنطری ےک انم ےس اسیعویئں ےناشعئ یک
۔ اس ےک رد یم ہی اتکب
یھکل اس یم تمصع اایبنء،وبنت دمحم،رقآن دیجم اک اسہقب
اتکوبں ےس اموخذ ہن وہانوک اثتب یک ےہ۔5
۔
اغیپم دمحمی : اپدری اھٹرک داس (م1919
)ء
ک اتک ب
دعم رضورت رقآن
ےک رد
یم یھکل ےہ
۔ اس یم افکرہ اور ثیلثت اور اک ابلط وہان، یحیسم اور
االسیم وقانین اک اقتلب ےہ ،اقح تین رقآن وااحدثی، ابلبئ ک
طبض اور تحص درتس ںیہن ےہ ہی اسیعویئں ےک دینی ادب اثتب ک
ےہ ۔زین آپ ےن ابلبئ ےک
انتاضق ت وک اثمولں ےس واحض یک
اس ےہ۔ اتکب
اک ارگنیی
رتہمج
یھب وموجد ےہ۔
وشنم ر دمحمی ےک انم ےس
اکی اابخر اک ارجاء یھب آپ ےن
اکوپنر یم
ک سج یم
ر د اسیعتیئ
رپ فلتخم اضمنیم اشعئ وہےت ےھت۔اس ےک اسھت اسیعویئں
ےک اقعدئ ےک
ابلط وہےن
وک وخب اظرہ یک
اجات اھت
۔ ا س
اابخ ر یم اپدرویں وکیئک رمہبت انمرظوں ک خب
چ لی تش
دی
یئگ نج ےک وبقل رکےن ےس اپدری رتکاےت ےھت۔ سج ےس
ایسفنیت
ارثاملسمنں رپ ااھچ ڑپاھ۔27
اس ےک العو ہ نشمویں
ک فلتخم ی یشظ
م میتی اخےن انب رک اس یم وچبں وک اسیعیئ انب رےہ ےھت۔
رضحت
ومیگنی
ےن اسیعویئں ےک اقمےلب
ےیلےک
اکوپنر یم میتی اخہن االسہیم
ک اینبد ریھک ۔
سج یم املسمن وچبں ک
میلعت اور
تعنص ورحتف اک اامتہم یک
اجاتاھت۔
ومالان دیمح ادلنی رفایہ ( م1930
)ء
آپ اک ہلسلس بسن ےہدیمح ادلنی نب دبعارکلمی نب رقابن
ربنق نب اتج یلع نب اقمئ یلع ےہ۔ دیمح ادلنی ک دیپاشئ1863
ء وک
وییپ ےک
رہش امظع ڑگھ ےک اگؤں رھپ اہی
یم ۔ وہیئ آپ اک اتکب انم املعمل دبعادیمحل رفایہ ےہ وج آپ اتکوبں یم انھکل دنسپ رکےت
ےھت۔28دیمح ادلنی
رفایہ ےن میلعت اک آاغز رھگ رپ یک10
اسل ک
رمع یم
ظفح رقآن یک ۔
ظفح رقآن ےک دعب افریس یم اہمرت
احلص ک ۔
14
اسل ک رمع یم رھپاہی
ں
ےس امظع ڑگھ دمرہس یم دا
لخ وہےئ
۔ در
س اظنیم ک بتک امظع ڑگھ یم
ڑپھ ۔
درس اظنیم لمکم
وہےن
رپ ؤنھکل
ور ےئگا
رفیگن لحم ےک دمرہس ےس ہقف ک میلعت
احلص ک۔زین رعب ادب ک میلعت آپ ےن
الوہر
ےس احلص ک
۔
دینی میلعت لمکم
ےک دعب ارگنیی میلعت ک وصحل ےک
رطف وتمہج وہےئ۔ یلع ڑگھ اکجل یم داہلخ ایل ا ، ورہفسلف
ربعاین اور اقنن یم اہمرت احلص ک۔29
1897
ءیم رکایچ
ےک ردم ۃس االالسم یم
رعب اور افریس
ےک ااتس
د
رقمر
وہےئ ۔دس
اسل دعب آپ اک
ابتدہل
یلع ڑگھ اکجل یم اٹنٹسس تیثیحب رپورسیف
۔ وہا
یلع ڑگھ اکجل
یم آپ دو اسل کت
رےہ۔اس ےک دعب آپ اک رقتر اہٰل آابد وی
وینریٹس یم وہا ۔
رپ اہجں6
اسل دترسی
ودترسی ا ور فینصت واتفیل یم
ے زگار ۔
1914
ء یم امثعہین ویوینریٹس دیحر آابد ےک
ایقم رپ آپ وک ویوینریٹس اک رپلپسن رقمر یک ایگ
۔1919ء وک آپ ےن المزتم
سے
ٰ اسی عفي
د ای
اور ونط واسپ
رشتفی ےل ےئگ۔ونط یم دمرہس اال
الصح ےس واہتسب وہےئ
۔
اس ےک ےیلآپ ےن ےئن
صنب اک
ارجاء ۔یک
ومالان رفایہ دمرۃس اال
الصح ےک اسھت ےس واہتسب ےھت نکیل المزتم ےک دوران وتق ہن وہےن رپ آپ
طخ واتکتب رپ
راہطب اجری رےتھک ےھت ۔30
آپ ےک اسا وہشمر هو ی
لکتش
ذتہ می ںعيبد الحی(م1886ء)
، ومالان یلبش امعنین( م1914ء)
،ومالان ضیف
انسحل اہسروپنری(م1887ء)
رپورسیف آرڈلن(م
1930ء ) اشلم ںیہ
۔
آپ ےک وہشم ر
اشرگدوں یم دبع ارلنمح
رگنایم(م1926ء)
،ارتخ انسح االصیح(م1958ء)
،مجن ادلنی االصیح ، دبعاالسلم رفایہ
انیم اور انسح االصیح(م1997ء )
اشلم ںیہ۔ ومالان دیمح ادلنی وک رقآن دیجم ےس وصخ یص فغش اھت اس اینبد رپ آپ ک ارثک اتکںیب ریسفت
اور اوصل ریسفت
ےس لعتم
ےہ۔
نکیل رد اسیعتیئ رپ یھب آپ ےن اکی دمعہ اتکب یھکل:1
۔ :صنری ےک رظنہی افشع اور افکرہ اک رد ہی اتکب ارگنیی وک ریمس رقار دای اور اسیعتیئ وک االخیق رقار رک ےک اس ےک دعب دوابرہ ریمس رشتعی (ینعی االسم رماد ےہ) ک اعمد وک
ریغ رضوری
رقار دای
۔ اس اتک ب یم اس رفموےض اک دملل رد یک۔3
۔
دعف الي ليبی شا ت : اپدری امعد ادلنی ( م1919
)ء
ک اتکب اقیلعتت
کے
رد یم یھکل
ےہ۔ا س یم اابثت وبنت دمحمی
اور
اانلیج ک رحتفی وک اثتب یک ےہ۔4
۔ رتاہن اجحزی
:ہی اتکب اپدری امعد
ادلنی
(م1900
)ء
اکرحتریی انمرظہ ےہ
۔وج اپدری ذموکر ےن الہ عیشت اعمل
ےک اسھت
وطخط ےک لکش یم ک یھت اور اس وک مغن
وبنطری ےک انم ےس اسیعویئں ےناشعئ یک
۔ اس ےک رد یم ہی اتکب
یھکل اس یم تمصع اایبنء،وبنت دمحم،رقآن دیجم اک اسہقب
اتکوبں ےس اموخذ ہن وہانوک اثتب یک ےہ۔5
۔
اغیپم دمحمی : اپدری اھٹرک داس (م1919
)ء
ک اتک ب
دعم رضورت رقآن
ےک رد
یم یھکل ےہ
۔ اس یم افکرہ اور ثیلثت اور اک ابلط وہان، یحیسم اور
االسیم وقانین اک اقتلب ےہ ،اقح تین رقآن وااحدثی، ابلبئ ک
طبض اور تحص درتس ںیہن ےہ ہی اسیعویئں ےک دینی ادب اثتب ک
ےہ ۔زین آپ ےن ابلبئ ےک
انتاضق ت وک اثمولں ےس واحض یک
اس ےہ۔ اتکب
اک ارگنیی
رتہمج
یھب وموجد ےہ۔
وشنم ر دمحمی ےک انم ےس
اکی اابخر اک ارجاء یھب آپ ےن
اکوپنر یم
ک سج یم
ر د اسیعتیئ
رپ فلتخم اضمنیم اشعئ وہےت ےھت۔اس ےک اسھت اسیعویئں
ےک اقعدئ ےک
ابلط وہےن
وک وخب اظرہ یک
اجات اھت
۔ ا س
اابخ ر یم اپدرویں وکیئک رمہبت انمرظوں ک خب
چ لی تش
دی
یئگ نج ےک وبقل رکےن ےس اپدری رتکاےت ےھت۔ سج ےس
ایسفنیت
ارثاملسمنں رپ ااھچ ڑپاھ۔27
اس ےک العو ہ نشمویں
ک فلتخم ی یشظ
م میتی اخےن انب رک اس یم وچبں وک اسیعیئ انب رےہ ےھت۔
رضحت
ومیگنی
ےن اسیعویئں ےک اقمےلب
ےیلےک
اکوپنر یم میتی اخہن االسہیم
ک اینبد ریھک ۔
سج یم املسمن وچبں ک
میلعت اور
تعنص ورحتف اک اامتہم یک
اجاتاھت۔ واادیمدنری 31 دنہواتسن
می
رباطن ی
دور ادتقار یم
ملسم یملکتم ک
مشن ی
سے
زمامح
ن زاب یم ضعب اسیعیئ راوبہں ک رظنہی افشع اور افکرہ ک رتددی یم یھکل ےہ۔2
۔ :وبنت
اس اتکب یم نج
اکنت
رپ ثحب ک
یئگ ان ک لیصفت ہی ےہ۔اایبنء ک
رضورت،یبن ک رعتفی،یبن اور اس ےک اویتمں یم اممتلث،یبن ک رطفت اور اصمحل،یبن ک
ااطع اتم ےک ےیل،تمصع اایبنء،اجمدہل،
افشع،زجعمہ،افشع،وضحر ﷺ ک وصخایصت۔
( ومالان رشف اقحل دصیقی دولہی م1936
ء )
رشف اقحل دصیقی
اک ہلسلس بسن ہی ےہ
۔
رشف اقحل نب الجل ادلنی
نب دبع اینغل نب خیش دبعارکلمی ےہ۔ ہلسلس بسن آےئگ اجرک
ؓاوب رکب دصقی
ےس لم اجات ےہ ۔31
رشف اقحل دصیقی ک دیپاشئ ہلحم وچڑی والن،
دیلہ یم1867
ءوک وہیئ ۔ رقآن ک میلعت
لمکم رکےن رپآپ
وکاولگنی
رعب وکسل دیلہ یم دالخ یک ایگ۔آپ ےنا طل ف نیسح احیل
(م1915
)ء ےس افر یس ک اتکںیب
ڑپھ
۔اس ےکدعب
یشنم افلض اک ااحتمن دای اور اجنپب یم اول وپزنشی احلص ک
۔ یشنم افلض
ک ااحتمن ےک دعب رصف او
ر
نح
ےک ےیل
دمرہس االالسم یم داہلخ
وہےئ۔ دویدنب اجرک یھب آپ ےن یئک بتک
میلعت احلص ک۔اذخ
دحثی ےک ےیلردیشادمح
وگنگیہ (م1905
)ء ےک درس یم رشکی وہےئ
۔ درایست ےکلیمکت ےک دعب
ذایت ذوق ےک اینبد رپ رد اسیعتیئ وک اانپ وموضع انب
ایل اور ہکم رکمہم اجرک رمح اہلل ریک
انی ےس نف انمرظہ ک میلعت احلص ک ۔اس دوران ہکم رکمہم یم
ادماد اہلل
اہمرج کم
(م1898
)ء
ےس تعیب وہےئ
۔ دنہواتسن آےن
ےک دعب
آپ ےن
رد اسیعتیئ یم وپری زدنیگ زگاری ۔ومالان رشف اقحل
وک اھٹ
زابنں رپ وبعر احلص اھت سج یم ربعاین ،
رکسنست،ویانین، ،ارگنیی، وتشپ،رعب، رتک اور
افریس اشلم یھت۔28
ونجری
1936ء وک لگنم
ےک دن
آپ ک واف ت وہیئ۔32
آپ ےک
مشہو ر
ااسذتہ یم اطلف نیسح احیل( م1915ء)،،ومالان ردیش
ادمح
وگنگیہ(م1905ء)اشلم ےہ۔ آپ ےک افینصتت ہی ےہ:1
۔ ا اصیتسل دنی وسیعی اقمبہلب دنی دمحمی2
۔ داعف التبه ي ا ن بب ش شزن ه
ارلنمح3
۔ انمرظہ دیحر آابد4
۔ انمرظہ اغزی وپر5
۔ انمرظہ اککل
رد ۔ اسیعتیئ یم آپ ےن
بہ
انمرظے ےیکسج یم دنچ ہی
ہے۔1
۔ انمرظہ اغزی وپر1883
ء2
۔ انمرظہ دیحر آ
ابد1891۔ء3
۔ انمرظہ دلھ1891
ء انمرظہ وپہن1893
ء۔
( ومالان انثء اہلل ارمرستی م1948
)ء لہنایسرراویوھاضر دنہواتسن
می
رباطن ی
دور ادتقار یم
ملسم یملکتم ک
مشن ی
سے
زمامح
ن زاب یم ضعب اسیعیئ راوبہں ک رظنہی افشع اور افکرہ ک رتددی یم یھکل ےہ۔2
۔ :وبنت
اس اتکب یم نج
اکنت
رپ ثحب ک
یئگ ان ک لیصفت ہی ےہ۔اایبنء ک
رضورت،یبن ک رعتفی،یبن اور اس ےک اویتمں یم اممتلث،یبن ک رطفت اور اصمحل،یبن ک
ااطع اتم ےک ےیل،تمصع اایبنء،اجمدہل،
افشع،زجعمہ،افشع،وضحر ﷺ ک وصخایصت۔
( ومالان رشف اقحل دصیقی دولہی م1936
ء ) قید
لحقرسبلسلہاےہندلان
لحقرم
لکربد
ارندبینغل نخیش
گئےسبلسلہے دنہواتسن
می
رباطن ی
دور ادتقار یم
ملسم یملکتم ک
مشن ی
سے
زمامح اتیوبیوبرظہافافردییھکے (ومالان انثء اہلل ارمرستی م1948
)ء
انثء اہلل
ک والدت ارمرست یم1868ء وہیئ وک ۔
وادل اک انم خیش رضح اھت ۔
نج اک آابیئ العہق
ریمشک، رسی رگن اھت ۔
اکروابر ےک ےلسلس
یم ارمرست آےئ اور
رھپ رست ارم
یم میقم وہےئگ
۔انثء
اہلل بج7
اسل ےک
وہےئوت
آپ ےک وادل
ااقتنل رک ےئگ ۔ڑبے اھبیئ
ےنروف رگی اک ہشیپ ھکسای
۔آپ ےن روفرگی
هی علت م
ت
ےک اس
اجری ریھک۔ ارمرست یم
میلعت احلص رکےن ےک دعب
آپ ےن دیلہ اک
رخ یک۔
دیلہ
ےس داراولعلم
دویدنب
اجرکیھب میلعت احلص ک۔اس ےک دعبدمرہس ضیف اعم اکوپنر
ےس وقعمالت ک اتکںیب
ڑپھ۔1892
ءوک آپ ک داتسر دنبی
وہیئ اور
ایس ہسلج یم دنوۃ ااملعلء ک اینبد یھب
ریھک یئگ ۔33
تکمیلتعلیم
رپ دمرہس اتدیئ
االالس م ارمرست یم
دمروہسےئ۔
دمرہس اتدیئ االالسم ےس
یفعتسم وہےن ےک دعب دمرہس االسہیم وکلٹ یم درس دےتی
رےہ
۔اس ےکدعب دترسی
ےس اگل وہ ےئاورارمرستواسپ ےلچ ےئگ
۔ آپ ابلط
ونتفں ےک اعتبق یم
فینصت و اتفیل
اورانمرظے رکےت رےہ۔34
1947
ء یم
اپاتسکن ےننب ےک اجنپب یم اسفدات رشوع وہےئ
۔ سج یم آپ اک اولکات اٹیب دیہش
وہا ےسسج آپ دربلداہتش وہ
ےئاور
اپاتسکن رجہت رک ےک آےئگ
۔ رفوری1948ء یم آپ
رپ افجل ےن
آکیٹ
یک سج ےک وہج
زمکور ےتہ ےئگ ۔15
امرچ1948
ء وک ااقتنل رکےئگ۔35
آپ ےک
وہشمر ااسذتہ یم دیس ذنری
(م نیسح2
0
19
)ء
احظف دبع اانملن
(م1916
)ء ،ومحمد انسحل (م1921
)ء ،ومالان ادمح اہلل (م1930
)ء ، ومالان ادمح نسح اکوپنری
(م1322
ء) اشلم
ںیہ ۔ آپ
ےن فلتخم وم
وضاعت رپ150
تک
اصتفین یھکل ںیہ۔36
اسیعتیئ ےک رد یم آپ ک اصتفین ک لیصفت ہی ےہ:1
۔ اعمرف
ارقلآن : ہی ااپندرویں
ےک رد یم
یھکل
ےہ وہنجں ےنرضحت یسیعؑ وک وضحرﷺ رپ وفتیق دےنی ک ادتسالل رق آن اور تنس
سے ک
۔
سج ےک رد یم فنصم ےن ہی اتکب یھکل
۔2۔ :اابثت اوتلدیح
ي لی ث ’’ےک رد یم
اکی اپدری ک اتکب‘‘ اابثت الی آپ ےن
لکھی 32 ا رمقل، دلج6
، امشر ہ1
، (ونجر ی–
امر چ 3
202
)
لطایتااحمدےویوسیحیی (
j
p )
5Muhammad Asʻad Thānvī, Research Review of Religious and Academic Services of Moulānā
Qāsim (Karachī: Karachi University, 2005), 29-34 ۔ Qāsim (Karachī: Karachi University, 2005), 29-34 ۔
6Thānvī, Research Review of Religious and Academic Services of Moulānā Qāsim, 34-51. ,
g
Qāsim (Karachī: Karachi University, 2005), 29-34 ۔
6Thānvī, Research Review of Religious and Academic Services of Moulānā Qāsim, 34-51. References 1Abū al-Hassan Nadvī, Seerat Muhammad ʻAlī Moungeerī (Karachi: Majlis Nashriyāt-e-Islām,
1998), 37-38. 1Abū al-Hassan Nadvī, Seerat Muhammad ʻAlī Moungeerī (Karachi: Majlis Nashriyāt-e-Islām,
1998), 37-38. )
2 Aseer Adrawī, Mujāhid Islām Rahmatullah keyranwī (Lahore: Dār al-kutub, 2015), 52-60. )
2 Aseer Adrawī, Mujāhid Islām Rahmatullah keyranwī (Lahore: Dār al-kutub, 2015), 52-60. 3 Molānā Habīb-ur-Raḥmān Aʻzmī,”Mahnāma.” Maʻārif 21, no. 4 (AʻzamGarh): 147-158. ḥ
,
,
(
4Imdādulla Sābrī, Āsār-e-Raḥmat (Madāris: Marifat Tāj book Depū), 2-18 ۔ ḥ
(
j
p )
5Muhammad Asʻad Thānvī, Research Review of Religious and Academic Services of Moulānā
Qāsim (Karachī: Karachi University, 2005), 29-34 ۔ 33 دنہواتسن
می
رباطن ی
دور ادتقار یم
ملسم یملکتم ک
مشن ی
سے
زمامح دنہواتسن
می
رباطن ی
دور ادتقار یم
ملسم یملکتم ک
مشن ی
سے
زمامح 7Adrawī, Mujāhid Islām Rahmatullah keyranwī, 84. 7Adrawī, Mujāhid Islām Rahmatullah keyranwī, 84. j
y
8Adrawī, Mujāhid Islām Rahmatullah keyranwī, 84-96. j
y
8Adrawī, Mujāhid Islām Rahmatullah keyranwī, 84-96. j
y
9 Adrawī, Mujāhid Islām Rahmatullah keyranwī, 78. j
y
9 Adrawī, Mujāhid Islām Rahmatullah keyranwī, 78. j
y
10Adrawī, Mujāhid Islām Rahmatullah keyranwī, 126. j
y
10Adrawī, Mujāhid Islām Rahmatullah keyranwī, 126. j
y
11 Adrawī, Mujāhid Islām Rahmatullah keyranwī, 132-258. 11 Adrawī, Mujāhid Islām Rahmatullah keyranwī, 132-258. j
y
12 Altāf Hussain Ḥālī, Ḥayāt-e-Jāwed (Kashmīr: Arslān book), 1:1-142. 12 Altāf Hussain Ḥālī, Ḥayāt-e-Jāwed (Kashmīr: Arslān book), 1:1-142. 13 Ḥālī, Ḥayāt-e-Jāwed, 170-384. y
14 Iʻnāyat Rasool ChiryaKūtī, Bushrā (Dhaka, 1938), 18. 14 Iʻnāyat Rasool ChiryaKūtī, Bushrā (Dhaka, 1938), 18. 15ChiryaKūtī, Bushrā, 19-26. 15ChiryaKūtī, Bushrā, 19-26. y
16 ChiryaKūtī, Bushrā, 26. 17 ChiryaKūtī, Bushrā, 25-26. 18Sābrī, Farangyio kā Jāl, 520-521. 18Sābrī, Farangyio kā Jāl, 520-521. 19Sābrī, Farangyio kā Jāl, 521-525. 20Sābrī, Farangyio kā Jāl, 526. gy
J
21Abd al-Hayī Hasnī, Nizahut al-Khawātar (Beirūt: Dār Ibn Hazam 1999), 8:1264. gy
21Abd al-Hayī Hasnī, Nizahut al-Khawātar (Beirūt: Dār Ibn Hazam 1 22Nadvī, Seerat Muhammad ʻAlī Moungeerī, 6. 22Nadvī, Seerat Muhammad ʻAlī Moungeerī, 6. 23 Nadvī, Seerat Muhammad ʻAlī Moungeerī, 7-26. 23 Nadvī, Seerat Muhammad ʻAlī Moungeerī, 7-26. 24Nadvī, Seerat Muhammad ʻAlī Moungeerī, 115-290 6Nadvī, Seerat Muhammad ʻAlī Moungeerī, 417. 27 Nadvī, Seerat Muhammad ʻAlī Moungeerī, 37-79. g
28Maulānā Sharaf al-Dīn, Zikr-e-Farāhī (Lahore: Dār al-Tazkīr, 2002), 1:39. 28Maulānā Sharaf al-Dīn, Zikr-e-Farāhī (Lahore: Dār al-Tazkīr, 2002), 1:39. 28Maulānā Sharaf al-Dīn, Zikr-e-Farāhī (L 29Sharaf al-Dīn, Zikr-e-Farāhī, 111-195. 30Anas Nazr, Ḥameed al-Dīn Farāhī and the Research and Comparative Study of Principles of 30Anas Nazr, Ḥameed al-Dīn Farāhī and the Research and Comparative Study of Principles of
Democracy, 46-61. Democracy, 46-61. 31Sābrī, Farangyio kā Jāl, 495. 32Sābrī, Farangyio kā Jāl, 392 - 406. 33Fazal-ur-Raḥmān Ibn Miān Muhammad, Ra’ees al-Munāzreen Maulānā Sanāullah Amrtasrī 33Fazal-ur-Raḥmān Ibn Miān Muhammad, Ra’ees al-Munāzreen Maulānā Sanāullah Amrtasrī
(Lahore: Muttabaʻt al-ʻArabiya, 1987), 60:68. 33Fazal-ur-Raḥmān Ibn Miān Muhammad, Ra’ees al-Munāzr (Lahore: Muttabaʻt al-ʻArabiya, 1987), 60:68. (
y
)
34Abd al-Majeed, Sīirat Sanā'ī (Lahore: Maktaba Qudūsiya, 1989 (
y
)
34Abd al-Majeed, Sīirat Sanā'ī (Lahore: Maktaba Qudūsiya, 1989), 1:121. j
(
y
)
35Ibn Miān Muhammad, Ra’ees al-Munāzreen Maulānā Sanāullah Amrta 35Ibn Miān Muhammad, Ra’ees al-Munāzreen Maulānā Sanāullah Amrtasrī, 279-283. 36Abd al-Majeed, Sīirat Sanā'ī, 244. 36Abd al-Majeed, Sīirat Sanā'ī, 244. دنہواتسن
می
رباطن ی
دور ادتقار یم
ملسم یملکتم ک
مشن ی
سے
زمامح j
37Ibn Miān Muhammad, Maulānā Sanāullah Amrtasrī, 183. j
37Ibn Miān Muhammad, Maulānā Sanāullah Amrtasrī, 183. 38Raḥmatullah keyranwī, Azālat al-Shkūk, ed. Maulana ʻAtāq Ahmad Bastavī, 166. 39keyranwī, Azālat al-Shkūk, 165. 34
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Stradivari’s Varnish Revisited: Feature Improvements Using Chemical Modification
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Article
Stradivari’s Varnish Revisited: Feature Improvements Using
Chemical Modification Maduka L. Weththimuni 1,2,*
, Giacomo Fiocco 2,3, Chiara Milanese 1
, Alberto Spinella 4,
Maria Luisa Saladino 5
, Marco Malagodi 2,3
and Maurizio Licchelli 1,2,* 1
Department of Chemistry, University of Pavia, Via T. Taramelli 12, 27100 Pavia, Italy; chiara.milanese@unipv.it
2 Department of Chemistry, University of Pavia, Via T. Taramelli 12, 27100 Pavia, Italy; chiara.milanese@unipv.it
2
Research Center for the Conservation of Cultural Heritage (CISRiC), University of Pavia, Via A. Ferrata 3,
27100 Pavia, Italy; giacomo.fiocco@unipv.it (G.F.); marco.malagodi@unipv.it (M.M.) p
y
y
y
2
Research Center for the Conservation of Cultural Heritage (CISRiC), University of Pavia, Vi
27100 Pavia, Italy; giacomo.fiocco@unipv.it (G.F.); marco.malagodi@unipv.it (M.M.) Research Center for the Conservation of Cultural Heritage (CISRiC), University of Pavia, Via A. Ferrata 3,
27100 Pavia, Italy; giacomo.fiocco@unipv.it (G.F.); marco.malagodi@unipv.it (M.M.) 3
Department of Musicology and Cultural Heritage, University of Pavia, Corso Garibaldi 178,
26100 Cremona, Italy 4
Advanced Technologies Network Center, University of Palermo, Viale delle Scienze, Ed. 18,
90128 Palermo, Italy; alberto.spinella@unipa.it 5
Department of Biological, Chemical, and Pharmaceutical Sciences and Technologies (STEBICEF), 5
Department of Biological, Chemical, and Pharmaceutical Sciences and Technologies (STEBICEF),
University of Palermo, Viale delle Scienze, Ed. 17, 90128 Palermo, Italy; marialuisa.saladino@unipa.it University of Palermo, Viale delle Scienze, Ed. 17, 90128 Palermo, Italy; marialuisa.saladino@unipa.it
Correspondence: madukalankani.weththimuni@unipv.it (M.L.W.); maurizio.licchelli@unipv.it (M.L.) y
y
p
*
Correspondence: madukalankani.weththimuni@unipv.it (M.L.W.); maurizio.licchelli@unipv.it (M.L. Abstract: The most widespread varnish formulations used by master violin-makers of the “Italian
Golden Age”, including Antonio Stradivari, were based on mixtures of siccative oils (e.g., linseed
oil) and natural resins (e.g., colophony). Similar formulations are still used for the finish of con-
temporary instruments. Although most precious violins made by Stradivari and other Cremonese
Masters are kept in museums, several instruments are still played and their finish may undergo
deterioration due to contact with the players. Moreover, the decay of the traditional varnish may
occur due to mechanical stress and natural aging caused by environmental agents (e.g., exposure to
uncontrolled light, humidity, and temperature changes). The main aim of this research work is to
investigate the possible improvement of varnish resistance to the decay induced by different aging
processes. For this purpose, the traditional varnish (linseed oil/colophony 3:1 w/w) was recreated
in the laboratory following an ancient recipe and then it was functionalized with a cross-linking
agent (3-Glycidyloxypropyltrimethoxysilane, GLYMO). Citation: Weththimuni, M.L.; Fiocco,
G.; Milanese, C.; Spinella, A.;
Saladino, M.L.; Malagodi, M.;
Licchelli, M. Stradivari’s Varnish
Revisited: Feature Improvements
Using Chemical Modification. Polymers 2023, 15, 3652. https://
doi.org/10.3390/polym15173652
Academic Editors: Jesús-María
García-Martínez and Emilia P. Collar
Received: 7 August 2023
Revised: 28 August 2023
Accepted: 30 August 2023
Published: 4 September 2023 Citation: Weththimuni, M.L.; Fiocco,
G.; Milanese, C.; Spinella, A.;
Saladino, M.L.; Malagodi, M.;
Licchelli, M. Stradivari’s Varnish
Revisited: Feature Improvements
Using Chemical Modification. Polymers 2023, 15, 3652. https://
doi.org/10.3390/polym15173652 Keywords: Stradivari; violin varnishes; linseed oil/colophony; SEM-EDS; NMR Academic Editors: Jesús-María
García-Martínez and Emilia P. Collar Received: 7 August 2023
Revised: 28 August 2023
Accepted: 30 August 2023
Published: 4 September 2023 Article
Stradivari’s Varnish Revisited: Feature Improvements Using
Chemical Modification Plain and functionalized varnishes under-
went artificial aging (UV light, temperature, and humidity variations), and their properties were
comparatively studied using different techniques. All the results suggest that the functionalized
varnish displays improved resistance to the aging process and particularly enhanced photostability
and increased hardness (resistance to scratches). 1. Introduction String musical instruments are traditionally coated with natural varnishes (of animal
or vegetable origin), which protect them from light, dust, humidity, and climatic changes
and improve the mechanical properties and aesthetical appearance of their surface [1]. Moreover, the existence of a direct relationship between the composition of the varnish and
the quality of the sound produced by the instruments is almost certain [2,3]. 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/). q
y
p
y
Ancient instrument-makers used three main varnish types, commonly named spirit
varnishes, essential oil varnishes (volatile oil), and oil varnishes (fixed oil-non-volatile) [1,4]. Since the 16th century, the principal binders for the preparation of varnishes have been
siccative vegetable oils because of their unique physico-chemical properties and good
performances in the surface finish (e.g., high gloss and transparency) [5]. Siccative oils are
mainly made by triglycerides of unsaturated fatty acids, i.e., oleic, linoleic, and linolenic,
which contain one, two, and three double bonds, respectively [6]. Upon exposition to https://www.mdpi.com/journal/polymers Polymers 2023, 15, 3652. https://doi.org/10.3390/polym15173652 Polymers 2023, 15, 3652 2 of 22 air, they undergo a chemical reaction, known as auto-oxidation [7], involving the sponta-
neous addition of oxygen to the unsaturated acyl chains to form unstable hydroperoxide
compounds. Radical species originating from hydroperoxide decomposition promote a
variety of further chemical processes, including intermolecular couplings, which induce
the formation of a polymeric network responsible for the drying of the oil films [6,8]. Several research studies suggest that the most widespread varnish formulations used
by master violin-makers, including Stradivari, for the finishing of bowed musical instru-
ments were based on mixtures of linseed oil (the most popular siccative oil) and natural
resins, especially Pinaceae resins (e.g., colophony), in the period between the 17th and 18th
century (the so-called Golden age of the Italian luthiers) [1,9,10]. Colophony is a natural product mainly consisting of various resin acids, especially
abietic acid [11,12], which has been widely used as a component of varnishes due to its
excellent properties (fast drying, excellent solubility and compatibility with other resins
and oils, and ready availability). Fascinated by the uniqueness of instruments made
by the great Masters of the past, particularly Stradivari’s violins, contemporary luthiers
have rediscovered the goodness of historical varnishes, also experimenting with oil/resin
mixtures of different compositions. These mixtures have been studied in recent years, and
the results have shown that the composition of 3:1 (oil:resin) would still provide a varnish
displaying the highest quality and durability, especially in the case of the ancient musical
instruments still played today [13]. Some researchers have focused their interest on this field to address some conservation
issues concerning historical musical instruments. Several of them, particularly the precious
violins made in the 17th and 18th centuries (e.g., by Stradivari and other Cremonese Mas-
ters), even if kept in controlled museum environments, are still played, and may undergo
deterioration due to different factors: (i) contact with the violinist’s skin and perspiration
(pH < 7), (ii) exposure to sunlight and in particular to UV components, humidity, and
temperature changes. As a consequence, these phenomena may cause drastic damage
(exfoliation, particle removal, fading, or browning) to the varnish layer [4]. Therefore, appropriate solutions or methods to overcome varnish declines and to
better protect these precious musical instruments are highly desirable. The historical
instruments under consideration represent an invaluable heritage from a cultural, artistic,
and economical point of view. In addition, research studies addressing how to avoid or
reduce the weaknesses of Stradivari’s varnish could be of great help to contemporary
violin-makers who use the traditional finishing method. In fact, the main aim of this research work is to perform chemical modifications of the
so-called Stradivari’s varnish to improve some of its properties (e.g., hardness, photostabil-
ity) without affecting its good finish performance. In particular, the effect of additional cross-
linking induced using an epoxysilane derivative (3-Glycidyloxypropyltrimethoxysilane,
GPTMS also known as GLYMO) has been investigated. g
GLYMO is a bifunctional organosilane containing a reactive organic epoxide and
hydrolyzable methoxysilane groups. The dual nature of its reactivity allows it to chemically
bind to both inorganic and organic compounds, thus acting as a cross-linking agent and/or
surface modifier. In the present work, Stradivari’s varnish was prepared in the laboratory following an
ancient recipe [14,15], which also involves the heating of linseed oil and colophony at quite
a high temperature (270 ◦C). The mixture was then treated with GLYMO to obtain chemical
functionalization. Furthermore, both plain linseed oil and colophony were also reacted
separately with the same epoxysilane to better understand the possible reaction(s) taking
place in the mixture. After application to wood specimens in the laboratory, the properties
and performances of the modified varnish were studied in comparison with the traditional
one using a wide range of techniques, such as chromatic variations and contact angle
measurements, micro Fourier-transform infrared spectroscopy (µ-FTIR), optical microscopy
(OM), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS),
thermogravimetric analysis and differential scanning calorimetry (TGA and DSC), nuclear Polymers 2023, 15, 3652 3 of 22 magnetic resonance spectroscopy (NMR), and hardness measurements. 2.1. Materials Linseed oil (n73100 Kremer, Kremer Pigmente GmbH & Co. KG, Aichstetten, Germany)
containing Mn-based catalyst (to accelerate the drying process) and colophony were pur-
chased from Kremer-Pigmente (Aichstetten, Germany). Both products were used without
any further purification for the preparation of the investigated varnish (75/25 w/w, linseed
oil/colophony). Moreover, 3-glycidyloxypropyltrimethoxysilane (GLYMO, ≥98%), dibutyltindi-
laurate (C32H64O4Sn, DBTDL, 95%), anhydrous ethanol (C2H5OH, ≥99.8%,) and deuterated
chloroform (CDCl3, 99.8 atom% D) were supplied by Sigma-Aldrich (St. Louis, MO, USA);
dichloromethane (CH2Cl2, 99.5%, stabilized with about 0.2% C2H5OH) was provided by Carlo
Erba (Milan, Italy); and n-octane (C8H18, 95%) was purchased from Sigma-Aldrich (St. Louis,
MO, USA) and used for cleaning the brushes between paint applications. 3 g
p
pp
The maple wood specimens (5 × 5 × 0.5 cm3) used in the present work were kindly
provided by Civica Scuola di Liuteria (Milan, Italy), while the brushes (Habico-204 AK,
Cremona tools, Cremona, Italy) with wild boar bristles for the application of paints on
wood and glass slides were purchased at Cremona-Tools. Abrasive papers (P400 and P800
Abranet ®, Mirka Italia srl, Tuscany, Italy) were used for cleaning the wood specimens. The behavior of
varnishes (traditional and modified) was investigated after exposing coating films and
coated wood specimens to artificial aging processes induced by UV irradiation and thermal
treatment. A proper comparison of results obtained on aged and unaged materials suggests
that the addition of limited amounts (about 5%) of GLYMO to the traditional varnish
induces considerable improvements in its performance, including enhanced resistance to
mechanical decay and to aging. 2.2.1. Preparation and Application of Varnishes and Other Materials 2.2.1. Preparation and Application of Varnishes and Other Materials Stradivari’s varnish (72/25: linseed oil, L/colophony, C) was prepared in the labora-
tory following an ancient recipe [14,15] as previously described [4] and named varnish LC. The silane-modified material named varnish LCS was prepared by reacting varnish LC
with GLYMO according to the following procedure, which was selected after conducting
several trials and according to our previous experience with different varnish modifica-
tions [16]: 3-glycidyloxypropyltrimethoxysilane (1.5 mL) was added into a round-bottom
flask containing varnish LC (30.0 mL) and then the reaction mixture was heated until 60 ◦C
in the presence of dibutyltindilaurate (DBTDL, 0.09 mL), which acts as a catalyst. The
reaction was carried out under an N2 atmosphere for 24 h. At the end of the reaction,
varnish LCS was collected and stored in a glass bottle. In addition to the preparation of the two main varnishes (LC and LCS), pure com-
ponents of LC were also reacted with epoxysilane to study its possible reactions with oil
components as well as with colophony. The resulting compounds were named LS and
CS, respectively. Material LS was obtained by reacting plain oil L with epoxysilane according to a
similar procedure to that conducted for LCS. For the preparation of functionalized colophony (CS), colophony (5.0 g) was dissolved
in anhydrous ethanol (1:2 volume), then GLYMO (0.25 mL) was added, and the reaction
was performed according to the experimental conditions described for LCS. The solid
product was recovered after solvent evaporation at room temperature under a fume hood
for several days. Varnish films (LC and LCS) were prepared on glass slides (microscope slides) using
flat brushes made from boar hair because these kinds of varnishes are self-leveling. In order
to obtain the correct thickness of the film, five consecutive applications (five layers) were
performed after drying each layer. The full drying of films of varnishes was obtained after Polymers 2023, 15, 3652 4 of 22 7 days at room temperature (20 ± 2 ◦C) and 2 days under a UV lamp (UV box: two lamps
of Philips TL-D 36W BLB with an emission peak at 360 nm). Thirty-six varnish films were
prepared in both kinds of varnishes, and for each experimental analysis, three films of the
same treatment were used to obtain the correct results. 2.2.2. Instruments and Techniques Infrared spectra were collected using both a PerkinElmer Spectrum 100 FT-IR in-
strument (Perkin-Elmer, Waltham, MA, USA), equipped with a universal ATR accessory
(Diamond crystal), and a Nicolet iN10 Thermo Fischer µ-FT-IR spectrometer (Thermo-
Fisher, Waltham, MA, USA) in ATR mode (Germanium crystal). The thermogravimetric
measurements (TGA) were performed with a Q5000 (TA Instruments, New Castle, DE,
USA) system under an air flow (10 mL/min) in a platinum sample holder. Here, a few mil-
ligrams of samples were heated from 25 ◦C to 1000 ◦C at a rate of 10 ◦C/min. Moreover, the
scanning differential calorimetry analysis (DSC) was performed with a Q2000 instrument
(TA Instruments) by heating a few milligrams of sample in an open aluminum crucible
from −90 ◦C to 300 ◦C, at a speed of 10 ◦C/min, under air flow (50 mL/min). Chromatic
variations were measured using a Konica Minolta (Konica Minolta, Inc., Tokyo, Japan)
CM-2600d spectrophotometer, determining the L*, a*, and b* coordinates of the CIELAB
space and the global chromatic variations ∆E* according to the UNI EN 15886 protocol [19]. Five measurements on each wood specimen were carried out, and the collected results are
average values from fifteen measurements (three different wood specimens were used for
each treatment) taken before treatment, after treatment, and after aging. Water contact angle measurements were carried out using a Lorentzen and Wettre
instrument (Zurich, Sweden) according to the UNI EN 15802 Protocol [20]. The results
were collected as average values by taking 15 different measurements for each kind of
treatment (three different wood specimens were used for each coating). The optical mi-
croscope observations were conducted using a light polarized microscope, equipped with
an Olympus TH4-200 lamp (visible light). Scanning electron microscopy (SEM) images
(backscattered electron) and energy dispersive X-ray spectra (EDS) were collected by using
a Tescan FE-SEM, MIRA 3XMU series (Tescan, Brno, Czech Republic) equipped with a
Schottky field emission source, operating in both low and high vacuums and located at
the Arvedi Laboratory, CISRiC-University of Pavia, Italy. Before SEM analysis, samples
were gold-sputtered using a Cressington sputter coater 208HR (Ted Pella, Inc., Redding,
CA, USA). Hardness measurements were determined by pencil test according to the ISO standard
(ISO 15184:1998) [21], and the obtained results were average values from three different
measurements (three different wood samples of each treatment were used for the test). 2.2.1. Preparation and Application of Varnishes and Other Materials Furthermore, both varnishes (LC and LCS) were applied to previously cleaned (using
two kinds of abrasive papers: P 400 and P 800) maple wood specimens (48 wood samples)
using the same brushes (flat brushes made from boar hair) according to the real cases,
as explained clearly in our previous papers [17,18]. To obtain the correct thickness of
the coating, eight applications were carried out in perpendicular directions for the wood
specimens, and the drying process was conducted in the same way as in the varnish
film preparation. Moreover, three different wood specimens of each treatment (LC and
LCS) were used for each experimental analysis in order to calculate the average value of
the measurements. 2.2.3. NMR Experiments For liquid-state NMR, all samples were dissolved in deuterated chloroform (CDCl3). The spectra were recorded at 300 K by means of a Bruker Avance II 400 spectrometer
operating at 400.15 and 100.63 MHz for the 1H and 13C nuclides, respectively, equipped
with an inverse broadband (BBI) probe. 1H spectra were acquired using a 12.15 µs pulse, a delay time of 3 s, and 16 scans. Polymers 2023, 15, 3652 5 of 22 Solid-state NMR experiments were performed using the spectrometer described above
using a 4 mm H-X CPMAS probe. 13C CPMAS spectra were acquired with an MAS spinning
speed of 8 kHz, 1024 scans, a contact time of 1.5 ms, a delay time of 3 s, and a 1H pulse
of 4.5 µs. The optimization of the Hartmann–Hahn condition was obtained using an
adamantane standard sample. This compound was also used as an external chemical shift
reference. All samples were placed in zirconia rotors sealed with KEL-F caps. The 1H spin-lattice relaxation time in the rotating frame (T1ρH) was obtained using
the variable spin lock (VSL) sequence using spin-lock pulses ranging from 0.1 to 7.5 ms
with a spin lock B1 field of 57.6 kHz and a contact time of 1.5 ms. p
The 13C spin-lattice relaxation time in the rotating frame (T1ρC) was obtained using
the variable spin lock (VSL) sequence using spin-lock pulses ranging from 0.4 to 30 ms and
a contact time of 1.5 ms. 2.2.4. Artificial Aging of the Samples When completely dried, the treated wood samples (36 specimens; 18 from each treat-
ment) and glass slides (24 varnish films; 12 from each treatment) were subjected to two dif-
ferent types of artificial aging processes (UV irradiation and thermal/humidity treatment). UV aging was conducted (for 720 h) using an irradiation system (Helios Italquartz, Milan,
Italy) equipped with two mercury lamps (power: 15 W), as previously reported [18,22,23],
and thermal/humidity aging was conducted in a climatic chamber at a temperature (T) of
45 ◦C with 28% relative humidity (RH) for 720 h (1 month) [2,24]. 3.1. Preparation of Plain and Modified Stradivari’s Varnishes 3.1. Preparation of Plain and Modified Stradivari’s Varnishes Different investigations carried out over the past few decades suggested that linseed
oil and colophony (or equivalent compounds) were the main components of varnishes
applied by the greatest violin-makers of the past, and particularly by Stradivari, to their
precious instruments [1,12]. Among the different composition ratios proposed based on
the experimental results for investigated varnishes, the 75/25 w/w (oil/resin) ratio has
been hypothesized as the one providing the most homogeneous coating. Such a mixture
composition roughly corresponds to a 1:1 stoichiometric ratio, if tri-linolenic glyceride (1)
and abietic acid (2, Scheme 1) are taken as representative compounds for linseed oil and
colophony, respectively [25]. Scheme 1. Formulae of tri-linolenic glyceride (1) and abietic acid (2). Scheme 1. Formulae of tri-linolenic glyceride (1) and abietic acid (2). According to ancient recipes [14,15], blending linseed oil with colophony requires
prolonged heating (up to 3 h) at quite a high temperature (up to 270 ◦C), which induces
chemical modifications of diterpenic acids in colophony and (poly)unsaturated triglycerides
in linseed oil [26–28], including isomerization and oxidation as well as the partial hydrolysis
of triglycerides [29]. In particular, oxidation processes may generate ketones, aldehydes,
alcohols, and carboxylic acid functionalities [6,8,30–32]. Moreover, esterification involving
carboxylic derivatives of resin and diglycerides formed by the partial hydrolysis of oil
components has been reported [33–36]. Following this “cooking step,” varnish can be used for application on the wood
surface. Its exposition to air and light induces the well-known curing process due to a quite Polymers 2023, 15, 3652 6 of 22 complex radical mechanism that involves unsaturated chains of fatty acids and dioxygen
and provides the final coating [6,8,33,35]. complex radical mechanism that involves unsaturated chains of fatty acids and dioxygen
and provides the final coating [6,8,33,35]. The introduction of the silane coupling agent into the siccative oil/resin mixture
mainly aims to induce an additional cross-linking in the varnish matrix besides the above-
mentioned curing process. It is expected that such an additional chemical process provides
an improvement of specific varnish features, such as hardness and resistance to aging. Hence, epoxysilane can promote two different reactions involving the varnish com-
ponents: (i) epoxy groups of silane undergo oxyrane ring opening by carboxylic groups,
with the consequent formation of an ester bond (Scheme 2). 3.2. Characterization of Varnish Materials before and after Aging 3.2. Characterization of Varnish Materials before and after Aging 3.1. Preparation of Plain and Modified Stradivari’s Varnishes In the oil/resin mixture, free
COOH functions can be provided by colophony components (e.g., abietic acid, 1) by free
fatty acids, and by the other possible oxidized compounds formed during the heating of
the oil/colophony mixture; (ii) hydrolysis/condensation of the trimethoxysilane groups
(Scheme 3). Reaction (i) is performed in quite mild experimental conditions (60 ◦C, 24 h)
in the presence of a catalyst (DBTDL) and allows us to introduce trimetoxysilane groups
into different chemical components of the mixture. It should be noted that this reaction
is carried out in the N2 atmosphere in order to prevent the oxygen-induced curing of the
varnish at this stage. Scheme 2. Possible reaction involving carboxylic groups of varnish components with epoxysilane
(RCOOH = abietic acid or similar component of resin, unsaturated fatty acid, or oxidation product of
triglycerides). Scheme 2. Possible reaction involving carboxylic groups of varnish components with epoxysilane
(RCOOH = abietic acid or similar component of resin, unsaturated fatty acid, or oxidation product of
triglycerides). In principle, the hydrolysis of trimethoxysilane groups and their further condensation
can occur, at least partly, during the functionalization step, although the availability of
water in such an experimental environment should be very limited. Therefore, it is expected
that silane-induced cross-linking mainly occurs after the exposition of varnish to air (and
ambient humidity), when the conventional curing process takes place. Moreover, epoxy
groups that possibly have not reacted in the functionalization step are still allowed to inter-
act with carboxylic groups that form due to oxidation processes during the conventional
curing step. The products formed owing to alcoxysilane condensation that cause additional cross-
linking can be countless. The process illustrated in Scheme 2 represents just an example. In
addition, a further reaction between methoxysilane side-chains and -OH groups belonging
to cellulose or lignin cannot be ruled out after the application of varnish on the wood
surface [36]. 7 of 22 Polymers 2023, 15, 3652 Scheme 3. Example of possible condensation reaction involving trimethoxysilane groups introduced
on different components of the varnish (RCOO groups deriving from abietic acid or similar component
of resin, unsaturated fatty acid, or oxidation product of triglycerides). 3.2.1. Optical Microscopy Analyses Films of the original Stradivari varnish (LC) and of the modified one (LCS) were
prepared on glass slides according to the traditional curing procedure (see Section 2.2.1)
and observed using an optical microscope before and after the artificial aging process
to point out the possible morphological differences between the two materials and any
variations induced by aging processes. Before aging, the films of the two different varnishes
are comparable to each other both when observed by the naked eye and by microscope. In particular, it was not possible to notice any differences from the homogeneity point of
view (Figure 1a,b). However, the films of varnish LC show extensive scratches on the entire
surface after exposition to UV irradiation (Figure 1c), and a more drastic deterioration was
observed after the thermal aging (Figure 1e). In this last case, the varnish film displays
a particularly inhomogeneous aspect with the presence of several aggregates. On the
contrary, the films of functionalized varnish LCS do not undergo significant alterations as
its the surface still appears smooth and homogeneous even after aging (Figure 1d,f). Polymers 2023, 15, 3652 8 of 22 Figure 1. Optical microscope images of varnish LC and varnish LCS films (left and right side,
respectively): (a,b) before aging, (c,d) after UV aging and (e,f) after thermal aging. Figure 1. Optical microscope images of varnish LC and varnish LCS films (left and right side,
respectively): (a,b) before aging, (c,d) after UV aging and (e,f) after thermal aging. Figure 1. Optical microscope images of varnish LC and varnish LCS films (left and right side,
respectively): (a,b) before aging, (c,d) after UV aging and (e,f) after thermal aging. 3.2.2. FTIR Investigations The FTIR analyses were carried out on coating films to highlight the chemical mod-
ifications induced by functionalization on the original components (L and C) and on
varnish LC. Figure 2 compares the spectra of linseed oil and linseed oil after the reaction with
epoxysilane (L and LS, Figure 2a); of pure rosin and functionalized rosin (C and CS,
Figure 2b); and of the two varnishes (LC and LCS, Figure 2c). Looking closely at the
fingerprint area, some differences can be observed, particularly in the region between 1100
and 1000 cm−1. The new peaks in the spectra of silane-functionalized samples, which do not
appear in the corresponding plain materials, could be ascribed to the trimethoxysilyl groups
(Si-O-C bonds) or to possible siloxane functions (Si-O-Si bonds) [37–40]. The spectrum of
plain epoxysilane (GLYMO) is reported in Figure S1 for comparison. 9 of 22 Polymers 2023, 15, 3652 Figure 2. FTIR spectra of original and functionalized materials: (a) L and LS, (b) C and CS, and
(c) varnish LC and varnish LCS. Figure 2. FTIR spectra of original and functionalized materials: (a) L and LS, (b) C and CS, and
(c) varnish LC and varnish LCS. Figure 2. FTIR spectra of original and functionalized materials: (a) L and LS, (b) C and CS, and
(c) varnish LC and varnish LCS. The presence of Si-O-C and Si-O-Si bonds would confirm the reaction between car-
boxylic groups and epoxide as well as the possible subsequent condensation of the alkoxysi-
lane groups in the presence of the DBTDL catalyst. 3.2.3. TGA and DSC Measurements The decomposition process observed for film L started at 101 ◦C and end at 475 ◦C, in
accordance with previously published data [4], while the degradation of the functionalized
sample LS is observed up to 500 ◦C (Figure S2). Moreover, the percentages of weight
loss after decomposition correspond to 94.8% and 86.4% for L and LS, respectively. The
plain colophony sample (C) decomposed between 122 ◦C and 300 ◦C, with a final weight
loss of 100.0%, in agreement with what was previously reported [4], while the heating of
CS induced two decomposition processes in succession: the first was more substantial,
in the 80–316 ◦C range, and generated a weight loss of 83.4%, while the second was
between 316 ◦C and 450 ◦C and showed an overall weight loss of 98.8% (Figure S3). The
two subsequent decompositions can be explained by considering that colophony is only
partially functionalized with GLYMO: the most abundant unreacted fraction behaves
similarly to plain C, while the less abundant silane-functionalized fraction is more resistant
to thermal decomposition. p
These data confirm that siccative oil and rosin react with GLYMO during the func-
tionalization process, both as isolate components and when they are blended to form the
so-called Stradivari varnish. In any case, the introduction of silane functionalities into their
structures significantly affects the thermal behavior of the resulting materials. In particular,
varnish LCS displays an improvement in resistance to heat, which can be ascribed to the
positive combination of the silane-induced cross-linking and the conventional siccative oil
curing process. Differential scanning calorimetry (DSC) analyses were also carried out on samples
of varnish films (LC and LCS) as well as on plain and functionalized components. In fact,
it is expected that structural modifications affect the phase transitions of the investigated
materials and particularly the glass transition temperature (Tg), i.e., the temperature at
which the varnish (or the resin) changes from a rigid glassy material to a soft one. The
Tg values are summarized in Table 1. It should be noted that all materials obtained after
the GLYMO-induced modification are characterized by Tg values significantly higher
than the corresponding unmodified compounds. Specifically, varnish LCS undergoes
glass transition at about 40 ◦C, while the traditional varnish LC has a Tg value of about
17 ◦C, indicating that the varnish modification provided a relevant increment to the glass
transition temperature (the value more than doubled) of the not-modified varnish. 3.2.3. TGA and DSC Measurements Film samples of the considered varnishes (LC and LCS), prepared using the traditional
curing process, were also investigated using TGA and DSC to assess the possible variations
caused by functionalization on the thermal behavior of the Stradivari varnish and of
its modified analog. Thermogravimetric analysis (temperature increasing from 25 ◦C
to 1000 ◦C) showed that varnish LC undergoes degradation in the 102–460 ◦C range,
suffering an overall weight loss of 93.5%, while the functionalized LCS material decomposes
between 106 ◦C and 500 ◦C, with a mass loss of 86.9% (Figure 3). These data suggest that
the introduction of GLYMO into the varnish matrix caused a small improvement in the
resistance to heat degradation (around a 7% difference in weight loss), which could be
related to the additional curing process promoted by the epoxysilane additive. TGA
experiments were also performed on film samples obtained by plain linseed oil (L) and
colophony (C), as well as on their derivatives obtained after the reaction with GLYMO (LS
and CS, respectively), in order to verify if the single components of the Stradivari varnish
are affected by the functionalization with epoxysilane. 10 of 22 10 of 22 Polymers 2023, 15, 3652 Figure 3. Thermogram (TGA) of varnishes LC and LCS. The decomposition process observed for film L started at 101 ◦C and end at 475 ◦C, in
accordance with previously published data [4], while the degradation of the functionalized
sample LS is observed up to 500 ◦C (Figure S2). Moreover, the percentages of weight
loss after decomposition correspond to 94.8% and 86.4% for L and LS, respectively. The
plain colophony sample (C) decomposed between 122 ◦C and 300 ◦C, with a final weight
loss of 100.0%, in agreement with what was previously reported [4], while the heating of
CS induced two decomposition processes in succession: the first was more substantial,
in the 80–316 ◦C range, and generated a weight loss of 83.4%, while the second was
between 316 ◦C and 450 ◦C and showed an overall weight loss of 98.8% (Figure S3). The
two subsequent decompositions can be explained by considering that colophony is only
partially functionalized with GLYMO: the most abundant unreacted fraction behaves
similarly to plain C, while the less abundant silane-functionalized fraction is more resistant
to thermal decomposition. Figure 3. Thermogram (TGA) of varnishes LC and LCS. Figure 3. Thermogram (TGA) of varnishes LC and LCS. 3.2.3. TGA and DSC Measurements This
means that the traditional varnish material becomes a little bit hard (reduced softness)
due to the chemical modification. Similarly, the material obtained by curing plain linseed
oil experiences a distinct increase in Tg due to the functionalization with epoxysilane
(Tg = −6.8 and 31.5 ◦C for L and LS, respectively). Pure rosin, C, showed a Tg of 60 ◦C, in
agreement with the literature [41], while the corresponding silane-modified material, CS,
undergoes glass transition at 75 ◦C. 11 of 22 Polymers 2023, 15, 3652 Table 1. Results of DSC measurements. Table 1. Results of DSC measurements. Films
Tg (◦C)
Pure linseed oil (L)
−6.8
Functionalized linseed oil (LS)
31.5
Colophony (C)
60
Functionalized colophony (CS)
75
Varnish LC
17.4
Varnish LC after UV aging (LC_UV)
−28.3
Varnish LC after thermal aging (LC_T)
−26.2
Varnish LCS
40.3
Varnish LCS after UV aging (LCS_UV)
33.5
Varnish LCS after thermal aging (LCS_T)
25.5 The study using thermal analyses was also performed on varnishes LC and LCS
after their artificial aging. The most interesting results come from DSC experiments and,
in particular, from the glass transition temperatures. In fact, the LC material undergoes
a drastic Tg decrease (by about 40 ◦C, see Table 1) after the aging induced both by UV
irradiation and temperature/humidity variation cycles. This result suggests that the
conventional varnish is strongly affected by the aging process, which makes the material
softer. The modified varnish, LCS, also experiences a decrease in Tg after aging, although
the observed variations are distinctly more restrained than in the case of LC. In fact, for the
modified varnish, Tg decreases from 40.3 to 33.5 ◦C after UV-induced aging and to 25.5 ◦C
after thermal aging. Hence, TGA and DSC measurements clearly indicate that the structural modification
induced by GLYMO in the varnish matrix provides a more resistant and less soft material,
which is also able to better preserve its properties after artificial aging. 3.2.4. NMR Analyses The varnishes and their components were also investigated using nuclear magnetic
resonance (NMR) spectroscopy both in CDCl3 solution (L, LS, and varnishes LC and
LCS before curing) and in the solid phase (L, LS, C, CS, and varnishes LC and LCS after
curing) in order to gain a better insight into the structural modifications induced by the
cross-linking process following the epoxysilane introduction. g p
g
p
y
The liquid-state 1H NMR spectra of the original and functionalized materials are reported
in Figure 4. Enlargements of the most relevant spectral regions are reported in Figure S4. Figure 4. 1H NMR spectra in CDCl3 of plain and functionalized materials. Figure 4. 1H NMR spectra in CDCl3 of plain and functionalized materials. Polymers 2023, 15, 3652 12 of 22 12 of 22 The signals due to linseed oil are present in all spectra. In particular, the resonances
between 5.2 and 5.5 ppm are due to the CH of glycerol moiety in triglycerides and to
vinyl protons of unsaturated fatty acid chains. Methylene protons of glycerol moiety in
triglycerides (and possibly in 1,2- and 1,3-diglycerides) resonate between 4.1 and 4.4 ppm,
while the CH2 between the double bonds in unsaturated chains resonates at 2.8 ppm. The
signals at 2.3, 2.0, and 1.6 can be assigned to CH2 in the α position to carbonyl groups,
to CH2 bonded to one double bond, and to CH2 in the β position to the carbonyl groups,
respectively. The intense peaks centered at 1.3 are due to all the other methylene groups
in aliphatic chains, while the terminal methyl groups of the fatty acid residues resonate
between 0.9 and 0.8 ppm. pp
The spectra of LC and LCS additionally display other signals due to the colophony
component. Selected spectral regions of the two varnishes are reported in Figure S5. The
spectrum of plain colophony is also reported for comparison. In particular, sharp peaks
between 0.75 and 1.25 ppm can be ascribed to methyl groups, while low intensity signals
between 4.7 and 6.0 ppm and between 6.8 and 7.3 ppm can be due to the protons of endo-
and exo-cyclic double bonds of abietic acid and other similar structures. y
Spectra of the silane-functionalized materials (LS and LCS) display a singlet at 3.7 ppm
due to unreacted methoxy groups of epoxysilane. 3.2.4. NMR Analyses On the other hand, signals ascribable
to an epoxy ring, such as the multiplet at about 3.4 ppm (see spectrum of plain GLYMO
in Figure S6), cannot be observed. These results strongly suggest that the epoxy ring
of GLYMO has exhaustively reacted with linseed oil or with the linseed oil/colophony
mixture, while the methoxysilane functions are still unaltered (at least partially) within the
experimental conditions used for functionalization. The different splitting of multiplets between 2.25 and 2.4 ppm in the spectra of the
examined materials is also worth noting (Figure 5). As already mentioned, this signal is
ascribed to methylene in the α position to ester carbonyl and is expected to be very sensitive
to any change undergone by the ester group. The variations observed both when linseed oil
is combined with colophony (LC) and with GLYMO (LS and LCS) suggest that the carboxyl
group of fatty acids has been partially involved in the reaction with resin components
and/or the epoxysilane cross-linker. Figure 5. 1H NMR spectra in CDCl3 of plain and functionalized materials. Enlargement of the signals
between 2.25 and 2.40 ppm. Figure 5. 1H NMR spectra in CDCl3 of plain and functionalized materials. Enlargement of the signals
between 2.25 and 2.40 ppm. 13C CPMAS NMR spectra of the original and functionalized materials measured in the
solid state are reported in Figure 6. In the case of L, LS, LC, and LCS, spectra were recorded
after the curing process. 13 of 22 13 of 22 Polymers 2023, 15, 3652 Figure 6. 13C CPMAS NMR spectra of the original and cross-linked solid products. Figure 6. 13C CPMAS NMR spectra of the original and cross-linked solid products. Figure 6. 13C CPMAS NMR spectra of the original and cross-linked solid products. No significant differences in the chemical shift and in the shape of signals can be
observed when comparing the spectra of both plain and functionalized linseed oil after
curing (L and LS). This can be explained taking into account the low % of added GLYMO. The only observable difference between C and CS is the different relative intensity of the
signals around 20 ppm, probably due to the higher proton density provided by the GLYMO. 3.2.4. NMR Analyses This phenomenon causes, at the Polymers 2023, 15, 3652 14 of 22 14 of 22 macroscopic level, an increase in the glass transition temperature, as already observed in
the DSC experiments. macroscopic level, an increase in the glass transition temperature, as already observed in
the DSC experiments. 3.3. Characterization of Coated Wood Specimens 3.2.4. NMR Analyses g
pp
p
y
g
p
y p
y
It is worth noting that in the spectra of samples LC and LCS the signal at 186 ppm, due
to the carboxylic groups of the colophony, are not present. This is in agreement with the
hypothesis of a “trans-esterification” process involving colophony and diglyceride species
during the heating treatment [25,33]. The values of the spin-lattice relaxation times in the rotating frame T1ρH and T1ρC
of varnishes LC and LCS are reported in Table 2. For the determination of both T1ρH and
T1ρC spin-lattice relaxation in the rotating frame, the signals at 38 and 30 ppm were chosen
in order to obtain the best results in the fitting process. These two signals are due to the C-1
and C-14 of the abietic acid (colophony component) and to the methylene groups of the
linseed oil fatty acid chains, respectively. Table 2. Values of the spin-lattice relaxation times in the rotating reference systems T1ρH and T1ρC
of the varnishes. Samples
LC
LCS
LC
LCS
Ppm
T1ρH (ms)
T1ρC (ms)
38 (colophony)
0.9 ± 0.1
1.3 ± 0.1
6.4 ± 0.1
9.6 ± 0.1
30 (oil)
0.8 ± 0.1
1.2 ± 0.1
2.3 ± 0.1
3.8 ± 0.1 Table 2. Values of the spin-lattice relaxation times in the rotating reference systems T1ρH and T1ρC
of the varnishes. The two components in both materials have equal values of T1ρH. This indicates that
the spin diffusion process makes the two material mixtures homogeneous in the tens of the
´ The two components in both materials have equal values of T1ρH. This indicates that
the spin diffusion process makes the two material mixtures homogeneous in the tens of the
´Å scale. On the contrary, the relaxation time values in the rotating reference system T1ρC
observed for varnish LC and LCS are considerably different. The spin-lattice relaxation
times in the rotating reference system T1ρC, unlike those of the proton (T1ρH), are not
influenced by the spin diffusion phenomenon, and therefore they reflect local mobility at
the level of a few monomer units [42]. From the analysis of the T1ρC values, an increase in
the values for both components of the mixtures in the presence of the cross-linking agent
(GLYMO) is clearly evident. This indicates that local rotational motions of the monomer
units are less favored in the presence of the cross-linker. 3.3. Characterization of Coated Wood Specimens The properties of LC and LCS were also comparatively investigated after their appli-
cation to wood specimens and further curing. Particularly, their behavior was examined
before and after artificial aging to evaluate the effect of GLYMO functionalization in terms
of post-application performance. For this purpose, the following measurements were
performed before and after the aging processes: chromatic variation and contact angle
measurements, SEM-EDS, micro-FTIR, NMR, and hardness. 3.3.1. Color Measurements The chromatic variations of the samples were investigated to analyze any differences
in color due to the modification of the Stradivari varnish before and after aging. The
colorimetric measurements were carried out on the coated wood specimens. The parameters
L*, a*, b*, and the global color variation ∆E* were obtained for the varnishes before and after
aging cycles, and all the given results were average values from 15 different measurements,
as explained in the Section 2. p
After the application of varnishes, the wood surfaces undergo quite comparable overall
chromatic changes (∆E* referred to the original wood color = 30.0 ± 0.9 and 32.2 ± 0.8
for LC and LCS, respectively), suggesting that the modification induced by GLYMO and
the consequent additional cross-linking do not significantly alter the original chromatic
properties of the Stradivari varnish. GLYMO is a colorless clear liquid and the addition of a
small amount as a cross-linking agent is not expected to significantly change the original
color of Stradivari’s varnish. The color variation is mainly due to the strong increase in the
b* coordinate (related to the blue-yellow changes), while coordinates L* (lightness) and a*
(green-red changes) experience more limited variations (Figure 7). Figure 7. Chromatic variations induced by varnish when applied to wood specimens (changes
compared to untreated wood). Figure 7. Chromatic variations induced by varnish when applied to wood specimens (changes
compared to untreated wood). Aging processes induce mainly an increased yellowing of the coated specimens, which
is more noticeable in the case of varnish LC and can be due to the degradation of the original
components (e.g., oxidation of linseed oil and colophony). In fact, after irradiation by UV
as well as after heat treatment, the coated wood surface undergoes further variations of
chromatic coordinates, which are larger in the case of LC than the LCS varnish. Specifically,
the values of ∆b* (changes referred to the unaged coated surface, directly related to the Polymers 2023, 15, 3652 15 of 22 15 of 22 yellowing) observed for LC (∆b*UV~18; ∆b*heat/hum~22) are more than double (>100%) if
compared to LCS (∆b*UV~9; ∆b*heat/hum~7, Table 3). Consequently, the overall chromatic
change, determined as the ∆E* value and taken as an indicator of the aging suffered by the
varnishes, is much lower in the case of the modified varnish LCS, in agreement with the
experimental results obtained from the analyses performed on varnish film samples. Table 3. 3.3.1. Color Measurements Chromatic variations of treated wood samples after two different aging cycles (changes
compared to unaged specimens). Samples
UV-Aged (720 h)
Heat/Humidity-Aged (720 h)
∆E*
∆L*
∆a*
∆b*
∆E*
∆L*
∆a*
∆b*
Varnish LC
21.9 (±5.8)
−9.1 (±5.5)
8.1 (±2.6)
18.0 (±3.1)
23.5 (±0.4)
−5.9 (±1.2)
6.0 (±0.9)
21.9 (±0.2)
Varnish LCS
11.9 (±2.3)
−5.5 (±2.9)
4.8 (±0.8)
9.2 (±1.3)
15.2 (±0.1)
−9.7 (±0.2)
9.3 (±0.3)
6.9 (±0.5) Table 3. Chromatic variations of treated wood samples after two different aging cycles (changes
compared to unaged specimens). 3.3.2. Contact Angle Measurements The results of the contact angle (α) measurements (average values from 15 measure-
ments) showed that varnish LCS exhibits distinctly higher hydrophobic properties than
varnish LC. In fact, the observed α increases by 34◦(+34.7%) due to the structural modifi-
cations induced by GLYMO (Table 4). The more pronounced water-repellent behavior of
modified varnish is preserved even after aging processes. In fact, the contact angle for the
LC material drops at values lower than 90◦after UV exposition or heat/humidity cycles,
while α is still around 110◦after both aging processes for LCS, indicating that the hydropho-
bic behavior of the surface is nearly preserved [43,44]. Hence, the coating obtained by
varnish LCS provides a better performance, even in terms of protection from water. Table 4. Contact angle measurements α (◦) on coated wood specimens before and after aging processes. Samples
Before Aging
UV-Aged
Heat/Humidity-Aged
(720 h)
(720 h)
Varnish LC
98.5 (±8.0)
86.6 (±6.0)
82.0 (±2.7)
Varnish LCS
132.5 (±3.8)
110.8 (±1.6)
110.3 (±7.2)
3 3 3 SEM EDS E
i
t Table 4. Contact angle measurements α (◦) on coated wood specimens before and after aging processes. 3.3.3. SEM-EDS Experiments SEM-EDS analyses were performed to examine the surface morphology and chemical
composition of aged and unaged samples. SEM images of the surface of varnishes LC and
LCS before starting the aging processes are reported in Figures 8a and 8b, respectively. The surfaces of both samples are quite homogeneous, with inclusions of small aggregates,
probably due to dust deposits on the specimens. EDS spectra (insets in Figure 8a,b) are in
agreement with the expected elemental compositions. In fact, only peaks of C and O are
observed in the spectrum of varnish LC, while the peak of Si is also present in the case of
LCS, due to the silane cross-linker being incorporated into the modified varnish. The pres-
ence of silicon, detected by the semi-quantitative analysis, is further confirmed by the EDS
mapping experiment (Figure 8c), which shows the quite homogeneous dispersion of the Si
element in the entire examined area of the film surface. The regular distribution of silicon is
an indication that GLYMO has been homogeneously dispersed into the varnish matrix and
that the cross-linking induced by epoxysilane has evenly affected the coating components. 16 of 22 Polymers 2023, 15, 3652 Figure 8. SEM-EDS images of coated wood specimens before aging processes: (a) varnish LC (EDS
spectrum in the inset); (b) varnish LCS (EDS spectrum in the inset; (c) EDS mapping for element Si of
varnish LCS (same area as (b)). Figure 8. SEM-EDS images of coated wood specimens before aging processes: (a) varnish LC (EDS
spectrum in the inset); (b) varnish LCS (EDS spectrum in the inset; (c) EDS mapping for element Si of
varnish LCS (same area as (b)). Figure 9a reports the SEM image of the LC coating after exposition under a UV lamp. The polymer layer is damaged and displays several inhomogeneous areas (Figure 9b),
as also observed by the optical microscope. Varnish composition (C and O only) was
confirmed in not-damaged areas, while in damaged areas EDS spectra showed the presence
of different elements (e.g., Na, Mg, K, Ca) that could be ascribed to the wood substrate,
whose surface is partially uncovered after artificial aging (inset in Figure 9b). SEM observations showed the presence of several inhomogeneous areas on the speci-
mens coated with LC, also after thermal aging (Figure 9c). 3.3.3. SEM-EDS Experiments In this case, too, the damaged
areas display an elemental composition that includes some elements related to the uncov-
ered wood surface (Figure 9d and corresponding inset). On the contrary, the surface of specimens coated with LCS appears quite homogeneous
after both UV and thermal aging processes (Figure 9e,f). The elemental composition of
varnish is preserved in all the observed areas and is similar to that before aging. These results confirm that LC varnish undergoes considerable degradation during ag-
ing, which compromises its protecting properties. On the contrary, the silane-modified LCS
varnish is more resistant to degradation induced by artificial aging, with the consequent
preservation of its aesthetical as well as protecting features. 17 of 22 17 of 22 Polymers 2023, 15, 3652 Figure 9. SEM images of coated wood specimens after aging processes: (a) varnish LC_UV (
spectrum in the inset); (b) magnification of damaged areas of LC_UV surface (EDS spectrum in
inset); (c) varnish LC_T (EDS spectrum in the inset); (d) magnification of damaged areas of L
surface (EDS spectrum in the inset); (e) varnish LCS_UV (EDS spectrum in the inset); (f) var
LCS_T (EDS spectrum in the inset). Figure 9. SEM images of coated wood specimens after aging processes: (a) varnish LC_UV (EDS
spectrum in the inset); (b) magnification of damaged areas of LC_UV surface (EDS spectrum in the
inset); (c) varnish LC_T (EDS spectrum in the inset); (d) magnification of damaged areas of LC_T
surface (EDS spectrum in the inset); (e) varnish LCS_UV (EDS spectrum in the inset); (f) varnish
LCS_T (EDS spectrum in the inset). 3.3.4. Micro-FTIR Measurements Wood samples coated with LC and LCS varnish were also investigated by µ-FTIR
(attenuated total reflection, ATR mode) before as well as after aging processes. The two
varnishes applied on wood surfaces display very similar spectra (Figure 10) when examined
before any aging. On the contrary, the spectra of plain and modified varnish measured at
the end of the artificial aging cycles display significant differences, in agreement with their
already observed different behaviors towards artificial weathering. In particular, the spectra
of LC recorded in correspondence with the damaged areas show several changes, partic- Polymers 2023, 15, 3652 18 of 22 18 of 22 ularly after the thermal aging cycle. For instance, several bands of the fingerprint region
decreased their intensity or disappeared, while the carbonyl band centered at 1720 cm−1
became broader or was split into more bands due to the formation of different carbonyl
derivatives (Figure 10a). In some cases, the spectra obtained on strongly damaged areas are
very similar to the spectrum taken on the surface of untreated wood (for comparison, see
Figure S7), confirming that weathering can strongly compromise the protecting properties
of the plain LC coating and that some wood areas become uncovered (and unprotected) at
the end of the thermal aging cycle. Figure 10. Micro-FTIR spectra of two different varnishes before and after aging processes: (a) varnish
LC (different areas); (b) varnish LCS. Figure 10. Micro-FTIR spectra of two different varnishes before and after aging processes: (a) varnish
LC (different areas); (b) varnish LCS. The spectra taken on different areas of the LCS surface after both artificial aging
processes are very similar to the spectrum of the unaged material (Figure 10b), as the
differences, if any, are very limited. Results provided by the µ-FTIR analyses are in good agreement with the above-
mentioned data obtained using optical microscope, SEM-EDS, and TGA-DSC experiments,
confirming that the chemically modified varnish, LCS, displays an enhanced resistance to
decay when compared with the traditional “Stradivari varnish” (LC). 4. Conclusions This research work focused on the improvement of the properties of Stradivari’s var-
nish using proper chemical modification. Limited amounts of the cross-linking agent
3-glycidyloxypropyltrimethoxysilane (GLYMO) were added to the traditional linseed
oil/colophony mixture (75:25), which is still used to prepare the varnish for contemporary
bowed musical instruments. Epoxysilane reacts through the epoxide ring with both compo-
nents of the varnish, introducing reactive alcoxysilane groups into the oil/resin matrix. In
addition to the known curing of the varnish induced by the oxidation of fatty acid chains
and consequent radical reactions, a cross-linking due to the condensation of the silane
group, therefore, can take place. g
p
p
As a consequence, the modified Stradivari’s varnish is more resistant to mechanical
decay (e.g., scratches), as shown by pencil hardness tests (the hardness changes from B to
H). Moreover, increased resistance to decline induced by aging was demonstrated using
comparative investigations carried out on the plain and modified varnishes before and after
artificial laboratory aging (cycles of UV irradiation and temperature/humidity variations). Results obtained using different techniques (OM, SEM-EDS, TGA and DSC, FTIR, and
µ-FTIR) coherently showed that the GLYMO-modified material is definitely less affected
by aging than the traditional Stradivari’s varnish. For instance, the traditional varnish,
LC, undergoes a drastic Tg decrease after artificial aging (from 17.4 to −26.3 or −28.2,
depending on the aging process), while the Tg reduction suffered by the modified material,
LCS, was distinctly smaller (from 40.3 to 33.5 or 25.5, depending on the aging process). This suggests that the modifications induced by GLYMO make the varnish less soft than
the plain material and better able to preserve its properties after artificial aging. Interestingly, the chemical modification does not induce significant changes in the
chromatic properties of the varnish (∆E* was about 30 and 32 for LC and LCS, respectively,
referring to the original wood color) Moreover, the color changes, particularly the yellow-
ing, induced by artificial aging are definitely smaller in LCS than in LC. The modified
varnish displays increased water-repellence when compared to the traditional one (by about
34%), and the hydrophobic behavior is also preserved (contact angle value around 110◦)
after performing artificial aging. On the contrary, wood samples coated with traditional
Stradivari’s varnish display a quite hydrophilic surface (α < 90◦) after aging. 3.3.5. Pencil Hardness Test The hardness of wood coatings is an important parameter to evaluate the effectiveness
of their protection from mechanical decay such as scratching. To evaluate the hardness
properties of the investigated varnishes, pencil tests were performed on wood specimens
coated with LC and LCS as well as on the corresponding coating films (prepared on
glass slides) according to the standard method [21]. The considered scale for the test
ranged between 9H (hardest) and 9B (softer), and the intermediate hardness level was “F”. This test provides information about the performance of coatings concerning resistance
to mechanical decay and durability [45,46]. Table 5 reports the results of the pencil test
performed on LC and LCS both before and after artificial aging. All the reported results are
average values of three measurements performed on coated specimens and on coating films. Polymers 2023, 15, 3652 19 of 22 19 of 22 Table 5. Results of pencil hardness test. Samples
After Treatments
UV-Aged
Thermal-Aged
Varnish LC
B
3B-2B
4B-3B
Varnish LCS
H
F
HB Samples
After Treatments
UV-Aged
Thermal-Aged
Varnish LC
B
3B-2B
4B-3B
Varnish LCS
H
F
HB The results obtained from the tests carried out before the aging processes on the
specimens coated with LC and LCS show that the modified varnish film is harder (H)
than traditional varnish (B). This confirms again that cross-linking induced by epoxysilane
provides a varnish layer that is more resistant to scratches than the LC varnish. Although
a decrease in hardness level (softening) is observed after aging for both materials, the
resistance of the not-modified varnish is affected to a larger extent than the functionalized
one. In fact, the hardness of LC decreases by 2–4 levels depending on the aging process
(from B to 2B-3B and to 3B-4B after UV and thermal aging, respectively), while a reduction
by only 1–2 levels is observed for the hardness of LCS (from H to F and B after UV and
thermal aging, respectively). Interestingly, the modified varnish after aging displays a
hardness level (F or HB) that is still higher than the not-aged traditional varnish (B). Hence,
the results of pencil test confirm again that additional cross-linking induced by GLYMO in
the linseed oil/colophony mixture provides an improved resistance of the resulting varnish
to scratches, even after artificial aging, if compared to the traditional “Stradivari” varnish. References 1. Echard, J.P.; Benoit, C.; Peris-Vicente, J.; Malecki, V.; Gimeno-Adelantado, J.V.; Vaiedelich, S. Gas chromatography/mass
spectrometry characterization of historical varnishes of ancient Italian lutes and violin. Anal. Chim. Acta 2007, 584, 172–180. [CrossRef] [PubMed] 2. Lammlein, S.; Künniger, T.; Rüggeberg, M.; Schwarze, F.W.M.R.; Mannes, D.; Burgert, I. Frequency dependent mechanical properties of
violin varnishes and their impact on vibro-mechanical tonewood properties. Results Mater. 2021, 9, 100137. [CrossRef] 3. Fiocco, G.; Gonzalez, S.; Invernizzi, C.; Rovetta, T.; Albano, M.; Dondi, P.; Licchelli, M.; Antonacci, F.; Malagodi, M. Composi-
tional and Morphological Comparison among Three Coeval Violins Made by Giuseppe Guarneri “del Gesù” in 1734. Coatings
2021, 11, 884. [CrossRef] 4. Weththimuni, M.L.; Canevari, C.; Legnani, A.; Licchelli, M.; Malagodi, M.; Ricca, M.; Zeffiro, A. Experimental Characterization of
Oil-Colophony Varnishes: A Preliminary Study. Int. J. Conserv. Sci. 2016, 7, 813–826. 5. Van den Berg, J.D.J. Analytical Chemical Studies on Traditional Linseed Oil Paints. Ph.D. Thesis, University of Amsterdam,
Amsterdam, The Netherlands, 2002. Available online: https://pure.uva.nl/ws/files/3716376/20662_Thesis.pdf (accessed on
26 April 2002). p
)
6. Juita; Dlugogorski, B.Z.; Kennedy, E.M.; Mackie, J.C. Low temperature oxidation of linseed oil: A review. Fire Sci. Rev. 2012, 1, 3. [CrossRef] 7. Litwinienko, G. Autooxidation of Unsaturated Fatty Acids and Their Esters. J. Therm. Anal. Calorim. 2001, 65, 639–646. [CrossRef]
8. Quarantelli, A.; Righi, F.; Renzi, M.; Bonomi, A. Processi ossidativi negli alimenti di origine vegetale. Ann. Fac. Medic. Vet. Di
Parma 2003, 23, 181–202. 7. Litwinienko, G. Autooxidation of Unsaturated Fatty Acids and Their Esters. J. Therm. Anal. Calorim. 2001, 65, 639–646. [CrossRef]
8
Quarantelli A ; Righi F ; Renzi M ; Bonomi A Processi ossidativi negli alimenti di origine vegetale Ann Fac Medic Vet Di 7. Litwinienko, G. Autooxidation of Unsaturated Fatty Acids and Their Esters. J. Therm. Anal. Calorim. 2001, 65, 639–646. [CrossRef] inienko, G. Autooxidation of Unsaturated Fatty Acids a 8. Quarantelli, A.; Righi, F.; Renzi, M.; Bonomi, A. Processi ossidativi negli alimenti di origine vegetale. Ann. Fac. Medic. Vet. Di
Parma 2003, 23, 181–202. attuati-Derieux, A.; Gomes, S.; Tirat, S.; Thao-Hey, S.; Echard, J.P. New insights into molecular evolution
rnishes: Towards pyrolysis-gas chromatography/mass spectrometry-based quantitation. E-Preserv. Sci. 201 10. Daher, C.; Pimenta, V.; Bellot-Gurlet, L. Towards a non-invasive quantitative analysis of the organic components in museum
objects varnishes by vibrational spectroscopies: Methological approach. Talanta 2014, 129, 336–345. [CrossRef] j
y
p
p
g
pp
11. 4. Conclusions In conclusion, the modified Stradivari’s varnish represents a promising alternative to
the one prepared according to the traditional method of contemporary violin-makers. In Polymers 2023, 15, 3652 20 of 22 20 of 22 fact, it retains the unique aesthetical features of the historical finish, and, at the same time,
is more resistant to mechanical and physico-chemical decay. fact, it retains the unique aesthetical features of the historical finish, and, at the same time,
is more resistant to mechanical and physico-chemical decay. Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/polym15173652/s1, Figure S1. FTIR spectrum of 3-glycidyloxypropyl-
trimethoxysilane (GPTMS or GLYMO); Figure S2. Thermogram (TGA) of Linseed oil (L) and function-
alised Linseed oil (LS); Figure S3. Thermogram (TGA) of colophony (C) and functionalised colophony
(CS); Figure S4. 1H NMR spectra in CDCl3 of plain and functionalised materials: enlargements of
specific spectral regions; Figure S5. 1H NMR spectra in CDCl3 of LC and LCS (selected regions). Colophony spectrum is also reported for comparison; Figure S6. 1H NMR spectrum in CDCl3 of
3-glycidyloxypropyltrimethoxysilane (GPTMS or GLYMO); Figure S7. Micro-FTIR Spectrum (ATR
mode) of plain untreated wood. Author Contributions: Conceptualization, M.L.; Methodology, M.L.W. and G.F.; Validation, M.L.W. and M.L.; Formal analysis, G.F., C.M., A.S., M.L.S. and M.M.; Investigation, M.M. and M.L.; Supervi-
sion, M.L.; Resources, M.L.; Data curation, C.M., A.S., M.L.S. and M.L.W.; Writing—original draft,
M.L.W.; Writing—review and editing, M.L.W. and M.L. All authors have read and agreed to the
published version of the manuscript. Funding: The authors acknowledge support from the Ministero dell’Università e della Ricerca (MUR)
and the University of Pavia through the program “Dipartimenti di Eccellenza 2023–2027”. Funding: The authors acknowledge support from the Ministero dell’Università e della Ricerca (MUR)
and the University of Pavia through the program “Dipartimenti di Eccellenza 2023–2027”. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Data Availability Statement: The data presented in this research study are available in the present
article and in the related Supplementary Information. Acknowledgments: The authors gratefully acknowledge Claudio Canevari (Civica Scuola di Liuteria,
Milan, Italy) for kind guidance during the coating applications and providing maple wood specimens. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. [
]
12.
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Robinet, L.; et al. The nature of the extraordinary finish of Stradivari’s instruments. Angew. Chem. Int. Ed. Engl. 2010, 49, 197–201. [CrossRef] 21 of 22 21 of 22 Polymers 2023, 15, 3652 13. Brandmair, B.; Greiner, S.P. (Eds.) Stradivari Varnish: Scientific Analysis of His Finishing Technique on Selected Instruments; Serving
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people or property resulting from any ideas, methods, instructions or products referred to in the content.
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https://openalex.org/W2231935749
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https://link.springer.com/content/pdf/10.1007/JHEP06(2016)035.pdf
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English
| null |
Phase structure of N $$ \mathcal{N} $$ = 2* SYM on ellipsoids
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The Journal of high energy physics/The journal of high energy physics
| 2,016
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cc-by
| 21,754
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Open Access, c⃝The Authors.
Article funded by SCOAP3. Published for SISSA by
Springer Received: July 29, 2015
Accepted: June 3, 2016
Published: June 7, 2016 Received: July 29, 2015
Accepted: June 3, 2016
Published: June 7, 2016 Received: July 29, 2015
Accepted: June 3, 2016
Published: June 7, 2016 Keywords: Supersymmetric gauge theory, Matrix Models, AdS-CFT Correspondence Phase structure of N “ 2˚ SYM on ellipsoids JHEP06(2016)035 Daniele Marmiroli
Nordita, KTH Royal Institute of Technology and Stockholm University,
Roslagstullsbacken 23, SE-106 91, Stockholm, Sweden
E-mail: daniele.marmiroli@nordita.org Daniele Marmiroli
Nordita, KTH Royal Institute of Technology and Stockholm University,
Roslagstullsbacken 23, SE-106 91, Stockholm, Sweden Daniele Marmiroli
Nordita, KTH Royal Institute of Technology and Stockholm University,
Roslagstullsbacken 23, SE-106 91, Stockholm, Sweden E-mail: daniele.marmiroli@nordita.org Abstract: We analyse the phase structure of an N “ 2 massive deformation of N “ 4
SYM theory on a four-dimensional ellipsoid using recent results on supersymmetric local-
isation. Besides the ’t Hooft coupling λ, the relevant parameters appearing in the theory
and discriminating between the different phases are the hypermultiplet mass M and the
deformation (or squashing) parameter Q. Geometric deformation manifests itself as an
effective mass term, thus braking the conformal invariance of the theory with massless
hypermultiplets. The structure of perturbative corrections around the spherical geometry
is analysed in the details and a systematic computational procedure is given, together with
the first few corrections. The master field approximation of the matrix model associated
to the analytically continued theory in the regime Q „ 2M and on the compact space
is exactly solvable and does not display any phase transition, similarly to N “ 2 SUpNq
SYM with 2N massive hypermultiplets. In the strong coupling limit, equivalent in our
settings to the decompactification of the four-dimensional ellipsoid, we find evidence that
the theory undergoes an infinite number of phase transitions starting at finite coupling
and accumulating at λ “ 8. Quite interestingly, the threshold points at which transitions
occur can be pushed towards the weak coupling region by drifting Q to the value 2M. ArXiv ePrint: 1410.4715 Open Access, c⃝The Authors. Article funded by SCOAP3. Phase structure of N “ 2˚ SYM on ellipsoids doi:10.1007/JHEP06(2016)035 Contents
1
Overview
1
2
About the partition function of N “ 2˚ SYM on ellipsoids
3
2.1
Low energy theory and the β´function
5
3
Almost conformal theory in the weakly coupled regime
7
3.1
Nearly conformal case
7
3.2
Small deformations of the round geometry
8
3.3
Conformal perturbations
10
4
Exact solution in the M Ñ Q
2 limit
12
5
Decompactification limit
16
5.1
Asymptotic behaviour in the strongly coupled phase
16
5.2
Strong coupling master field for general Q and M
18
5.3
Q´driven phase transitions
21
6
Conclusions
24
A Barnes double zeta and related functions
25
B Mapping infinite products
27
C Kernel function and infinite series
29
D Analytic solution of the compact model in the M „ Q
2 limit
32
E Analytic continuation and behaviour of the solution at the boundary
36 Contents
1
Overview
1
2
About the partition function of N “ 2˚ SYM on ellipsoids
3
2.1
Low energy theory and the β´function
5
3
Almost conformal theory in the weakly coupled regime
7
3.1
Nearly conformal case
7
3.2
Small deformations of the round geometry
8
3.3
Conformal perturbations
10
4
Exact solution in the M Ñ Q
2 limit
12
5
Decompactification limit
16
5.1
Asymptotic behaviour in the strongly coupled phase
16
5.2
Strong coupling master field for general Q and M
18
5.3
Q´driven phase transitions
21
6
Conclusions
24
A Barnes double zeta and related functions
25
B Mapping infinite products
27
C Kernel function and infinite series
29
D Analytic solution of the compact model in the M „ Q
2 limit
32
E Analytic continuation and behaviour of the solution at the boundary
36 JHEP06(2016)035 E Analytic continuation and behaviour of the solution at the boundary 1
Overview The holographic principle states a precise equivalence between string theory and gauge the-
ory [1–3], though only in a few cases a neat formulation of such correspondence is known. Namely, for gauge theories with maximal supersymmetry in their respective space-time
dimension. On the other hand, the recent past bears witness of an increasing interest in
extending our knowledge of holography to theories with a lesser amount of symmetries. In particular, a massive deformation of N “ 4 SYM theory in four dimensions was con-
sidered in [4–9] in relation with its dual, type IIB strings on the so called Pilch-Warner
background [10]. This theory, known as N “ 2˚ SYM, displays a very interesting and
complicated phase structure in the large N limit, characterised by fourth-order [7] phase – 1 – transitions starting at finite values of the ’t Hooft coupling constant and accumulating
at infinite λ [6]. These transitions are associated to the blowing up of nearly massless
states that become dominant in the strong coupling phase and reproduce, in a somewhat
unexpected way, the beahviour predicted by the supergravity solution [10, 11]. Testing the holographic correspondence implicitly presumes some understanding of the
gauge theory at strong coupling. Luckily enough, for theories with sufficient supersymme-
try, localisation is a rather powerful tool that allows exact and direct computations in field
theory [12]. By “exact” we mean at any value of the coupling constant and by “direct”
we mean that solve the path integral without relying on any possible duality whatsoever. Indeed, the partition function of N “ 4 SYM on S4 and that of its N “ 2 supersym-
metric massive deformations have been localised to matrix integrals in [13]. Precisely on
these results, the aforementioned investigation of the phase structure of N “ 2˚ SYM put
its foundations, allowing highly non-trivial checks of an underlying holographic principle
for non-conformal theories. Far from being an isolated case, a similar structure of weak
phase transitions accumulating at infinite coupling has been observed in three-dimensional
massive Chern-Simons theories [14–16], five-dimensional SYM-CS [17] and in SQCD mod-
els [7, 14, 18]. JHEP06(2016)035 On the other hand, recent progresses in the formulation of gauge theories admitting
rigid supersymmetry on curved spaces [19–23] have allowed the authors of [24] to localise
to matrix integrals the partition functions of N “ 2 supersymmetric gauge theories on
the four-dimensional ellipsoid. 1
Overview Although we have
not succeeded in finding an analytical solution for generic deformations, we can focus on
certain corners of the parameter space where exact computations are viable. Under the
squashing of the four-sphere, the vacuum expectation value of hypermultiplets masses gets
shifted to mH
ij “
ˇˇˇai ´ aj ˘ Q
2 ¯ M
ˇˇˇ, meaning that massless modes appear at the thresholds of the coupling constant is out of our current reach, due to the number of parameters
involved in the game and the complicated phase structure of the theory. Although we have
not succeeded in finding an analytical solution for generic deformations, we can focus on
certain corners of the parameter space where exact computations are viable. Under the
squashing of the four-sphere, the vacuum expectation value of hypermultiplets masses gets
shifted to mH
ij “
ˇˇˇai ´ aj ˘ Q
2 ¯ M
ˇˇˇ, meaning that massless modes appear at the thresholds
2µ “ n
ˇˇˇQ
2 ´ M
ˇˇˇ, with n an arbitrary integer. Hence, fine tuning M and Q, all the thresh-
olds can be made arbitrary small, pushing phase transition close to λ “ 0. In the limit
where Q Ñ 2M, as we show in section 4, the theory is well described by the matrix integral
studied in [26], that in turn emerges from the large N limit of a 0`1 dimensional supersym-
metric matrix quantum mechanics of UpNq matrices. Quite interestingly, phase transitions
disappear in this limit, and the theory can be solved exactly at arbitrary coupling. The
absence of phase transitions in this regime is somewhat reminiscent of N “ 2 SUpNq SYM
theory on S4 with 2N massive hypermultiplets in the fundamental representation [7], but
we have not checked whether additional symmetries are recovered in these settings. JHEP06(2016)035 For general values of M and Q we provide numerical evidence that the phase structure
of the theory on the deformed geometry mimics that of the one on flat space. Indeed, the
pattern of repeating transitions is enhanced by the displacements of the effective masses
by ˘Q
2 terms. We analyse this complicated structure in section 5. The phenomenon
briefly outlined above has another interesting consequence. 1
Overview As it turns out, it is possible
to push the critical values of the coupling constant towards the weakly coupled region by
adjusting Q and M, or otherwise stated, phase transitions can appear at fixed coupling
in the flow from the flat geometry to the curved one. To our knowledge, this is a novel
and distinguishing feature of the N “ 2˚ theory on the ellipsoid that can be relevant for
determining a plausible holographic dual theory. 1
Overview The aim of this paper is to investigate the structure of the
massive N “ 2˚ SYM theory, defined in section 2, on such curved space, in particular in
light of a possible new generalisation of the holographic duality. In order to do so we avail
on standard methods for solving matrix models in the large N, master field approxima-
tion. Solving the problem then amounts to computing the density function ρpaiq of the
eigenvalues ai of the matrix field, after which the expectation value of any supersymmetric
observable compatible with the localisation procedure can be determined by the classical
average over the density ρpaiq itself. For the conformal theory defined on the round four-
sphere, the master field approximation of ρ is well known. At weak coupling it behaves at
the boundary of the eigenvalue support C “ r´µ, µs according to an inverse square root law
ρpaq „ 1{
a
µ2 ´ a2 being µ “
? λ{2π. The most natural generalisation of this result is to
understand the theory in the nearly-conformal and nearly-flat approximation. It is known
that, as the hypermultiplet is given a small mass M, the maximum eigenvalue receives a
contribution proportional to M2 [7]. In section 3 we show that adding a small deformation
of the background, that turns it into an ellipsoid with eccentricity
? 1 ´ b4, amounts to a
redefinition of the mass M2 Ñ M2 ´ pb ´ 1q2 for b „ 1. At strong coupling, the large N density of eigenvalues obeys the same equation of the
Gaussian case [13], up to a rescaling of the coupling constant proportional to the square
mass and deformation, similarly to [5]. The solution is therefore Wigner semi-circle law
ρpaq „
a
µ2 ´ a2 with µ “
1
2π
c
λ
´
Q2
4 ´ M2
¯
, where we introduced the squashing param-
eter Q “ b ` b´1. This regime has also been investigated in [25] in the M “ 0 case, in
which limit we find perfect agreement. Finding a solution for the density ρ at finite values – 2 – of the coupling constant is out of our current reach, due to the number of parameters
involved in the game and the complicated phase structure of the theory. 2
About the partition function of N “ 2˚ SYM on ellipsoids Availing on supersymmetric localisation, the authors of [24] derived the matrix model
encoding the partition function of SYM theories with at least N “ 2 supersymmetry on
the ellipsoid defined by x2
0
R2
0
` x2
1 ` x2
2
R2
1
` x2
3 ` x2
4
R2
2
“ 1
(2.1) (2.1) As on flat space, the massive N “ 2 theory of our present interest is obtained by giving
a mass term M to the hypermultiplet of N “ 4 SYM and Yukawa couplings dictated by
supersymmetry. We refer to this theory as N “ 2˚ SYM on the ellipsoid, as in [13, 27]. In the strongly coupled regime this theory was, to some extent, the interest of [25]. The
partition function of the massive theory reads ZN“2˚ “
ż
dˆa0 e
´ 8π2
g2 Trˆa2
0ZN“2˚
1´loop
ˇˇZN“2
inst
ˇˇ2
(2.2) (2.2) being ZN“2˚
1´loop “
Zvec
1´loop
Zhyp
1´loop
(2.3) ZN“2˚
1´loop “
Zvec
1´loop
Zhyp
1´loop (2.3) – 3 – the ratio of the one-loop vector multiplet partition function and massive hyper multiplets
partition function. The geometric deformation acting on the round sphere S4 reflects
into an algebraic deformation of the one-loop determinants by a term proportional to the
squashing parameter Q. For the vector multiplet one has Zvec
1´loop ”
ź
αP∆`
1
pˆa0 ¨ αq2 Υbpˆa0 ¨ αqΥbp´ˆa0 ¨ αq
(2.4) (2.4) where the product is restricted to the positive roots α of the Cartan subalgebra ∆of the
gauge group. For any massive hypermultiplet in some representation R one has a factor of ”
Zhyp
1´loop
ı´1
”
ź
ρPR`
Υ
ˆQ
2 ` ˆ
M ` ˆa0 ¨ ρ
˙
Υ
ˆQ
2 ` ˆ
M ´ ˆa0 ¨ ρ
˙
(2.5) JHEP06(2016)035 (2.5) and again the product is restricted to positive weights ρ. The notation is as follows: a0 is
a Cartan subalgebra valued real matrix, ˆa0 “ ?R1R2 a0, M is the hypermultiplet mass,
ˆ
M “ ?R1R2 M, b “
a
R1{R2 and Q “ b ` 1
b is the deformation parameter. The infinite
products have been regularised using the Υb function defined in appendix (A.12) which
differs from the one used in [24] only by a normalisation factor. 2
About the partition function of N “ 2˚ SYM on ellipsoids In terms of the eigenvalues
ˆa0 ¨ α it is straightforward to write down the contribution of each adjoint vector multiplet
and each adjoint or fundamental hyper multiplet ź
iăj
1
pˆai ´ ˆajq2 Υbpˆai ´ ˆajqΥbp´ˆai ` ˆajq
ź
iăj
Υ
ˆQ
2 ` ˆ
M ` ˆai ´ ˆaj
˙
Υ
ˆQ
2 ` ˆ
M ´ ˆai ` ˆaj
˙
t
ź
i
Υ
ˆQ
2 ` ˆ
M ` ˆai
˙
(2.6) adjoint vector multiplet
ź
iăj
1
pˆai ´ ˆajq2 Υbpˆai ´ ˆajqΥbp´ˆai ` ˆajq
adjoint hyper multiplet
ź
iăj
Υ
ˆQ
2 ` ˆ
M ` ˆai ´ ˆaj
˙
Υ
ˆQ
2 ` ˆ
M ´ ˆai ` ˆaj
˙
fundamental hyper multiplet
ź
i
Υ
ˆQ
2 ` ˆ
M ` ˆai
˙
(2.6) (2.6) Note in particular that in (2.2) the instanton contribution
ˇˇZN“2
inst
ˇˇ2 is given by Nekrasov
partition function [28] counting (anti-)self-dual instantons localised at the north (south)
pole of the ellipsoid where the theory approaches the Ω´deformed theory with ϵ1 “ 1{R1
and ϵ2 “ 1{R2. In the following discussion we will totally forget about the non-perturbative
contributions to the partition function, which in the large N limit assume the form Zk´inst „ e´ 8π2kN
λ
(2.7) (2.7) being k P N the instanton number. Although the classical instanton action can eventually
be renormalised and the instanton moduli space might blow up quicky enough to produce
a finite instanton contribution to the total action, as it was indeed discussed in [7], in-
stantons are always exponentially suppressed in the large N limit throughout the whole
phase diagram of the N “ 2˚ theory on the round four-sphere. We reasonably expect the
same to hold true in the present case. In appendix (B) we argue that (2.6) are indeed
the correct massive deformations of the massless N “ 2 theory on the ellipsoid and they
give back smoothly N “ 4 on the round sphere when both the massive and the geometric
deformations are removed. – 4 – 2.1
Low energy theory and the β´function From the direct analysis of the partition function along the lines of [13], one can harvest
some information about the low energy dynamics of the theory. Because of the nature of the
space-time there are various scales that can be chosen as natural energy scales: R0, R1, R2 or
M. The description of the theory at low energies in terms of a running coupling constant
brakes down whenever the energy of interactions becomes comparable with the on-shell
mass of hypermultiplets. Also, hard deformations of the four-sphere act as an infrared
cut-offalong certain directions, altering the dynamics at very low energies. Hence we can
say this description roughly holds at energy scales Λ such that 1{R ! Λ ! M. When the
deformation parameter takes its minimum, meaning b “ 1, the one-loop determinant factor
reproduces the theory on the round S4, see appendix (B). It is clear that when the mass M
is much larger than 1, relations (A.10), (A.12) and the asymptotic expansion (A.7) tell us
that the coupling constant gets renormalised by a β´function similar to the one of N “ 2˚
SYM on S4 and which shall equate the latter in the b Ñ 1 limit. To this end let’s consider
the parameter β “ R1´R2
2R2
so that for R1 Á R2 we have Q “ 2`β2. 2.1
Low energy theory and the β´function Expanding Zhyp
1´loop for large values of the argument through (A.7) and keeping
terms that are proportional to piˆa0 ¨ αq2, we have the leading order asymptotic beahviour log
"
Υ
ˆQ
2 ` iM ` iˆa0 ¨ α
˙
Υ
ˆQ
2 ` iM ´ iˆa0 ¨ α
˙*
(2.11)
“ piˆa0 ¨ αq2 log
ˆQ2
4 ` M2
˙
´
ˆQ2
4 ` M2
˙
log
ˆQ2
4 ` M2
˙
` Opiˆa0 ¨ α logpiˆa0 ¨ αqq (2.11) – 5 – – 5 – Opposedly to the S4 case, vector multiplets do contribute here, being their 1-loop
partition function affected by the large Q deformation log tΥ piˆa0 ¨ αq Υ piˆa0 ¨ αqu “
“ ´ log rΓ2 piˆa0 ¨ αq Γ2 p´iˆa0 ¨ αqs ` 1
2rpiˆa0 ¨ αq2 ` Q2s log Q2 ` subleading
(2.12) (2.12) The term Γ2 piˆa0 ¨ αq Γ2 p´iˆa0 ¨ αq needs further attention, as we can see from the last
line of (2.12). Using the expansion of the double Gamma for small values of one of the
parameters (A.9) we have JHEP06(2016)035 log rΓ2 piˆa0 ¨ αq Γ2 p´iˆa0 ¨ αqs “ ´1
2 piˆa0 ¨ αq2 log b2 ` piˆa0 ¨ αq2 `
` 2ζ1
Rp´1q ´ ζ1
H
ˆ
´1, iˆa0 ¨ α
b
˙
´ ζ1
H
ˆ
´1, ´iˆa0 ¨ α
b
˙
` log Γ
ˆiˆa0 ¨ α
b
˙
` log Γ
ˆ
´iˆa0 ¨ α
b
˙
´
1
12b2
ˆ
ψ
ˆiˆa0 ¨ α
b
˙
` ψ
ˆ´iˆa0 ¨ α
b
˙
` 2γE
˙
` log 2π ` Cr
(2.13) (2.13) The remainder Cr can’t be computed exactly in the general case, but it can be quickly
estimated as b Ñ 8 The remainder Cr can’t be computed exactly in the general case, but it can be quickly
estimated as b Ñ 8 Cr “
8
ÿ
k“2
p´1q2k´1B2k
2kp2k ´ 1q
ˆ
ζH
ˆ
2k ´ 1, iˆa0 ¨ α
b
˙
´ ζRp2k ´ 1q
˙
„ iˆa0 ¨ α
3240 b
(2.14) (2.14) which is more than acceptable for our purposes. 2.1
Low energy theory and the β´function Then proceeding along
the lines of (B.3) and following, we have for the vector multiplet JHEP06(2016)035 Zvec
1´loop“
ź
ną0
“
pn ´ 1 ` iˆa0 ¨ αqpn ` 1 ` β2 ´ iˆa0 ¨ αqpn ´ 1 ` iˆa0 ¨ αqpn ` 1 ` β2 ´ iˆa0 ¨ αq
‰n
(2 8) (2.8) which at first non-trivial order in β reads which at first non-trivial order in β reads ź
ną0
rpn ´ 1 ` iˆa0 ¨ αqpn ` 1 ´ iˆa0 ¨ αqpn ´ 1 ` iˆa0 ¨ αqpn ` 1 ´ iˆa0 ¨ αqsn
ˆ
„
1 ` nβ2
2n ` 2
pn ` 1q2 ´ piˆa0 ¨ αq2
(2.9) ź
ną0
rpn ´ 1 ` iˆa0 ¨ αqpn ` 1 ´ iˆa0 ¨ αqpn ´ 1 ` iˆa0 ¨ αqpn ` 1 ´ iˆa0 ¨ αqsn (2.9) ną0
ˆ
„
1 ` nβ2
2n ` 2
pn ` 1q2 ´ piˆa0 ¨ αq2
(2.9) and so by definition of Barnes G-function and so by definition of Barnes G-function and so by definition of Barnes G-function Zvec
1´loop “ p1 ` 2β2qpiˆa0 ¨ αq2Gp1 ` iˆa0 ¨ αqGp1 ´ iˆa0 ¨ αqGpiˆa0 ¨ α ` 1qGpiˆa0 ¨ α ´ 1q (2.1 Here we have used the fact that the behaviour of the infinite product is essentially deter-
mined by the large n terms, so that the equation above boils down to a rescaling of Zvec
1´loop
by 1`2β2. Analogously the contribution of the hyper multiplet gets rescaled by
1
1`β2 . We
are interested in the effect of geometric deformations to the running coupling, thus we need
to expand for large hyper’s masses M Ñ 8 keeping the ratio M{Q fixed. Note that in
doing so, the latter might be big as well, meaning that we are not constraining Q to acquire
small values. 2.1
Low energy theory and the β´function The expression in (2.13) gets highly
simplified for large values of b, leading to og rΓ2 piˆa0 ¨ αq Γ2 p´iˆa0 ¨ αqs “ ´1
2 piˆa0 ¨ αq2 log b2 ` piˆa0 ¨ αq2 ´ 2 log
ˆiˆa0 ¨ α
b
˙2
` Op1q
(2 15) (2.15) At this point we must collect all relevant contributions from the expansions above. Terms which are quadratic in a0 ¨ α contribute to the Gaussian integration, renormalising
the coupling constant and giving rise to the running coupling. So, with respect to the
undeformed case, for large values of the mass M and the deformation Q the β´function
gets modified to 1
λRpΛq “ 1
λ ´ C2
8π2 log
Q2
4 ´ M2R2
Q
(2.16) (2.16) Note we now have dimensionful quantities, having restored powers of the radius R (as
a short notation for ?R1R2) and being C2 the second Casimir of the gauge group. The
energy scale Λ is identified with the inverse size of the ellipsoid 1{Λ „ ?R1R2. Linear and
logarithmic terms should not alter the convergence of the integral, and terms independent
of a0 can be discarded in the overall divergent constant (which does not affect quantum
averages). Note that for the massless theory the beta function boils down to a Q-dependent
rescaling of the coupling constant. – 6 – 3
Almost conformal theory in the weakly coupled regime 3 There are several different regimes in which the theory can be understood analytically. To
this end, it is often useful to separate the scales that appear in the game. Besides the
hypermultiplet’s mass M which is set by hand into the theory, we must consider the width
of the eigenvalue distribution µ , which is small at weak ’t Hooft coupling and increases at
strong coupling, and the geometric deformation parameter Q. As anticipated, this gives
rise to a rich ensemble of different behaviours. In section 5 we derive a saddle point equa-
tion in the decompactification limit that takes into account all these effects simultaneously,
though we are not able to solve such equation exactly, and we must rely on numerical
results. In the present section we present analytical results obtained in different corners of
the parameter space of the theory. JHEP06(2016)035 3.1
Nearly conformal case In the following we mostly adopt conventions in which the Coulomb moduli ai’s and the
mass M are dimensionless. As they appear ubiquitously in (2.2) and following equa-
tions, the moduli and masses are rescaled by the square root of the inverse product of the
equivariant parameters ζ “ ?R1 R2. However, in some circumstances it will turn out to
be convenient to strip the hat offof such variables and make them dimensionless. This
amounts to rescaling Q as well pai, x
M “ ζai, ζM Ñ ai, M;
Q Ñ qQ “ Q
ζ “ 1
R1
` 1
R2
(3.1) (3.1) with the effect that the nearly round case (Q Á 2) can be treated as a perturbation in qQ,
assuming that R1, R2 " 1. Exponentiating the one-loop partition function (2.3) and then
differentiating with respect to the Coulomb modulus one has the following saddle point
equation for the matrix integral (2.2) /
ż µ
´µ
dˆaj ρpˆajq
„
Kpˆai ´ ˆajq ` Kp´ˆai ` ˆajq ´ K
ˆQ
2 ` ˆ
M ` ˆai ´ ˆaj
˙
´ K
ˆQ
2 ` ˆ
M ´ ˆai ` ˆaj
˙
“ 16π2
g2
Y M
(3 (
)
where µ is the width of the eigenvalues distribution and we have defined the Kernel function Kpxq “ d
dx log Υpx|b, b´1q
(3.3) (3.3) in (C.1). At weak coupling the eigenvalue distribution approaches the Wigner semi-circle
law in the same way as it does in the N “ 2˚ theory on the round sphere [5] ρpxq “
2
πµ2
a
µ2 ´ x2
with
µ “
? λ
2π fpQ, Mq
(3.4) (3.4) hence the argument of the first and second contributions in (3.2) above becomes small
ˆai ´ ˆaj „ ζµ Ñ 0 as λ Ñ 0, given that ζ remains finite. We want to determine the function
fpQ, Mq, at least perturbatively in Q and M under the assumptions that fpQ „ 2, M „
0q “ f1pQq ` M2f2pQq, and f2pQq “ a constant at lowest order. The additive structure – 7 – can be inferred directly from the computations of [5], whereas the factorisation of small M
corrections is implied by the fact that on the hard deformed ellipsoid the contribution of
light fields must still be perturbative in the mass. 3.1
Nearly conformal case To this end, we then use the asymptotic
expansion of the double gamma function (A.8), at first order in ai ´aj we can approximate can be inferred directly from the computations of [5], whereas the factorisation of small M
corrections is implied by the fact that on the hard deformed ellipsoid the contribution of
light fields must still be perturbative in the mass. To this end, we then use the asymptotic
expansion of the double gamma function (A.8), at first order in ai ´aj we can approximate Kpˆai ´ ˆajq ` Kp´ˆai ` ˆajq
(3.5)
“ ´ d
dˆai
rlog Γ2pˆai ´ ˆajq ` log Γ2pQ ´ ˆai ` ˆajq ` log Γ2p´ˆai ` ˆajq ` log Γ2pQ ` ˆai ´ ˆajqs (3.5) Kpˆai ´ ˆajq ` Kp´ˆai ` ˆajq “ ´ d
dˆai
rlog Γ2pˆai ´ ˆajq ` log Γ2pQ ´ ˆai ` ˆajq ` log Γ2p´ˆai ` ˆajq ` log Γ2pQ ` ˆai ´ ˆajqs with the leading contribution coming from the singularity in ai “ aj JHEP06(2016)035 “
2
ˆai ´ ˆaj
´ d
dˆai
plog Γ2pQ ´ ˆai ` ˆajq ` log Γ2pQ ` ˆai ´ ˆajqq
“
2
ˆai ´ ˆaj
` Opˆai ´ ˆajq (3.6) where the derivative contributions in the second to last line are at most Opˆai ´ ˆajq because
of anti-symmetry. As expected, this gives rise to the undeformed saddle point equation only
as long as Q is not big enough for Γ2pQq terms above to become non-negligible on the l.h.s. of (3.2). In that case, and at first order in µ, they should be treated as a constant that renor-
malises the r.h.s. of (3.2), along with akin contributions from KpQ{2˘Mq. This is the same
procedure that accounts for the running of the coupling constant in the N “ 2 theory on the
four-sphere. Besides small mass corrections, we are interested in the contribution of small
deformations of the S4. In the compact case, at zero mass M and Q “ 2 the theory is confor-
mal (2.16), therefore we can regard small corrections as perturbations around the conformal
theory. On the other hand, the general Q case is hardly manageable, though interesting
information can be extracted in certain peculiar limits, namely by means of fine tuning the
hypermultiplets masses and the deformation parameter as in the upcoming section 4. 3.2
Small deformations of the round geometry The distribution of eigenvalues evidently deviates from the Wigner-Dyson law when we
include subleading contributions at weak coupling. From the first and second summand
in (3.2) one can extract corrections proportional ai ´ aj which in turn are given by higher
powers of the argument in (A.8). Note that K in the equation cited above implicitly
depends on Q through (C.1) even though its argument does not explicitly depend on it. The structure of corrections in this regime obeys ´ d
dai
rlog Γ2pai ´ ajq ` log Γ2p´ai ` ajqs “
2
ai ´ aj
` Akpai ´ ajqk
(3.7) (3.7) In order to determine the coefficients Ak one has to show particular consideration to
the regularisation of infinite products in Υ functions. It turns out to be convenient to get
rid of the divergent contributions to K regularising the infinite products that appear in
the Υ function as in (C.1), (C.2). This procedure leaves the partition function unaltered. Indeed, one is allowed to multiply Υpxq by a Gaussian factor to ensure convergence, without – 8 – affecting the finite, x-dependent part. Setting affecting the finite, x-dependent part. Setting Υpxq Ñ
ź
mn
pmb`nb´1 `xqe´ αmnx2
2
´βmnx ź
pq
ppb`qb´1 `Q´xqe´ αpqpQ´xq2
2
´βpqpQ´xq (3.8) (3.8) the extra x dependent terms coming from the exponentials in Z1´loop cancel between the
vector and the hypermultiplet contributions. Moreover, accounting on (3.8), the derivative
in (C.2) gets modified to ÿ
mn
pmb ` nb´1 ` xq´1 ´ x
ÿ
mn
αmn ´
ÿ
mn
βmn
(3.9) (3.9) JHEP06(2016)035 so that eventually the ζRp1q divergences appearing in the x „ 0 region are cancelled
by choosing αmn “ β2
mn “ pmb ` nb´1q´2. Interestingly, all the coefficients Ak can be
determined re-writing (3.7) through (C.3) as 8
ÿ
m,n“0
„
1
mb ` nb´1 ` aij
´
1
mb ` nb´1 ´ aij
` 2aij
ÿ
m`ną0
1
pmb ` nb´1q2
“ ´2aij
«
8
ÿ
m,n“0
1
pmb ` nb´1q2 ´ a2
ij
´
ÿ
m`ną0
1
pmb ` nb´1q2
ff
“ 2
aij
´ 2aij
8
ÿ
k“1
˜
´1
k
¸
p´1qka2k
ij
ÿ
m`ną0
1
pmb ` nb´1q2k`2
(3.10) (3.10) and since A1 “ 0 once the regularisation has been taken into account, they bring no linear
correction to the saddle point equation. 3.2
Small deformations of the round geometry In order to compute corrections due to small
deformations we set b “ 1 ` β and b´1 “ 1 ´ β so that Q “ 2 ` β2 and hence to second
order in β ! 1 we see that all coefficients can be determined exactly as (C.4) and following A2k “ 0
A2k`1 “ 2p´1qk`1
˜
´1
k
¸"
ζRp2k`1q`ζRp2k`2q´β2
„
pk`1q pζRp2k`1q`ζRp2k`2qq
´pk`1qp2k`3q
3
rζRp2kq ` 4ζRp2k ` 1q ` 5ζRp2k ` 2q ` 2ζRp2k ` 3qs
*
(3.11) A2k “ 0 for k ě 1. The second subleading contribution to (3.5) can be computed the same way. Indeed, the small argument asymptotics of Γ2 cannot be employed in the compact case
since Q is not, strictly speaking, a small quantity. Using again (C.2) and proceeding as
above, one finds for the unrenormalised coefficients Bkpai ´ ajqk “ ´2pai ´ ajq
8
ÿ
m,n“0
1
pmb ` nb´1 ` Qq2
„
1 `
pai ´ ajq2
pmb ` nb´1 ` Qq2
`
ÿ
hą3
Bhpai ´ ajqh
(3.12) (3.12) Remarkably the leading contribution in ai ´ aj can be determined nonperturbatively
in b in this case. Once the regularization term has been subtracted from (3.12), the – 9 – divergencies annihilate and we are left with the finite contribution B1 “ ´2
`
b2 ` b´2˘
ζRp2q
(3.13) (3.13) Higher orders in the weak coupling expansion can be easily computed in the b „
1 approximation. It is sufficient to note that for k ą 1, Bk’s can be mapped to Ak’s
by identification of m, n Ñ m ` 1, n ` 1 and subtraction of the two terms with indices
tm, nu “ t0, 1u, t1, 0u A2k`1 ` B2k`1 “ 2p´1qk`1
˜
´1
k
¸ ”
b2k`2 ` b´2k´2ı
ζRp2k ` 2q
(3.14) (3.14) JHEP06(2016)035 Although the expression for Ak given above has been obtained perturbatively in β „ 0
and in the weak coupling regime, the relation (3.14) is an identity to all orders in both
β and ai ´ aj. We can then re-sum the expansion and conclude that the first half of the
kernel accounts for Kpˆai ´ ˆajq ` Kp´ˆai ` ˆajq “ bπ cotppˆai ´ ˆajqbπq ` b´1π cotppˆai ´ ˆajqb´1πq
(3.15) (3.15) where we have restored the dimensional dependence of the moduli. 3.2
Small deformations of the round geometry Now, since ˆa “ ζa,
bζ “ R1 and b´1ζ “ R2, the latter equation suggests that the ρpaiq is discontinuous at every
point in the Coulomb moduli space where |aij| “
n
R1 , m
R2 with n, m arbitrary integers. Al-
though this equality cannot be fulfilled at weak coupling where the radia are of order unity
and the eigenvalues are small, one can have a hint about the consequences of (3.15) in the
decompactification limit. Namely as the R’s grow, also the size of the eigenvalue support
grows, generating an infinite number of discontinuities. These first appear as cusps in the
eigenvalue density ρ at the ends of the support where |aij| „ 2µ is larger, moving towards
the origin as λ is increased, and triggering the transition of ρ from an inverse square root
shape at weak coupling to a Wigner-like law at strong coupling. This is precisely the phe-
nomenon described in [6, 7] for massive N “ 2 theories on S4, the exception being that in the
present case it emerges from geometrically altering the structure of space-time. It is then
natural to expect that further adding a mass term for hypermultiplets will contribute with a
second generation of discontinuities in the eigenvalue density at sufficiently large coupling. 3.3
Conformal perturbations Next we must determine the M-dependent contribution to the saddle point equation coming
from KpQ{2 ` M ` ai ´ ajq ` KpQ{2 ` M ´ ai ` ajq in the kernel of (3.2). There are again
two different regimens in which analytic computations are viable. The first is the nearly
conformal case of M „ 0, the second is the large mass limit, which being equivalent to the
decompactification limit will be treated separately. In the first case the mass M is, for a
compact ellipsoid, much smaller than Q and comparable to µ, so using the definition of K
and proceeding as in (3.12), one has ´Ckpai ´ ajqk “ KpQ{2 ` M ` ai ´ ajq ` KpQ{2 ` M ´ ai ` ajq – 10 – “ ´2pai ´ aj ` Mq
8
ÿ
m,n“0
1
´
mb ` nb´1 ` Q
2
¯2
»
—–1 `
pai ´ aj ` Mq2
´
mb ` nb´1 ` Q
2
¯2
fi
ffifl
´2pai ´ aj ´ Mq
8
ÿ
m,n“0
1
´
mb ` nb´1 ` Q
2
¯2
»
—–1 `
pai ´ aj ´ Mq2
´
mb ` nb´1 ` Q
2
¯2
fi
ffifl
´4pai ´ ajq
8
ÿ
m`ną0
1
pmb ` nb´1q2 ` O
`
|ai ´ aj|3 ` |M|3˘
(3.16) ´4pai ´ ajq
8
ÿ
m`ną0
1
pmb ` nb´1q2 ` O
`
|ai ´ aj|3 ` |M|3˘
(3.16) (3.16) JHEP06(2016)035 To the lowest non-trivial order in perturbation theory around b „ 1, M „ 0, it is easy
to determine the behaviour of the regularised sums. First order terms in M cancel so that
to lowest non-trivial order the M´corrected contribution to the kernel is ´C1pai ´ ajq
(3.17)
“ ´4pai ´ ajq
ÿ
regularised
1
´
mb ` nb´1 ` Q
2
¯2 ´ 12M2 pai ´ ajq
8
ÿ
m,n“0
1
´
mb ` nb´1 ` Q
2
¯4 (3.17) where the regularised series is intended as subtracted of the last line of the second to last
equation above. 3.3
Conformal perturbations Setting again b “ 1 ` β and expanding around β “ 0, it is straightforward
to determine the first few orders in the β expansion (see appendix C), C1 “ 4ζRp2q ´ 4β2 p4ζRp2q ` 3ζRp3qq ` 12M2
„
ζRp3q ` 1
2β2
ˆ
ζRp3q ´ 4ζRp5q
˙
(3.18) (3.18) Quite interestingly, after the manipulations shown in (C.9) in appendix, one can re-sum
the series over the indices m, n and rewrite it Quite interestingly, after the manipulations shown in (C.9) in appendix, one can re-sum
the series over the indices m, n and rewrite it ÿ
regularised
1
´
mb ` nb´1 ` Q
2
¯2 “
8
ÿ
n“1
b2p´1qn`1ψp1qpb2n ` 1q ´ ψp1qp2q
(3.19) (3.19) being ψpnq polygamma functions. We can now approximate this series with its dominant
contribution. As n grows, the ψp1qpb2n ` 1q is asymptotically
1
b2n`1 and the series resums
to the Hurwitz-Lerch Phi function. Eventually we get to ´ 4pai ´ ajq
ÿ
regularised
1
´
mb ` nb´1 ` Q
2
¯2 “ ´4pai ´ ajq
“
Φp´1, 1, b´2 ` 1q ` 1 ´ ζRp2q
‰
(3 20) (3.20) (
)
As we take the large deformation limit we see that this part of the kernel contributes
with the asymptotic value As we take the large deformation limit we see that this part of the kernel contributes
with the asymptotic value ´ 4pai ´ ajq
“
log 2 ` 1 ´ ζRp2q ´ 2b´2ζRp2q ` O
`
b´3˘‰
(3.21) (3.21) therefore we conclude that for b large enough and M „ 0 the qualitative behaviour of
the saddle point equation is entirely determined by (3.15). In principle one should be – 11 – able to determine higher orders in M and Q, though we see already from the expression
above that the mass and the deformation parameter mix beyond the first non-trivial order. Moreover, none of these sums is expected to receive contributions to linear order in a small
β expansion, due to the b Ø b´1 symmetry of the problem. We can now gather from (3.14)
and (3.18) all M2 and β2 terms that are proportional to ai and hence appear on the r.h.s. 3.3
Conformal perturbations of the saddle point equation accounting for a rescaling of the coupling constant 8π2
λR
ai “
„8π2
λ
` pA1 ` B1 ` C1q
ai
(3.22) (3.22) from which JHEP06(2016)035 fpQ, Mq “ 1 ` λ3ζRp3q
“
M2 ´ β2‰
8π2
(3.23) (3.23) In the latter equation the relative sign between M2 and β2 should not be suspicious,
remember indeed that in our conventions M is purely imaginary, while β is real at this
stage. Note also that for b “ 1 the latter reproduces, to this order, the result of [7]. Also,
the structure of higher order corrections in (3.17) agrees with what we have assumed at
the beginning of this section based on general physical requirements on the gauge theory. We conclude this section with a comment. In (3.15) we have pointed out that, whenever
Rpai ´ ajq equals an integer n (R is either of the radia), the leading contribution to the
kernel of the saddle point equation is of the Hilbert kind. In addition note that the first
correction is still linear in ai, and reads A1 ` B1 “ ´2ζRp2qb2
(3.24) (3.24) where we have assumed b " 1 for simplicity. In this regime the latter term dominates
the r.h.s. of (3.22) as the corrections due to (3.20) are bounded to approach a constant. Henceforth λR „
8π2λ
b2p2ζRp2qq
for b " 1
(3.25) (3.25) can be interpreted as the statement that the theory squashed to the two-dimensional sphere,
and in the large radius limit, inevitably flows to the weakly coupled region. To make this
point clearer, note that the resummation of perturbative contributions to first half of the
kernel holds at finite values of ai´aj, hence at finite coupling, while the contribution of the
second half of the kernel is inversely suppressed as ai ´ aj grows. Moreover for R Ñ 8 the
condition Rpai ´ ajq “ n is fulfilled by an infinity of points in the Coulomb moduli space
accumulating around n “ 8. It follows that the dominant and first subleading behaviour
of the rescaled coupling are entirely determined by the geometric collapse of the dimensions
along the shrunk radia. 4
Exact solution in the M Ñ Q
2 limit Although an analytic solution seems not to be viable for arbitrary hypermultiplet mass and
deformation, one can think to extend one’s comprehension of the dynamical properties of
the theory beyond the small perturbation regime through a suitable fine tuning of Q and – 12 – M. It turns out to be interesting to analytically continue M to real values and consider the
limit in which M „ Q{2. Physical insight suggests that in this case the order of the λ Ñ 0
and M Ñ Q{2 limits actually counts. More precisely, there must exist two different phases
according to which of λ and m “ Q{2 ´ M is smaller. We first assume that both λ and m
are small compared to the other quantities entering the problem; then using the properties
of Barnes double zeta functions, we can expand the kernel appearing in the saddle point
equation according to K
ˆQ
2 ` M ´ ai ` aj
˙
“
1
ai ´ aj ` Q
2 ´ M
´ d
dai
log Γ2
ˆQ
2 ` M ´ ai ` aj
˙
` subleading
(4.1) JHEP06(2016)035 K (4.1) (
)
and similarly for K
´
Q
2 ` M ` ai ´ aj
¯
. The log Γ2 contributions that persist in these
formulas are again worked out by means of (3.12), and since M „ Q{2, exactly cancel the
akin contribution coming from Kpai ´ajq`Kp´ai `ajq. It is straightforward to show that
subleading terms also cancel and that the saddle point equation considerably simplifies to and similarly for K
´
Q
2 ` M ` ai ´ aj
¯
. The log Γ2 contributions that persist in these
formulas are again worked out by means of (3.12), and since M „ Q{2, exactly cancel the
akin contribution coming from Kpai ´ajq`Kp´ai `ajq. It is straightforward to show that
subleading terms also cancel and that the saddle point equation considerably simplifies to /
ż µ
´µ
daj ρpajq
„
1
ai ´ aj
´ 1
2
1
ai ´ aj ` m ´ 1
2
1
ai ´ aj ´ m
“ 8π2
λ ai
(4.2) (4.2) which holds up to corrections of order ai
´
Q
2 ` M
¯´1
to the right hand side and that can
be reabsorbed in a redefinition of the coupling constant. 4
Exact solution in the M Ñ Q
2 limit Quite interestingly the integral
equation (4.2) emerges from the large N approximation of the 0`1 dimensional supersym-
metric matrix quantum mechanics of UpNq matrices interacting with a gauge field and its
fermionic superpartner [26]. In turn, such theory is realised as the dimensional reduction
of N “ 1 SYM from four to 0 ` 1 dimensions and describes the low energy dynamics of
N supersymmetric degrees of freedom probing a codimension-3 subspace of the full four-
dimensional space. The solution to (4.2) for m2 “ ´1 has been determined analytically
in [26], in parametric form, developing a method originally proposed by J. Hoppe. Since
Q is real and M is purely imaginary we need to analytically continue M to real values
to define m. Physical values of the masses are then given by the analytic continuation of
m “ im1 which though implies that Q acquires unphysical values. The analytic continu-
ation is perfectly well defined in the present case thanks to the analyticity properties of
double gamma functions. In appendix (D) we review the solution of [28] in some details
and add the dependence on m, emphasising certain aspects which are particularly rele-
vant in our context. The solution of (4.2) is given by a set of parametric expressions for
the rescaled ’t Hooft coupling g2 “ m12λ
8π2 , the maximum eigenvalue µ and the distribution
ρpaiq. Introducing by standard notation (D.11) the incomplete/complete elliptical integrals
of first and second kind Fpγ, lq, Epγ, lq, Kplq “ Fpπ
2 , lq and Eplq “ Epπ
2 , lq, and the ratio which holds up to corrections of order ai
´
Q
2 ` M
¯´1
to the right hand side and that can
be reabsorbed in a redefinition of the coupling constant. Quite interestingly the integral
equation (4.2) emerges from the large N approximation of the 0`1 dimensional supersym-
metric matrix quantum mechanics of UpNq matrices interacting with a gauge field and its
fermionic superpartner [26]. In turn, such theory is realised as the dimensional reduction
of N “ 1 SYM from four to 0 ` 1 dimensions and describes the low energy dynamics of
N supersymmetric degrees of freedom probing a codimension-3 subspace of the full four-
dimensional space. The solution to (4.2) for m2 “ ´1 has been determined analytically
in [26], in parametric form, developing a method originally proposed by J. Hoppe. 4
Exact solution in the M Ñ Q
2 limit Since
Q is real and M is purely imaginary we need to analytically continue M to real values
to define m. Physical values of the masses are then given by the analytic continuation of
m “ im1 which though implies that Q acquires unphysical values. The analytic continu-
ation is perfectly well defined in the present case thanks to the analyticity properties of
double gamma functions. In appendix (D) we review the solution of [28] in some details
and add the dependence on m, emphasising certain aspects which are particularly rele-
vant in our context. The solution of (4.2) is given by a set of parametric expressions for
the rescaled ’t Hooft coupling g2 “ m12λ
8π2 , the maximum eigenvalue µ and the distribution
ρpaiq. Introducing by standard notation (D.11) the incomplete/complete elliptical integrals
of first and second kind Fpγ, lq, Epγ, lq, Kplq “ Fpπ
2 , lq and Eplq “ Epπ
2 , lq, and the ratio – 13 – “ Eplq
Kplq, one finds (D.20), (D.31) g2plq “ m12
π4 χ2plqK4plq
µplq “ m1
π rKplqEpγ, lq ´ EplqFpγ, lqs
νp1qplq “ m12
12 ´ 2m1K2plqθplq
“
5θplq
`
θplq ` l ´ 2
˘
` pl ´ 6ql ` 6q ` p2 ´ lqpl ´ 1q
‰
5π2“
θplq
`
3θplq ` 2l ´ 4
˘
´ l ` 1
‰
(4.3) (4.3) π
νp1qplq “ m12
12 ´ 2m1K2plqθplq
“
5θplq
`
θplq ` l ´ 2
˘
` pl ´ 6ql ` 6q ` p2 ´ lqpl ´ 1q
‰
5π2“
θplq
`
3θplq ` 2l ´ 4
˘
´ l ` 1
‰ being the modulus 0 ă l ă 1, the modular angle sin2 γ “ Kplq ´ Eplq
l Kplq
(4.4) JHEP06(2016)035 (4.4) and the shorthand χ2plq “ lp1 ´ 2θplqq ´ 3θ2plq ` 4θplq ´ 1
3
(4.5) (4.5) Also, the momenta of the eigenvalue distribution are defined by νpnq “
ş
dxρpxqx2n “
1
N
Trˆa2n
0
, and can be determined recursively up to (in principle) arbitrary order (D.29). In particular νp1q is related to the derivative of the free energy with respect to the coupling Also, the momenta of the eigenvalue distribution are defined by νpnq “
ş
dxρpxqx2n “
1
N
Trˆa2n
0
, and can be determined recursively up to (in principle) arbitrary order (D.29). 4
Exact solution in the M Ñ Q
2 limit Doing so one would get M2 ´ β2 “ 1 ´ 2 ˜m ` Opm2q
(4.9) (4.9) and therefore fpQ, Mq would depend on one single parameter fpM “ Q{2 ´ ˜mq “ 1 ´ λ3ζp3q p1 ´ ˜mq
4π2
(4.10) (4.10) in agreement with (D.23), even though the precise expansions of µpλq obtained in the two
different ways above do not coincide due to the evident non-commutativity of the limits
considered. JHEP06(2016)035 Secondly, in the region where the ’t Hooft coupling is much smaller then the mass, the
second and last terms under integral sign in (4.2) become subleading, due to the fact that
ai ´ aj ! m1, meaning that the equation simplifies further to /
ż µ
´µ
daj ρ0pajq
1
ai ´ aj
“ 8π2
λR
ai
λR “ λ ´
λ2
8π2m12 ` O
`
λ3{m14˘
(4.11) (4.11) In this regime the exact solution is simply Wigner semicircle ρ0pxq “
2
πµ2
a
µ2 ´ x2
µ “
?λR
2π
(4.12) (4.12) Increasing the ratio of the ’t Hooft coupling against the effective mass one reaches a
region in which the solution (4.3) is the only good description. Further on, and since the
kernel of the saddle point equation is only sensitive to the rescaled coupling, one can let
λ take large values imposing the scaling limit λ m12 ! 1. In these settings also m1 ! µ
holds, meaning that the kernel above is approximately minus m2 times the one-dimensional
discrete Laplace operator acting on
1
pai´ajq /
ż µ
´µ
daj ρ8pajq
1
pai ´ ajq3 “ ´
8π2
m2g2
Y M
ai
(4.13) (4.13) In this regime the maximum eigenvalue should be better described by (D.28), but
there exsist alternative descriptions of the master field solution, as shown in appendix E. 4
Exact solution in the M Ñ Q
2 limit In particular νp1q is related to the derivative of the free energy with respect to the coupling νp1q “ g4
N2
BF
Bg2
(4.6) (4.6) The asymptotic analysis of (4.3) is carried out in the following way. In the limit where
l Ñ 0 the coupling goes also to zero, hence one can expand g2 in powers of l, invert the series
and substitute in the small l expansion of the other relevant quantities. This produces a gen-
uine weak coupling expansion. Analogously, the strong coupling asymptotics is computed
by expanding g2 around l “ 1. For the maximum eigenvalue this produces (D.23), (D.28) µpλq “
$
’
’
’
&
’
’
’
%
m1
? λ
2π ´ m1
? 2
ˆ λ
4π2
˙ 3
2
` O
`
λ5˘
λ ! 1
m1 3? 3π2λ
4π
` O
`
λ0˘
λ " 1
(4.7) (4.7) Due to the nature of the correspondence with our original problem, we are interested
in the weakly coupled expansion of µ in the range where the effective mass is bounded
by 0 ă m ă 1. By weak coupling here we mean small g2, in such a way that µ “
? 2g ` Opg3q ! 1, so not necessarily small λ. At weak coupling the density of eigenvalues
behaves at the endpoints of the cut according to a square root law ρpxq „
a
µ2 ´ x2 with
µ2 increasing linearly as the ’t Hooft coupling and m12, which indeed plays the role of an
effective mass term in an N “ 2˚-like theory on a round sphere. There are some interesting
considerations one can draw from this simplified version of the original problem. Firstly,
consider the M „ 0, b „ 1 expansion of section 3.1 and the relative expression (3.23) for the
function fpQ, Mq defined in (3.4) as the first order deviation of the maximum eigenvalue
from the round and massless background. Since the radius of convergence of the M´series
is M “ 1 one can formally consider values of M “ Q
2 ´ ˜m „ 1 ´ ˜m
(4.8) M “ Q
2 ´ ˜m „ 1 ´ ˜m (4.8) – 14 – with ˜m a now positive and small real number. 4
Exact solution in the M Ñ Q
2 limit In particular one can notice that the analytic continuation from real to imaginary values
of m involves a phase transition from a Wigner-like distribution ρRpxq „
b
µ2
R ´ x2
µR „ λ1{4
(4.14) (4.14) to solution with inverse square root behaviour at the boundary of the eigenvalue support uare root behaviour at the boundary of the eigenvalue support ρIpxq „
1
b
µ2
I ´ x2
µI „ λ1{4
(4.15) (4.15) Matrix models transitions of this kind are ususally well understood, and in the present
case a better understanding of the phenomenon involved here can be relevant for uncovering
the phase structure of the N “ 2˚ theory. – 15 – The analytic structure of the solution can be understood straightforwardly. As can be
already seen from (4.3), µ becomes purely imaginary when m1 is continued to give physical
values of the deformation parameter. In other words, sending m1 Ñ 0 the effective coupling
constant g2 goes to zero accordingly and the eigenvalue density gets squeezed in a region
of zero size around the origin. Further continuing m1 along the imaginary axes produces
negative values of g2, though the ’t Hooft coupling λ remains positive. Moreover, the
maximum eigenvalue µ becomes purely imaginary meaning that we entered an unphysical
region. So, in these settings there is no phase transition at finite values of the coupling as the
phenomenon of cut collision pointed out in [6] happens at zero coupling and zero cut size. Although the phase structure of the theory appears trivial in this fine tuned limit,
there is some evidence that it is not. Let us consider the first momentum of ρpaiq as given
in (4.3), its asymptotic expansions read at the first few orders JHEP06(2016)035 νp1qpλq “
$
’
’
&
’
’
%
1
12
`
m12 ´ m13˘
` λm13
8π2 ´ λ2m13
32π4 ` O
`
λ6˘
λ ! 1
3
5
ˆ 3
π
˙2{3
λ2{3m13 ` 6
3? 3
3? λm13
5π4{3
` O
`
λ0˘
λ " 1
(4.16) (4.16) It is immediately clear from the weak coupling expansion that for m1 large enough
one cannot consistently send λ Ñ 0 as the semi-positive definite quantity νp1q apparently
becomes negative, as can be seen in figure 1. 4
Exact solution in the M Ñ Q
2 limit Interestingly there exists a physical inter-
pretation of this fact, though, strictly speaking, it is not related to our original problem
because of the fact that for m1 of order unity, the integral equation (4.2) ceases to be a
good approximation of (3.2). However, when λ is large enough the width of the eigenvalue
distribution is much larger than m1, meaning that the integral (4.2) receives most of its con-
tribution from the singularities of the kernel due to modes of masses ai ´ aj ˘ n m1, n P N,
that have become massless. If we gradually diminish λ we will eventually reach 2µ “ m1,
after which point the kernel is a regular function on the support of the integral. In figure 1
this phenomenon is evident and the second momentum (in red) becomes positive when
the ratio of the maximum eigenvalue over the mass (in blue) approaches
µ
m1 “ 1
2. Further
decreasing λ, in the region where λ ! m1 the Hilbert part of the kernel becomes largely
dominant and the eigenvalues distribute according to the solution of the Gaussian model,
Wigner semicircle law. This quite unexpected phenomenon is not evident for m1 ă 1 [26]
since the value at which νp1q turns negative is g2 “ 1 and hence outside of the domain of
convergence of the weak coupling expansion. 5.1
Asymptotic behaviour in the strongly coupled phase Note that ˆa0 “ ?R1R2 a0 “ R1
b a0 appears ubiquitously in the matrix model, therefore
there are different scaling limits one can consider. One option is to keep R1 fixed and
send R2 Ñ 0; this produces b Ñ 8 and accordingly ˆa0 is always small. Henceforth in this
particular scaling limit the matrix model can be always threated as it lays in the weakly
coupled phase, assuming that the ’t Hooft coupling is bound to satisfy µ{b ! 1. We dub – 16 – Μ H 10 m L
2
4
6
8
10
Λ
-0.08
-0.06
-0.04
-0.02
0.02
0.04
Μ m
5
10
15
20
Λ
-1.0
-0.5
0.5
10 Μ m
5
10
15
20
25
30
35
Λ
-5
5
Figure 1. In the analytically continued model with m1 ě 1 the expectation value of x2 is not
a positive semidefinite quantity for all values of the ’t Hooft coupling λ. The red line represents
x2
as a function of λ, while the blue line represents the rescaled maximum eigenvalue
µ
m1 for
m1 “ 2, 15, 100 respectively from left to right. The onset of a phase in which none of the scalar
modes can be massless, otherwise stated 2µ ă m1, is signaled by
x2
turning negative. Note that
in the first (last) plot
µ
m1 is rescaled by
1
10 (10). Figure 1. In the analytically continued model with m1 ě 1 the expectation value of x2 is not
a positive semidefinite quantity for all values of the ’t Hooft coupling λ. The red line represents
x2
as a function of λ, while the blue line represents the rescaled maximum eigenvalue
µ
m1 for
m1 “ 2, 15, 100 respectively from left to right. The onset of a phase in which none of the scalar
modes can be massless, otherwise stated 2µ ă m1, is signaled by
x2
turning negative. Note that
in the first (last) plot
µ
m1 is rescaled by
1
10 (10). JHEP06(2016)035 this phase the hardly deformed compact phase. A second option is to let Q grow with
one of the radia of the ellipsoid in a suitable decompactification limit by letting R1 Ñ 8
and R2 be finite. In this case µ grows with λ. 5.1
Asymptotic behaviour in the strongly coupled phase We refer to this as the hardly deformed
decompactified phase, which in turn corresponds to the decompactification of the two-
dimensional theory obtained in the hardly deformed compact phase interacting with KK
modes that propagate on the small compact sphere of radius R2. The third option is to
consider the scaling limit R1{Q fixed, which also implies R2 „ 1{R1, and that therefore
describes the pure two dimensional theory on flat space. In the first case, we can use the asymptotic expansion of Barnes double gamma from
the very beginning and consider the scaling limit in which the difference between any
two eigenvalues is in modulus much smaller then the deformation (rescaled by the radius)
|ˆai ´ ˆaj| „ 2ˆµ ! Q, accordingly d
dˆai
log Γ2pQ ´ ˆaijq “ 1
2Q log Q ´ ˆaij log Q ´ Q ´ ˆaij ` Opˆa2q
d
dˆai
log Γ2pQ ` ˆaijq “ ´1
2Q log Q ´ ˆaij log Q ` Q ´ ˆaij ` Opˆa2q
(5.1) (5.1) For the mass dependent kernel functions we also have ´ K
ˆQ
2 ` M ` ˆaij
˙
´ K
ˆQ
2 ` M ´ ˆaij
˙
“ ´2ˆaij log
ˆQ2
4 ´ M2
˙
(5.2) (5.2) so that all the information coming from squashing and mass terms is rephrased in a rescaled
coupling constant on the r.h.s. of a saddle point equation of the form /
ż µ
´µ
daj ρpajq
1
ai ´ aj
“ ai
«
8π2
λ
` log
Q2
4 ´ M2
Q
ff
(5.3) (5.3) Note that this integral equation for ρpaiq is solved by Wigner semi-circle law, and
that the beta-function such obtained coincides with the result found in (2.16) by direct
inspection of the partition function. – 17 – On the other hand, in the second case pointed out above the contribution of Q and M
to the kernel of (5.13) becomes subleading with respect to the Hilbert part, and in turn
the saddle point equation can be approximated using (A.7) /
ż µ
´µ
dˆaj
ρpˆajq
ˆai ´ ˆaj
ˆQ2
4 ´ M2
˙
“ ˆai
16π2
λ
(5.4) (5.4) In [25] the theory with N “ 4 supersymmetry was studied in this regime and the two
descriptions agree when we send M Ñ 0 in the formula above. 5.1
Asymptotic behaviour in the strongly coupled phase The density of eigenvalues is
again of Wigner type; in the two limiting cases above, equation (5.13) is solved by (3.4) with JHEP06(2016)035 µpλ, Q, Mq “
$
’
’
’
’
’
’
’
’
&
’
’
’
’
’
’
’
’
%
1
2π
g
f
f
e
λ
1 `
λ
8π2 log
´
Q
4 ´ M2
Q
¯
2µ ! minpQ, |M|q
1
2π
d
λ
ˆQ2
4 ´ M2
˙
2µ " maxpQ, |M|q
(5.5) (5.5) The apparently implicit conditions on µ above can be worked out easily and set the
ranges of λ in which the respective expansions hold. We can then consider the limit m “
Q
2 ´M „ 0 and compare the asymptotic solutions with that in (4.3). As one can see from the
left-hand plot depicted in figure 2, the expansion at µ ! Q (in red) is a good approximation
of the exact solution (in orange) in the region where the maximum eigenvalue is itself smaller
than one and the approximation of section 4 holds. Quite interestingly the agreement be-
tween (4.3) and the first of (5.5) is significant all the way through the weakly coupled region
to the strongly coupled one, to some extent, thanks to the fact that the actual expansion
parameter is µ “ m
? λ
2π , (m “ 1
2 in the plot). For larger values of m2λ, the matrix model
in (4.2) ceases to be a good description of the problem and the asymptotic solution at larger
λ (in green) significantly differs from the exact solution of (4.2). In the right-hand plot of
figure 2 one can observe the behaviour of the asymptotic solutions (5.5) at different values
of Q and m. It can be noticed that the bulk of the solution evidently gets stiffer by either
increasing Q or m. This fact is symptomatic of the assumptions made in (4.2), in particular
of the fact that such description breaks down when Q´dependent contributions resurge in
the expansion (4.1) of the kernel of (3.2) at very big values of Q. In this region there is no
simple asymptotic description of the solution and one needs to study the complete saddle
point equation in the decompactification limit, which is the subject of the next section. 5.2
Strong coupling master field for general Q and M In order to get some insight in the case of general Q, M at large coupling we need to
compute the logarithmic derivative appearing in the kernel of (3.2). In order to do so, – 18 – M ~
Q
2
Μ << Q
Μ >> Q
20
40
60
80
100
Λ
2
5
10
20
50
100
Μ2
Q = 50HgL, 90HrL, 140HbL
m = 0.5HgL, 0.9HrL, 0.7HbL
500
1000
1500
2000
2500
Λ
100
1000
104
105
Μ2
Figure 2. On the left: µ2pλq (orange) as obtained solving exactly the fine tuned matrix model
of section 4 compared with the two asymptotic behaviours in the regions where µ ! Q (red)
and Q ! µ (green). The former matches the exact solution for Q „ 2M, while the latter
deviates significantly from it, due to large Q corrections that make the integrand of (4.2) a bad
approximation of the original problem. On the right: asymptotic expansions of the solution for
increasing values of the effective mass m “ Q
2 ´ M. One can observe that as either m or Q are
increased, the asymptotics deviates significantly from the simple behaviour depicted on the left. Note that ordinate axes are in logarithmic scale. M ~
Q
2
Μ << Q
Μ >> Q
20
40
60
80
100
Λ
2
5
10
20
50
100
Μ2
Q = 50HgL, 90HrL, 140HbL
m = 0.5HgL, 0.9HrL, 0.7HbL
500
1000
1500
2000
2500
Λ
100
1000
104
105
Μ2 JHEP06(2016)035 Figure 2. On the left: µ2pλq (orange) as obtained solving exactly the fine tuned matrix model
of section 4 compared with the two asymptotic behaviours in the regions where µ ! Q (red)
and Q ! µ (green). The former matches the exact solution for Q „ 2M, while the latter
deviates significantly from it, due to large Q corrections that make the integrand of (4.2) a bad
approximation of the original problem. On the right: asymptotic expansions of the solution for
increasing values of the effective mass m “ Q
2 ´ M. One can observe that as either m or Q are
increased, the asymptotics deviates significantly from the simple behaviour depicted on the left. Note that ordinate axes are in logarithmic scale. 5.2
Strong coupling master field for general Q and M consider the definition of such functions consider the definition of such functions Kpxq “ d
dx log Υpx|b, b´1q
“ d
dx
␣
log Γ´1
2 px|b, b´1q ` log Γ´1
2 pQ ´ x|b, b´1q
(
“ d
dx
␣
´ζ1
2p0; b, b´1|xq ´ ζ1
2p0; b, b´1|Q ´ xq
(
(5.6) (5.6) where where d
dsζ2ps; b, b´1|xq
ˇˇˇˇ
s“0
“ ´
ÿ
m,n
pmb ` nb´1 ` xq´s logpmb ` nb´1 ` xq
ˇˇˇˇ
s“0
“ ´ log
ź
m,n
pmb ` nb´1 ` xq
(5.7) (5.7) So deriving with respect to x So deriving with respect to x ´ d
dxζ1
2p0; b, b´1|xq “
ř
m,n
ś
i‰n,j‰npib ` jb´1 ` xq
ś
m,npmb ` nb´1 ` xq
“
ÿ
m,n
pmb ` nb´1 ` xq´1
(5.8) (5.8) one arrives to the simple relation one arrives to the simple relation Kpxq “ ζ2p1, b, b´1|xq ` ζ2p1, b, b´1|Q ´ xq
(5.9) (5.9) At this stage we need to reintroduce the dimensional dependence of x on R in order to
extract information about the decompactification limit R Ñ 8 (R can be chosen at one’s
liking). After inverting the limit and the sum operations, one can approximate the double At this stage we need to reintroduce the dimensional dependence of x on R in order to
extract information about the decompactification limit R Ñ 8 (R can be chosen at one’s
liking). After inverting the limit and the sum operations, one can approximate the double – 19 – sum with a double integral over µ “ m{R, ν “ n{R (note that this also rescales K by 1{R) 8
ÿ
m,ně0
1
mb ` nb´1 ` xR “
ż R
1{R
ż R
1{R
dµ dν
1
µb ` νb´1 ` x
“ R
“
Q log Q ` pb´1 ´ bq log b
‰
` x log Qx
R ´ x ` O
ˆ 1
R
˙
(5.10) (5.10) All of the four contributions to (3.2) can be worked out this way. Putting all the terms
together we see that pure divergences and linear terms globally cancel out. At the end of
the day the contribution of K’s to the saddle point equation reads All of the four contributions to (3.2) can be worked out this way. Putting all the terms
together we see that pure divergences and linear terms globally cancel out. 5.2
Strong coupling master field for general Q and M At the end of
the day the contribution of K’s to the saddle point equation reads JHEP06(2016)035 KpRaijq ` Kp´Raijq ´ KpQ{2 ` RM ` Raijq ´ KpQ{2 ` RM ´ Raijq
“
ÿ
m,n
"
1
Ω` Raij `
1
Ω` Q ´ Raij `
1
Ω´ Raij `
1
Ω` Q ` Raij
´
1
Ω` Q
2 `MR`Raij
´
1
Ω` Q
2 ´MR´Raij
´
1
Ω` Q
2 `MR´Raij
´
1
Ω` Q
2 ´MR`Raij
+
(5.11) (5.11) Though the latter gives rise to a particularly involved integral equation, let us point
out that derivatives of Kpxq are much simpler Though the latter gives rise to a particularly involved integral equation, let us point
out that derivatives of Kpxq are much simpler K1pxq “ log
ˆQ
Rx
˙
´ log
ˆQ
R
ˆQ
R ´ x
˙˙
K2pxq “ 1
x ´
1
Q
R ´ x
(5.12) (5.12) Hence we can differentiate twice the saddle point equation with respect to x and get
to Hence we can differentiate twice the saddle point equation with respect to x and g Hence we can differentiate twice the saddle point equation with respect to x and get
to to /
ż µ
´µ
dy ρpyq
„
2
x ´ y `
1
x ´ y ` pQ
`
1
x ´ y ´ pQ
´
1
x ´ y `
pQ
2 ` M
´
1
x ´ y `
pQ
2 ´ M
´
1
x ´ y ´
pQ
2 ` M
´
1
x ´ y ´
pQ
2 ´ M
ff
“ 0
(5.13) (5.13) where pQ “ Q{R. Note that for large radius and pQ „ 0, this equation is equivalent to the
undeformed case with mass M studied in [5–7, 9]. Indeed, this is the behaviour whenever
the deformation scales slower then R and is indeed killed by decompactification. On the
other hand, also the opposite scaling limit, in which Q is increased insanely faster then R,
returns an equation of the same kind of that in [6] with mass parameter Q. The solution
in these two limiting cases is known exactly and one can draw some information from that,
in particular we expect the solution to exhibit phase transitions in λ whenever a Coulomb
modulus becomes null and a new massless boson appears. 5.3
Q´driven phase transitions The phase structure of the theory at pQ “ 0 was understood in [5–7, 9]. In this case there
exists a sub-critical phase for values of the ’t Hooft coupling such that 2µpλq ă M where the
distribution of eigenvalues is dominated by an inverse square root law. For higher values
of the coupling the maximum eigenvalue overcomes a new thresholds every time 2µ “ nM,
with n P N. At each threshold point a bosonic mode with Coulomb moduli ai ´ aj “ nM
becomes massless and the theory enters a new phase. Eventually, for sufficiently large λ,
these contributions become dominant and the density of eigenvalues assumes the Wigner
semi-circle shape typical of the type IIB supergravity solution. Therefore, in order to
understand how the deformation of the space-time alters this picture and what one can
learn about a possible holographic dual candidate, we first consider small pQ perturbations
around the known solution. Later we will let pQ become larger and eventually overcome
M, which will allow us to gain a hint about the hardly deformed geometry. p For pQ „ 0 the eigenvalue density is substantially determined by the ratio of µ{M. Note that in this limit the coupling constant in (5.13) gets rescaled by a factor of two. One
can then expect to find a sub critical phase in which ρpxq still behaves as 1{
a
µ2 ´ x2 at
the boundary of integration. Figures 3 and 4 (top) show this is indeed the case, with a
maximum eigenvalue µ „ Mfµpλq
fµpλ ! M2q „ exp
„
´4π2
λ
` 3Q2
4M4
(5.16) (5.16) which grows linearly with M as dictated by dimensional analysis (figure 3 bottom-right). The function fµpλq is unknown for general values of M and Q, though under the present
approximations it is straightforward to see that it amounts to a rescaling of the coupling
constant w.r.t. the pQ “ 0 case. In figure 3 (top-right) one can note the square root
behaviour of µ for λ ă 4π2
M
(„ 40 in the picture), in agreement with the approximation
in (5.5), and its consequent blow-up as λ Ñ λc, being λc the critical value of the coupling
at the first threshold. As the deformation is increased, µ grows quickly and the theory encounters a phase
characterised by a highly oscillatory behaviour of ρ. 5.2
Strong coupling master field for general Q and M This corresponds to values of
the coupling for which the width of the eigenvalue support hits an integer number times
one of the mass shifts that appear in (5.13) 2µ “ nQ, n
ˇˇˇˇ
Q
2 ˘ M
ˇˇˇˇ
n P N
(5.14) (5.14) and we immediately note that the first of such phase transitions can be arbitrarily close
to λ “ 0 as 2M approaches Q. At weak coupling the eigenvalue distribution has inverse and we immediately note that the first of such phase transitions can be arbitrarily close
to λ “ 0 as 2M approaches Q. At weak coupling the eigenvalue distribution has inverse – 20 – square root singularities at the boundaries of the cut square root singularities at the boundaries of the cut ρ0pxq „
1
a
µ2 ´ x2
µ “
? λ
2π fpQ, Mq
for |x| „ µ
(5.15) (5.15) and the maximum eigenvalues is proportional to
? λ and to the function fpQ, Mq that
we computed in (3.23) for small values of the deformations. Introducing once more m “
Q
2 ´M, we expect the first resonance to appear at 2µ “ m or, extrapolating from the weak
coupling analysis, λc “ m2π2. As the coupling is further increased more resonances will
appear and eventually the solution for ρpxq will approach Wigner semi-circle distribution
ρpxq „
a
µ2 ´ x2 through the accumulation phenomenon pointed out in [6]. JHEP06(2016)035 5.3
Q´driven phase transitions In figures 4 top-right and bottom-left
this behaviour is depicted, respectively, just below and above the critical point. In par-
ticular, one can notice that above the critical point, where µ has overcome M ´
pQ
2 , the – 21 – M = 3
M = 1.5
M = 1
M = 0.8
-2
-1
0
1
2
1
2
3
4
5
ΡHxL at Λ=30
æ
æ
æ
æ
æ
æ
æ
æ æ æ æ æ æ æ æ æ æææææææ
æ
æ
ææææ
10
20
30
40
50
60
Λ
0.88
0.90
0.92
0.94
0.96
ΜHΛL at M=1
æ
æ æ
æ
æ
æ
æ
1.0
10.0
5.0
2.0
20.0
3.0
30.0
1.5
15.0
7.0
M
1.0
10.0
5.0
2.0
20.0
3.0
1.5
15.0
7.0
ΜHML at Λ=30
Figure 3. On the left: sub critical eigenvalue densities at different values of the hypermultiplet
mass M and negligible deformation Q reproduce the known results obtained from the theory on
the round S4. On the right: coupling dependence (on top) and mass dependence (bottom) of the
maximum eigenvalue µ. Dimensional analysis and asymptotic freedom imply that µ „ Me´8π2{λ
at weak coupling. The linear dependence on M is evident in the lower graph. In the intermediate
region the approximation done in (5.5) is in agreement with numerical results until λ „ 4π2
M . As the
theory approaches the first threshold the maximum eigenvalue blows up and all the approximations
done so far break down. æ
æ
æ
æ
æ
æ
æ
æ æ æ æ æ æ æ æ æ æææææææ
æ
æ
ææææ
10
20
30
40
50
60
Λ
0.88
0.90
0.92
0.94
0.96
ΜHΛL at M=1
æ
æ æ
æ
æ
æ
æ
1.0
10.0
5.0
2.0
20.0
3.0
30.0
1.5
15.0
7.0
M
1.0
10.0
5.0
2.0
20.0
3.0
1.5
15.0
7.0
ΜHML at Λ=30 M = 3
M = 1.5
M = 1
M = 0.8
-2
-1
0
1
2
1
2
3
4
5
ΡHxL at Λ=30 JHEP06(2016)035 Figure 3. On the left: sub critical eigenvalue densities at different values of the hypermultiplet
mass M and negligible deformation Q reproduce the known results obtained from the theory on
the round S4. On the right: coupling dependence (on top) and mass dependence (bottom) of the
maximum eigenvalue µ. Dimensional analysis and asymptotic freedom imply that µ „ Me´8π2{λ
at weak coupling. 5.3
Q´driven phase transitions In this regime the master field solution is – 22 – -2
-1
0
1
2
0.2
0.4
0.6
0.8
1.0
ΡHxL at Λ=30, M=3, QR=0.01
-3
-2
-1
0
1
2
3
0.2
0.4
0.6
0.8
1.0
ΡHxL at Λ=30, M=3, QR=0.05
-3
-2
-1
0
1
2
3
0.2
0.4
0.6
0.8
1.0
ΡHxL at Λ=30, M=3, QR=0.1
-6
-4
-2
0
2
4
6
0.05
0.10
0.15
0.20
ΡHxL at Λ=30, M=3, QR=10
Figure 4. Distribution of eigenvalues below the critical point, on the first line, and above the
critical point, on the second line. Below the critical point the density of eigenvalues obeys an
inverse square root law ρpxq „
a
µ2 ´ x2. Criticality is achieved by increasing Q in such a way that
µ can overcome the first threshold located at µ “ M ´ pQ{2. Below the critical point Q´oscillations
are visible as superimposed to the inverse square root behaviour. Slightly above the critical point
the first cusp-like transition appears as a coarse structure underling oscillations (dotted blue line). Further increasing Q, the maximum eigenvalue also increases, overcoming more thresholds and
driving ρ to a Wigner-like phase (dotted blue line). -2
-1
0
1
2
0.2
0.4
0.6
0.8
1.0
ΡHxL at Λ=30, M=3, QR=0.01 -3
-2
-1
0
1
2
3
0.2
0.4
0.6
0.8
1.0
ΡHxL at Λ=30, M=3, QR=0.05 JHEP06(2016)035 -3
-2
-1
0
1
2
3
0.2
0.4
0.6
0.8
1.0
ΡHxL at Λ=30, M=3, QR=0.1 -6
-4
-2
0
2
4
6
0.05
0.10
0.15
0.20
ΡHxL at Λ=30, M=3, QR=10 Figure 4. Distribution of eigenvalues below the critical point, on the first line, and above the
critical point, on the second line. Below the critical point the density of eigenvalues obeys an
inverse square root law ρpxq „
a
µ2 ´ x2. Criticality is achieved by increasing Q in such a way that
µ can overcome the first threshold located at µ “ M ´ pQ{2. Below the critical point Q´oscillations
are visible as superimposed to the inverse square root behaviour. Slightly above the critical point
the first cusp-like transition appears as a coarse structure underling oscillations (dotted blue line). Further increasing Q, the maximum eigenvalue also increases, overcoming more thresholds and
driving ρ to a Wigner-like phase (dotted blue line). asymptotically determined by (4.3) and behaves at the boundary as
a
µ2 ´ x2. 5.3
Q´driven phase transitions The linear dependence on M is evident in the lower graph. In the intermediate
region the approximation done in (5.5) is in agreement with numerical results until λ „ 4π2
M . As the
theory approaches the first threshold the maximum eigenvalue blows up and all the approximations
done so far break down. oscillatory behaviour of Q perturbations is superimposed to the cusp-triggered transition. The latter appears as a coarse structure (dotted blue line) underling smaller Q´oscillations
(solid red line) and reproduce the structure of ρ on flat space above the first threshold. The crucial point here is that the transition appears to be triggered by the increasing de-
formation and the coexistence of two phenomena: the increase of the maximum eigenvalue
µ together with the decrease of the effective masses |ai ´ aj| ´ |M ´ Q{2|. This fact is a
totally new feature w.r.t. the N “ 2˚ theory on flat space where transitions appear in the
flow from the weakly coupled to the strongly coupled region. In addition, as pQ is increased
further, the difference M ´
pQ
2 becomes smaller with respect to µ, meaning that an increasing
number of massless bosons blow up. Eventually, these modes become dominant, letting the
theory flow to a new phase with a Wigner-like distribution of eigenvalues characterised by fµpλ " M2q ”
? λfµpQ, Mq “
? λ
˜
1 ´
pQ2
8M2
¸
(5.17) (5.17) as obtained in (5.5). This phase is strongly reminiscent of the strongly coupled phase of
N “ 2˚ theory on flat space. The phenomenon of cusp-accumulation that produces the
transition is actually shared by the deformed and the un-deformed theories, though in the
former, the effect of increasing the deformation reduces the distance between consecutive
cusps at fixed coupling, while in the latter the coupling needs to be increased in order to
reach the threshold. We can speak, in this case, of Q´driven phase transitions. A way to have a deeper understanding of the Q´driven phase transitions is to consider
very large coupling λ and the limit Q Ñ 2M. 5.3
Q´driven phase transitions Figure 5
depicts how, far above the critical point, the simple Wigner behaviour is altered by this phe-
nomenon. For values of Q, M ! µ, on the left, the theory is dominated by massless modes
and ρpxq approaches Wigner semicircle distribution, perturbed by heavy modes of large
masses M `Q{2. As Q is decreased further, the latter eventually decouple and such oscilla-
tions disappear. On the right of 5, values of Q „ 2M, but still comparable with µ produce
a big Q´oscillation, visible as a peak right in the middle of Wigners distribution. We can
conclude that the ratio of µ{ pQ determines the entity of oscillations, while the ratio of M{Q
determines transition. These effects are particularly interesting in view of an holographic
interpretation, even more so as they happen at fixed values of the coupling constant. – 23 – -50
50
0.002
0.004
0.006
0.008
ΡHxL at Λ=6000, M=1, QR=10
-150
-100
-50
50
100
150
0.001
0.002
0.003
0.004
0.005
0.006
0.007
ΡHxL at Λ=6000, M=100, QR=201
Figure 5. Distribution of eigenvalues far above the first critical point. On the left, for µ „ 80
and pQ{2 ´ M “ 4, cusp-driven phase transitions have brought the master field solution from the
inverse square root behaviour to a Wigner-like distribution, deformed by Q´oscillations. On the
right, small values of pQ{2 ´ M imply that the theory lies deeply in the strongly coupled region,
while Q comparable with µ cannot produce oscillations, resulting in just one peak on top of Wigner
distribution. -50
50
0.002
0.004
0.006
0.008
ΡHxL at Λ=6000, M=1, QR=10 -150
-100
-50
50
100
150
0.001
0.002
0.003
0.004
0.005
0.006
0.007
ΡHxL at Λ=6000, M=100, QR=201 JHEP06(2016)035 Figure 5. Distribution of eigenvalues far above the first critical point. On the left, for µ „ 80
and pQ{2 ´ M “ 4, cusp-driven phase transitions have brought the master field solution from the
inverse square root behaviour to a Wigner-like distribution, deformed by Q´oscillations. On the
right, small values of pQ{2 ´ M imply that the theory lies deeply in the strongly coupled region,
while Q comparable with µ cannot produce oscillations, resulting in just one peak on top of Wigner
distribution. 6
Conclusions Evidently, N “ 2˚ SYM theory on an ellipsoid has an extremely rich structure. One
can identify various regimes beyond the weakly and strongly coupled ones depending on
the relative magnitude of the ’t Hooft coupling λ, the squashing parameter Q and the
hypermultiplets mass M. Relying on supersymmetric localisation [24], we have analysed in
the details the contribution of small deformations to the weakly coupled, nearly conformal
theory, and we have found agreement with the previously studied theory on the round S4 [6,
7]. Also, we have identified a regime in which the dynamics of the non-conformal theory
can be described in terms of a Q, M´dependent β´function. One can immediately think
of possible generalisations of these results to even less symmetric space-time manifolds. The next natural step would be to consider a squashed four-sphere, which in turn breaks
an additional Up1q symmetry. Though, Killing equations have been derived in [29] but
seem not to admit solutions in this specific case. On the strong coupling side, though yet unable to find an analytic solution for arbitrary
values of Q and M, we are able to provide numerical evidence for a variety of new features
that this theory exhibits. In some corner of the moduli space we are also able to provide
an analytic understanding of these phenomena, and match previously established results. To our knowledge, a new and distinguishing feature of the analytically continued model
is the presence of phase transitions at fixed coupling that are triggered by increasing the
squashing Q of the four-sphere. These mimic phase transitions at fixed Q, M discovered
in [6] and that accumulate at λ “ 8. The former seem to be particularly interesting in light
of a possible holographic description, even more so as they can accumulate at finite values
of the coupling constant when M is fined tuned to be 1
2Q{R. At present, the meaning of
such behaviour from the supergravity point of view is unclear, though it is clear that string
fluctuations must be taken into account in order to probe such regime. – 24 – A number of non-trivial cross checks have been performed to match the N “ 2˚ SYM
theory and its dual geometry [10, 11], including the computation of large circular Wilson
loops [5] and the free-energy of the theory on S4 [30]. Acknowledgments I gratefully acknowledge The Foundation Blanceflor Boncompagni-Ludovisi, Nordita, KTH
Royal Institute of Technology and Stockholm University for supporting my research. Also,
I am thankful to Konstantin Zarembo for many useful discussions and comments about
the draft of the present paper. 6
Conclusions The strong coupling behaviour of large
Wilson loops has also been reproduced in [31], where a supergravity solution sourced by
D5´branes wrapping an S2 has been considered. Although the squashed theory still lacks
a string description, it is of primary interest, in order to shed some light on the nature of
the latter, to consider similar observables at strong coupling from localisation, both in the
compact and decompactified phases. Lastly, it should be possible to make contact between
the theory in the compact phase under large deformations and two-dimensional Yang-Mills. This last aspect might be relevant in light of the known relations dictated by localisation
between supersymmetric Wilson loops in four dimensional SYM and two-dimensional YM
theory [32, 33]. JHEP06(2016)035 A
Barnes double zeta and related functions In this appendix we gather information about the special functions that appear throughout
the text. Barnes double Zeta function is defined for any real number x, real positive a, b,
natural numbers m, n such that ma ` nb ` x ą 0 and complex s, as the infinite sum ζ2px|a, b; sq “
8
ÿ
m,n“0
pam ` bn ` xq´s
(A.1) (A.1) The sum only converges for Re psq ą 2, but the function itself admits analytic contin-
uations in all the parameters. A related function is Barnes double Gamma function The sum only converges for Re psq ą 2, but the function itself admits analytic contin-
uations in all the parameters. A related function is Barnes double Gamma function log Γ2px|a, bq “ d
dsζ2px|a, b; sq
ˇˇˇˇ
s“0
` log ρ2pa, bq
(A.2) (A.2) being being log ρ2pa, bq “ ´ lim
xÑ0
ˆ d
dsζ2px|a, b; sq
ˇˇˇˇ
s“0
` log x
˙
(A.3) (A.3) Alternatively it is possible to show (Lerch formula) log Γ2px|a, bq “ d
ds rζ2px|a, b; sq ´ χps; a, bqss“0
(A.4) (A.4) where χ is doubled version of the Riemann zeta function χps; a, bq “
ÿ
m`ną0
pam ` bnq´s
(A.5) (A.5) – 25 – The notation m ` n ą 0 is intended as summing over all couples of non-negative
integers that are not simultaneously null pm, nq P N2
0 ´ tp0, 0qu. The function ζ2px|a, b; sq
has an analytic continuation in the complex s´plane with simple poles at s “ 1 and s “ 2. It follows that the Barnes double Gamma admits the product representation Γ2px|a, bq “ e´r01x` 1
2 pr02`r12qx2
x
8
ź
m`ną0
1
1 ` x
Ω
e
x
Ω´ x2
2Ω2
(A.6) (A.6) where Ω“ am ` bn and Rij encode the residues (i “ 1) and finite parts (i “ 0) of
ζ2p0|a, b; sq at the pole s “ j. Their explicit expressions evade the present scope, so we
avoid writing them. A
Barnes double zeta and related functions From the infinite product representation and the explicit form of the
coefficients Rij, it also follows the asymptotic expansion at large x JHEP06(2016)035 log Γ2px|a, bq “ ´ x2
2ab log x ` 1
2
ˆ1
a ` 1
b
˙
x log x ´
„1
4 ` 1
12
ˆa
b ` b
a
˙
log x
` 3x2
4ab ´ 1
2
ˆ1
a ` 1
b
˙
x ´ χ1p0|a, bq ` Opx´1q
(A.7) and at small x log Γ2px|a, bq “ ´ log x ´ r01x ` 1
2 pr02 ` r12q x2 `
8
ÿ
j“3
p´1qj
j
χpj; a, bq xj
(A.8) (A.8) holding in the entire complex plane with a cut on the negative real axis. The double
Gamma can be expanded also for small values of its parameters (as well as for large values,
but in our case there is no difference). For instance, for a Ñ 0 the following holds log Γ2px|a, bq „ b
a
” x
2b
´
1 ´ x
b
¯
plog b ´ 1q ´ ζ1
H
´
´1, x
b
¯
` ζ1
Rp´1q
ı
´ 1
2 log a´ 1
2
´
1´ x
b
¯
log b` 1
2 log Γ
´x
b
¯
` 1
2 log 2π´ a
12b
´
ψ
´x
b
¯
`γE
¯
´
8
ÿ
k“2
p´1q2k´1B2k
2kp2k ´ 1q
´a
b
¯2k´1 ´
ζH
´
2k ´ 1, x
b
¯
´ ζRp2k ´ 1q
¯
(A.9) (A.9) Two more formulas are of particular interest to us. The first relates the double Gamma
with unity parameters to the Barnes G-function Two more formulas are of particular interest to us. The first relates the double Gamma
with unity parameters to the Barnes G-function Gp1 ` xq “
Γpxq
Γ2px|1, 1q
(A.10) (A.10) while the second one is the shift formula while the second one is the shift formula Γ2px ` b|a, bq “
? A
Barnes double zeta and related functions 2π a
1
2 ´ x
a
Γpx
aq
Γ2px|a, bq
(A.11) (A.11) We further introduce the Γb and Υb function that appear in the one-loop regularized
determinants of [24] and whose usage is widespread in the context of Liouville CFT We further introduce the Γb and Υb function that appear in the one-loop regularized
determinants of [24] and whose usage is widespread in the context of Liouville CFT Υbpxq “ Γ´1
b px|, b, b´1qΓ´1
b pQ ´ x|, b, b´1q
Γb “
Γ2px|b, b´1q
Γ2pQ{2|b, b´1q
(A.12) (A.12) – 26 – – 26 – where Q “ b ` b´1. From the meromorphic properties of the Barnes double Gamma it
follows that the Υ function is an entire function of x, has zeroes at x “ ´Ω, Q ` Ω, and
is symmetric under the exchange b Ñ 1{b. The Υ function inherits its properties from the
double Gamma and Zeta functions ΥbpQ{2q “ 1
Υbpxq “ ΥbpQ ´ xq
Υbpx ` bq “ Γpbxqb1´2bx
Γpp1 ´ xqbq Υbpxq
Υbpx ` b´1q “ Γpx{bqb2x{b´1
Γpp1 ´ xq{bq Υbpxq
(A.13) (A.13) JHEP06(2016)035 Also, it admits an integral representation in the strip 0 ă Re pxq ă Q Υbpxq “ exp
ż 8
0
dt
t
»
–
ˆQ
2 ´ x
˙2
e´t ´
t
2 sinh2 ´
Q
2 ´ x
¯
sinh bt
2 sinh t
2b
fi
fl
(A.14) (A.14) We refer the reader to the appendix of [34] for a review of some of the properties of
these special functions. We refer the reader to the appendix of [34] for a review of some of the properties of
these special functions. B
Mapping infinite products In this section we provide evidence that the set of equations (2.6) represents the correct
higgsing of the massless theory on the ellipsoid. We do so by showing that they reproduce
N “ 2 theory on the ellipsoid, N “ 2˚ and N “ 4 on the round sphere when one removes
respectively the mass deformation, the geometric one or both. Let us start from the
contribution of the vector multiplet from [24] is Zvec
1´loop “
ź
αP∆`
1
pˆa0 ¨ αq2
ź
m,ně0
pmb ` nb´1 ` ˆa0 ¨ αqpmb ` nb´1 ` Q ´ ˆa0 ¨ αq
ˆpmb ` nb´1 ´ ˆa0 ¨ αqpmb ` nb´1 ` Q ` ˆa0 ¨ αq
(B.1) (B.1) In their conventions a0 is a Cartan subalgebra valued real matrix, ˆa0, ˆ
M habe been
rescaled by ?R1R2, b “
a
R1{R2 and Q “ b ` 1
b. The infinite product can be regularised
using the Υb function defined in (A.12) “
ź
αP∆`
1
pˆa0 ¨ αq2 Υbpˆa0 ¨ αqΥbp´ˆa0 ¨ αq
(B.2) (B.2) Note that there is difference of rΓ2pQ{2|b, b´1qs4 in the normalisation between their
conventions and the usual definition in CFT. It is of course unessential to keep track of it
as it gets reabsorbed by a redefinition of the infinite multiplicative constant in front of the
partition function. – 27 – The limit b Ñ 1 corresponds to the theory on a round S4. B
Mapping infinite products In this limit the unrenor-
malised product reads ź
m,ně0
pmb ` nb´1 ` ˆa0 ¨ αqpmb ` nb´1 ` Q ´ ˆa0 ¨ αq ź
m,ně0
pmb ` nb´1 ` ˆa0 ¨ αqpmb ` nb´1 ` Q ´ ˆa0 ¨ αq ˆ pmb ` nb´1 ´ ˆa0 ¨ αqpmb ` nb´1 ` Q ` ˆa0 ¨ αq
“
ź
n1ě0
“
pn1 ` ˆa0 ¨ αqpn1 ` 2 ´ ˆa0 ¨ αqpn1 ´ ˆa0 ¨ αqpn1 ` 2 ` ˆa0 ¨ αq
‰n1`1
“
ź
n2ą0
“
pn2 ´ 1 ` ˆa0 ¨ αqpn2 ` 1 ´ ˆa0 ¨ αqpn2 ´ 1 ´ ˆa0 ¨ αqpn2 ` 1 ` ˆa0 ¨ αq
‰n2
(B.3) (B.3) “
ź
n2ą0
“
pn2 ´ 1 ` ˆa0 ¨ αqpn2 ` 1 ´ ˆa0 ¨ αqpn2 ´ 1 ´ ˆa0 ¨ αqpn2 ` 1 ` ˆa0 ¨ αq
‰n2 JHEP06(2016)035 as there are n1 ` 1 ways to write a non-negative integer n1 as the sum of two non-negative
integers m, n. B
Mapping infinite products Rearranging the terms and using the z Ñ ´z symmetry of the Hpzq function,
it is easy to convince oneself that this infinite product is indeed equivalent to ¨ αq2Gp1 ` ˆa0 ¨ αqGp1 ´ ˆa0 ¨ αqGpˆa0 ¨ α ` 1qGpˆa0 ¨ α ´ 1q “ pˆa0 ¨ αq2 H2pˆa0 ¨ αq (B.4) (B.4) One can work out the contribution of an hypermultiplet of mass M in some represen-
tation R in the same way ”
Zhyp
1´loop
ı´1
“
“
ź
ρPR`
ź
m,ně0
ˆ
mb`nb´1` Q
2 ` ˆ
M `ˆa0 ¨ ρ
˙ ˆ
mb`nb´1` Q
2 ´ ˆ
M ´ˆa0 ¨ ρ
˙
ˆ
ˆ
mb ` nb´1 ` Q
2 ` ˆ
M ´ ˆa0 ¨ ρ
˙ ˆ
mb ` nb´1 ` Q
2 ´ ˆ
M ` ˆa0 ¨ ρ
˙
“
ź
ρPR`
Υ
ˆQ
2 ` ˆ
M ` ˆa0 ¨ ρ
˙
Υ
ˆQ
2 ` ˆ
M ´ ˆa0 ¨ ρ
˙
(B.5) (B.5) Note that, since Υpxq “ ΥpQ ´ xq, this is the same as
ź
Υ
ˆQ
2 ` ˆ
M ` ˆa0 ¨ ρ
˙
Υ
ˆQ
2 ´ ˆ
M ` ˆa0 ¨ ρ
˙
(B.6) Note that, since Υpxq “ ΥpQ ´ xq, this is the same as Note that, since Υpxq “ ΥpQ ´ xq, this is the same as
ˆQ
˙
ˆQ
˙ ź
ρPR`
Υ
ˆQ
2 ` ˆ
M ` ˆa0 ¨ ρ
˙
Υ
ˆQ
2 ´ ˆ
M ` ˆa0 ¨ ρ
˙
(B.6) (B.6) Since a0 is real also M is real. B
Mapping infinite products In the b Ñ 1 limit the expression above simply reads ź
ρPR
ź
n1ě0
”´
n1 ` 1 ` ˆ
M ` ˆa0 ¨ ρ
¯ ´
n1 ` 1 ´ ˆ
M ´ ˆa0 ¨ ρ
¯ın1`1
ˆ
”´
n1 ` 1 ` ˆ
M ´ ˆa0 ¨ ρ
¯ ´
n1 ` 1 ´ ˆ
M ` ˆa0 ¨ ρ
¯ın1`1
“ H
´
ˆ
M ` ˆa0 ¨ ρ
¯
H
´
´ ˆ
M ` ˆa0 ¨ ρ
¯
(B.7) ź
ρPR
ź
n1ě0
”´
n1 ` 1 ` ˆ
M ` ˆa0 ¨ ρ
¯ ´
n1 ` 1 ´ ˆ
M ´ ˆa0 ¨ ρ
¯ın1`1 ˆ
”´
n1 ` 1 ` ˆ
M ´ ˆa0 ¨ ρ
¯ ´
n1 ` 1 ´ ˆ
M ` ˆa0 ¨ ρ
¯ın1`1
“ H
´
ˆ
M ` ˆa0 ¨ ρ
¯
H
´
´ ˆ
M ` ˆa0 ¨ ρ
¯
(B.7) (B.7) Using the properties in (A.13), it is possible to get rid of the pˆa0¨αq´2 in the regularised
one loop partition function and to obtain an expression Z1´loop “
ś
αP∆` Υpˆa0 ¨ α ` bq Υp´ˆa0 ¨ α ` bq b´2
ś
hypers
ś
ρPR` ΥpQ{2 ` ˆ
M ` ˆa0 ¨ ρq ΥpQ{2 ` ˆ
M ´ ˆa0 ¨ ρq
(B.8) (B.8) – 28 – – 28 – for which the b Ñ 1 limit is straightforward. Then, availing on (A.6), one can write the
more compact expression for which the b Ñ 1 limit is straightforward. B
Mapping infinite products Then, availing on (A.6), one can write the
more compact expression Υ
ˆ
x ` Q
2
˙
“
ź
m,n
ˆΩ1
Ω
˙
2
´
1 ` x
Ω1
¯ ´
1 ´ x
Ω1
¯
epr01´ 1
ΩqQ´
´
r02`r12´ 1
Ω2
¯ˆ
x2` Q2
4
˙
(B.9) (B.9) through which the overall, a0´independent, multiplicative constant that makes (B.8) finite
can be computed straightforwardly exp
#˜
r02 ` r12 ´
ÿ
m,ně0
1
Ω2
¸
ˆ
«ˆ
ˆa0 ¨ α ` b ´ Q
2
˙2
`
ˆ
´ˆa0 ¨ α ` b ´ Q
2
˙2
´
´
ˆ
M ` ˆa0 ¨ ρ
¯2
´
´
ˆ
M ´ ˆa0 ¨ ρ
¯2
ff+
“ exp
#
2
˜
r02 ` r12 ´
ÿ
m,ně0
1
Ω2
¸ ˜ˆ
b ´ Q
2
˙2
´ ˆ
M2
¸+
(B.10) JHEP06(2016)035 where Ω1 “ Ω` Q
2 and we have assumed that the hypermultiplet comes in the same
representation of the vector multiplet. Again we se that in the limit of no deformation
b Ñ 1, Pestun’s result is readily recovered. C
Kernel function and infinite series The kernel of the saddle point equation (3.2) is conveniently defined through the function Kpxq “ d
dx log Υpx|b, b´1q
“ d
dx
␣
log Γ´1
2 px|b, b´1q ` log Γ´1
2 pQ ´ x|b, b´1q
(
“ ζ2p1; b, b´1|xq ` ζ2p1; b, b´1|Q ´ xq
(C.1) (C.1) the last equality holding by means of the definition of Barnes multiple gamma function d
dx log Γ2px|a, bq “ d
dx
d
ds rζ2px|a, b; sq ´ χps; a, bqss“0
“ d
ds
d
dx
8
ÿ
m,n“0
pam ` bn ` xq´s
ˇˇˇˇ
s“0
“ ´
8
ÿ
m,n“0
pam ` bn ` xq´1
“ ´ζ2px|a, b; 1q
(C.2) (C.2) When inverting the order of the x and s derivatives, we always think about operating
on the suitably regularised function, in which the ζRp1q divergences appearing in the x „ 0
region have been removed. See the discussion after (3.8) about regularising these infinite – 29 – sums. Using the equation above we can easily determine the small x expansion of K ´ d
dx log Γ2pQ ´ xq ´ d
dx log Γ2pQ ` xq
“ ´ζ2pQ ´ x|b, b´1; ´1q ` ζ2pQ ` x|b, b´1; ´1q
“
8
ÿ
m,n“0
"
1
mb ` nb´1 ` Q ` x ´
1
mb ` nb´1 ` Q ´ x
*
“ ´2x
8
ÿ
m,n“0
1
pmb ` nb´1 ` Qq2
„
1 `
x2
pmb ` nb´1 ` Qq2
` O
`
x3˘
(C.3) (C.3) JHEP06(2016)035 We can see from above that the coefficients of this expansions are typically infinite
series involving two indices. In the general case, the sums of these series are rather hard to
compute exactly, therefore we must exploit the dependence on the deformation parameter
b and expand in a neighbourhood of b in which they become treatable. C
Kernel function and infinite series The easiest of such
series, once expanded around b “ 1 ` β with β „ 0, reads 8
ÿ
m`ną0
1
pmb ` nb´1qs „
8
ÿ
m`ną0
„
1
pm ` nqs ´ sβ
m ´ n
pm ` nqs`1
` β2
8
ÿ
m`n“0
„
´s
n
pm ` nqs`1 ` sps ` 1q
2
pm ´ nq2
pm ` nqs`2
(C.4) (C.4) The constant term is obtained simply by setting k “ m`n and counting multiplicities 8
ÿ
m`ną0
1
pm ` nqs “
8
ÿ
k“1
k ` 1
ks
“ ζRps ´ 1q ` ζRpsq
(C.5) (C.5) The first order contribution is zero because of anti-symmetry in m Ø n, hence the first
β-dependent correction is at order β2, as suggested by the b Ø b´1 symmetry. The first
sum at this order is easily seen to be, after symmetrisation, ´ s
2 times the zeroth order. The second sum is computed with the trick of determining the range of the difference l
of two given numbers whose sum k is fixed, indeed l takes the values ´k, ´k ` 2, ´k `
4, . . . , k ´ 2, k, so 8
ÿ
m`ną0
pm ´ nq2
pm ` nqs`2 “
8
ÿ
k“1
1
ks`2
k{2
ÿ
l“´k{2
p2lq2 “ 1
3 rζRps ´ 1q ` ζRpsq ` 2ζRps ` 1qs
(C.6) (C.6) Given the small β expansion of (C.4) it is in principle possible to compute all the
contributions order by order in β, as the series appearing at higher order share this same
structure. As one can see, there are divergences emerging at s “ 2, but they precisely
cancel against the regularization terms. The second easiest sum is Given the small β expansion of (C.4) it is in principle possible to compute all the
contributions order by order in β, as the series appearing at higher order share this same
structure. As one can see, there are divergences emerging at s “ 2, but they precisely
cancel against the regularization terms. C
Kernel function and infinite series The second easiest sum is 8
ÿ
m,n“0
1
pmb ` nb´1 ` Qqs „
8
ÿ
m,n“0
„
1
pm ` n ` 2qs ´ sβ
m ´ n
pm ` n ` 2qs`1
` β2
8
ÿ
m,n“0
„
´s
n ` 1
pm ` n ` 2qs`1 ` sps ` 1q
2
pm ´ nq2
pm ` n ` 2qs`2
(C.7) (C.7) – 30 – – 30 – and can be computed in the same way as above by setting and can be computed in the same way as above by setting 8
ÿ
m,n“0
1
pm ` n ` 2qs “
8
ÿ
k“1
k
pk ` 1qs “
8
ÿ
k“1
k ´ 1
ks
“ ζRps ´ 1q ´ ζRpsq
(C.8) (C.8) The linear-in-β term is again zero for parity reasons, and so are all odd-in-β terms
in the expansion. To compute terms proportional to even powers of β we can use the
same technique of (C.4). Hence the first term appearing at order β2 is again a half of
´ζRps ´ 1q ` ζRpsq. Among the infinite sums appearing above, the last one which is
relevant for the case under study is JHEP06(2016)035 ÿ
regularised
1
´
mb ` nb´1 ` Q
2
¯2 “
8
ÿ
m,n“0
1
´
mb ` nb´1 ` Q
2
¯2 ´
8
ÿ
m`ną0
1
pmb ` nb´1q2
(C.9) (C.9) To this end it is convenient to employ the binomial expansion 8
ÿ
m,n“0
1
´
mb ` nb´1 ` Q
2
¯2 “
8
ÿ
m,n“0
8
ÿ
k“0
4b2
˜
´2
k
¸
pb2 ´ 1qkp2m ` 1qk
p2m ` 2n ` 2qk`2
(C.10) (C.10) and then substitute s “ m ` n as above and reorganise the series 8
ÿ
s“0
sÿ
m“0
8
ÿ
k“0
˜
´2
k
¸
2´kb2 pb2 ´ 1qkp2m ` 1qk
ps ` 1qk`2
(C.11) (C.11) Note that the sum in m is over all odd numbers. Proceeding as above for the contri-
bution coming from the regularisation we obtain a sum over even numbers. Considering
that the relative sign between these contribution is a minus, then we can write (C.9) as Note that the sum in m is over all odd numbers. Proceeding as above for the contri-
bution coming from the regularisation we obtain a sum over even numbers. C
Kernel function and infinite series Considering
that the relative sign between these contribution is a minus, then we can write (C.9) as pC.9q “
8
ÿ
s“0
2s
ÿ
m“0
8
ÿ
k“0
˜
´2
k
¸
2´kb2 p´1qm`1pb2 ´ 1qkmk
sk`2
“
8
ÿ
s“0
2s
ÿ
m“0
4b2p´1qm`1
p2s ` pb2 ´ 1qmq2
(C 12) (C.12) (
)
At this stage it is clear how divergent contributions annihilate against each other. To
this end note that the double sum can be reorganised as At this stage it is clear how divergent contributions annihilate against each other. To
this end note that the double sum can be reorganised as 8
ÿ
s“0
2s
ÿ
m“0
“
8
ÿ
m“0
8
ÿ
s1“2
´
8
ÿ
m“2
m
ÿ
s1“2
(C.13) (C.13) where s1 “ 2s, so that the region in the origin is removed. The series over s1 is (almost)
the definition of polygamma functions pC.9q “ 4b2
8
ÿ
m“1
p´1qm`1ψp1qpb2m ` 1q ´ 4ψp1qp2q
(C.14) (C.14) The series above is dominated by its tail, and in the m " 1 region the summand
behaves like
1 The series above is dominated by its tail, and in the m " 1 region the summand
behaves like
1 ψp1qpb2m ` 1q „
1
b2m ` 1
(C.15) (C.15) – 31 – – 31 – Therefore the last series can be rephrased using the definition of the Hurwitz-Lerch
Phi function, and considering that ψp1qp2q “ ζRp2q ´ 1, eventually we can write pC.9q “ 4
“
Φp´1, 1, b´2 ` 1q ` 1 ´ ζRp2q
‰
(C.16) (C.16) Lastly, note that the for large b the Phi function asymptotes a constant value
Φp´1, 1, 1q “ log 2. Lastly, note that the for large b the Phi function asymptotes a constant value
Φp´1, 1, 1q “ log 2. D
Analytic solution of the compact model in the M „ Q
2 limit The solution of the integral equation (4.2) was constructed in [26] availing on some older
ideas ascribable to J. Hoppe. We review such construction here and add the dependence
on the effective mass m. Later on, we analytically continue the result in the region where
parameters acquire physical values and draw our conclusions. We will try to keep the
notation as close as possible to the one in the original paper, and we refer the interested
reader to the latter for all the details that we will skip here. JHEP06(2016)035 Let us first remind about the notation. In our original problem the scalar fields take
purely imaginary values, which thing is required by the convergence of the path integral;
in our notation they are expressed as iˆa0. Up to this point a0 is a matrix in the Cartan
subalgebra of the gauge group and the hat is the operation of multiplication by the square
root of the inverse product of the two radia of the ellipsoid. It follows that the mass term
is also purely imaginary, but the deformation parameter Q is by definition real and we will
need to analytically continue over it. For the sake of neatness we hence choose the following
notation - eigenvalues of the matrix iˆa0 are here denoted as x, y, the hypermultiplet mass M
is purely imaginary and Q is real. The purpose of this section is to consider the particular
limit in which Q
2 ´ M1 “ m „ 0, which makes proper sense only when we continue to
M1 “ iM P R. In the limit we are considering the saddle point equation reads ż
m2 ρpyq dy
px ´ yq rpx ´ yq2 ´ m2s “ ´8π2
λ x
(D.1) (D.1) The problem where m2 “ ´1 was solved in [26], therefore we set m “ im1 and consider
real m1. To this end let us introduce the generalised resolvent The problem where m2 “ ´1 was solved in [26], therefore we set m “ im1 and consider
real m1. D
Analytic solution of the compact model in the M „ Q
2 limit To this end let us introduce the generalised resolvent Gpyq “ y2
g2 ` i
„
W
ˆ
y ` im1
2
˙
´ W
ˆ
y ´ im1
2
˙
Wpzq “
ż µ
´µ
dy ρpyq
z ´ y
(D.2) (D.2) which is related to the density of eigenvalues through which is related to the density of eigenvalues through lim
ϵÑ0 tWpy ` iϵq ´ Wpy ´ iϵqu “ ´2πiρpyq
(D.3) (D.3) Considering then the following equation Considering then the following equation G
ˆ
x ` im1
2
˙
“ G
ˆ
x ´ im1
2
˙
(D.4) (D.4) – 32 – for x belonging to the support of the eigenvalue distribution, by definition we get Wpx ` iϵq ` Wpx ´ iϵq ´ Wpx ` im1q ´ Wpx ´ im1q “ 2x
g2
for ϵ Ñ 0
(D.5) (D.5) that corresponds to (D.1) with the identification g2 “ m12λ
4π2 . The definition of Gpzq implies
it has two square root branch cuts over the intervals z P r˘µ ˘ im1{2s. Moreover one can
convince oneself that the function Gpzq is real on the real z´axis, the imaginary axis and
on the cuts. Thus, the problem of determining ζ “ Gpzq is equivalent to the problem of
finding the inverse map ζpzq that maps the upper half plane to the domain of reality of G. Since Gpzq is a holomorphic function on the upper-right quadrant deprived by the half cut
rim1{2, µ ` im1{2s, the inverse map is uniquely determined by its Hilbert transform JHEP06(2016)035 zpζq “ A
ż ζ
x1
dt
pt ´ x3q
a
pt ´ x1qpt ´ x2qpx4 ´ tq
(D.6) (D.6) where the turning points x1 ą x2 ą x3 ą x4 are the values of Gpzq respectively at
z “ 0, im1{2 ´ iϵ, µ ` im1{2, im1{2 ` iϵ. D
Analytic solution of the compact model in the M „ Q
2 limit Imposing these actual values on ζpzq one is able to
write a set of integral equations for the physical quantities m1
2 “ A
ż x1
x2
dt
pt ´ x3q
a
pt ´ x1qpt ´ x2qpx4 ´ tq
µ “ A
ż x2
x3
dt
px3 ´ tq
a
pt ´ x1qpt ´ x2qpx4 ´ tq
µ “ A
ż x3
x4
dt
px3 ´ tq
a
pt ´ x1qpt ´ x2qpx4 ´ tq
(D.7) (D.7) The remaining unknown quantities can be fixed by considering the large ζ asymp-
totics (D.6) and matching it with large z asymptotics of Gpzq in (D.2) A “ g
2
g
2
ż 8
x1
dt
1
?t ´ x1
“ ζp8q
x1 ` x2 ` x4 “ 2x3
x2
1 ` x2
2 ` x2
4 ´ 2x2
3 “ 6m12
g2
m13 ´ 12m1
ż
x2ρpxqdx “ 8g4a
(D.8) (D.8) The last equation is particularly useful to determine the second moment of the eigen-
value distribution in parametric form in terms of the quantity The last equation is particularly useful to determine the second moment of the eigen-
value distribution in parametric form in terms of the quantity a “ 1
40
`
´x3
1 ` px2 ` x4q x2
1 ` px2 ` x4q 2x1 ´ px2 ´ x4q 2 px2 ` x4q
˘
(D.9) (D.9) The sets of equations (D.7) and (D.8) fully solve the problem, albeit in a somewhat
implicit way. To get some clue on the nature of the solution it turns out to be helpful to – 33 – rephrase (D.7) in terms of elliptic integrals. Using the relations rephrase (D.7) in terms of elliptic integrals. Using the relations rephrase (D.7) in terms of elliptic integrals. Using the relations ż u
c
dt
t
a
pa ´ tqpb ´ tqpt ´ cq
“
2a
?a ´ c Fpγ, lq ´ 2
? a ´ c Epγ, lq
a ą b ě u ą c
ż u
c
dt
1
a
pa ´ tqpb ´ tqpt ´ cq
“
2
?a ´ c Fpγ, lq
a ą b ě u ą c
ż a
u
dt
t
a
pa ´ tqpt ´ bqpt ´ cq
“
2c
?a ´ c Fpδ, pq ´ 2
? D
Analytic solution of the compact model in the M „ Q
2 limit a ´ c Epδ, pq
a ą u ě b ą c
ż a
u
dt
1
a
pa ´ tqpt ´ bqpt ´ cq
“
2
?a ´ c Fpδ, pq
a ą u ě b ą c
(D.10) (D.10) JHEP06(2016)035 Fpγ, lq “
ż γ
0
dα
a
1 ´ l sin2 α
Epγ, lq “
ż γ
0
dα
a
1 ´ l sin2 α
(D.11) (D.11) are the incomplete elliptic integrals of, respectively, the first and second kind and γ “ arcsin
c
u ´ c
b ´ c
δ “ arcsin
c
a ´ u
a ´ b
l “ b ´ c
a ´ c
l2 ` p2 “ 1
(D.12) (D.12) are their moduli, one obtains from (D.7) the set of parametric equations p´x1 ` x2 ` x4qKplq ` 2px1 ´ x4qEplq “ 0
(D.13)
px3 ´ x1qFpγ, lq ` px1 ´ x4qEpγ, lq “ 2µ
g
?x1 ´ x4
(D.14)
2px4 ´ x3qKppq ` 2px1 ´ x4qEppq “ m1
g
?x1 ´ x4
(D.15) (D.15) In the first and last equation above we have used the fact that for γ “ π
2 incomplete
integrals become complete elliptic integrals Kplq “ Fpπ{2, lq and Eplq “ Epπ{2, lq. It is
convenient to introduce new variables In the first and last equation above we have used the fact that for γ “ π
2 incomplete
integrals become complete elliptic integrals Kplq “ Fpπ{2, lq and Eplq “ Epπ{2, lq. D
Analytic solution of the compact model in the M „ Q
2 limit It is
convenient to introduce new variables λi “
xi
x1 ´ x4
yi “ g xi
(D.16) (D.16) for which one can easily see that for which one can easily see that l “ λ2 ´ λ4
1 ´ l “ λ1 ´ λ2
(D.17) (D.17) The last λi is fixed by the third equation in (D.8), while equation (D.13) determines
parametrically
p q The last λi is fixed by the third equation in (D.8), while equation (D.13) determines
parametrically λ2 “ 1 ´ 2θplq “ 1 ´ 2 Eplq
Kplq
(D.18) (D.18) Using these relations it is also straightforward to express the modular angle in (D.14)
in parametric form Using these relations it is also straightforward to express the modular angle in (D.14)
in parametric form sin2 γ “ Kplq ´ Eplq
l Kplq
(D.19) (D.19) – 34 – – 34 – At this stage all quantities are fixed in terms of one single parameter, the elliptic
modulus l P r0, 1s, and it is just a matter of algebra to re-write (D.14) and (D.15) as g2plq “ m12
π4 χ2plqK4plq
µplq “ m1
π rKplqEpγ, lq ´ EplqFpγ, lqs
(D.20) (D.20) where the shorthand χ reads where the shorthand χ reads χ2plq “ 4λ2m1 ´ 3λ2
2 ´ 2λ2 ` 1
12
“ lp1 ´ 2θplqq ´ 3θ2plq ` 4θplq ´ 1
3
(D.21) (D.21) JHEP06(2016)035 The weak coupling expansion corresponds to taking l Ñ 0. Expanding g2plq and
inverting the series one finds a perturbative expression for the elliptic modulus The weak coupling expansion corresponds to taking l Ñ 0. Expanding g2plq and
inverting the series one finds a perturbative expression for the elliptic modulus “ 16
? 2g
m1
´ 64g2
m12 ` 164
? 2g3
m13
´ 576g4
m14 ` 695
? 2g5
m15
´ 1152g6
m16
` 1677g7
? 2m17 ´ 1760g8
m18
` 13531g9
8
? 2m19 `O
`
g10˘ (D.22) that can be plugged into the quantities of interest to obtain a genuine small coupling
expansion. Note that, as can be already understood from the first of (D.20), the coupling g
appears rescaled by the effective mass m1 at all orders of perturbation theory. This suggests
that, at weak coupling, the width of the eigenvalue distribution µ must be proportional to
the rescaled combination g “ m1? λ, as there are no energy scales other that m1 in (D.1). D
Analytic solution of the compact model in the M „ Q
2 limit Indeed one finds µpgq “
? 2g ´
g3
? 2m12 `
15g5
4
? 2m14 ´
165g7
8
`? 2m16˘ ` 8555g9
64
? 2m18 ` O
`
g11˘
g ! 1
(D.23) In order to extract information about the opposite regime one needs to determine the
asymptotics for l Ñ 1. Since the elliptic integral Kplq is logarithmically divergent at l “ 1
it is convenient to substitute variables in the following way In order to extract information about the opposite regime one needs to determine the
asymptotics for l Ñ 1. Since the elliptic integral Kplq is logarithmically divergent at l “ 1
it is convenient to substitute variables in the following way l “ 1 ´ e´L
(D.24) (D.24) The asymptotics of (D.20) as L Ñ 8 produces otics of (D.20) as L Ñ 8 produces The asymptotics of (D.20) as L Ñ 8 produces g2 “ m12pL ´ 3 ` 4 logp2qqpL ` 4 logp2qq2
12π4
(D.25) (D.25) which differs from the result in [26] only in the choice of notation for the logarithmic
divergence regulator L (the logp2q can be reabsorbed in the definition of L). After inverting
the series one has
1 L “ ˜g ` 1 ` 1
˜g ` O
`
˜g´2˘
(D.26) (D.26) being being
˜g “ π
4
3 2
2
3 3
1
3 g
2
3
m
2
3
(D.2 ˜g “ π
4
3 2
2
3 3
1
3 g
2
3
m
2
3
(D.27) (D.27) from which one obtains the strong coupling asymptotics from which one obtains the strong coupling asymptotics µp˜gq “ m1˜g
2π ` O
`
˜g0˘
(D.28) µp˜gq “ m1˜g
2π ` O
`
˜g0˘ (D.28) – 35 – – 35 – Momenta of the eigenvalue distribution. Comparing the large ζ asymptotics (D.6)
and large z asymptotics of Gpzq in (D.2) one can recursively compute even momenta of the
eigenvalue distribution νpnq “
ş
x2nρpxqdx (odd-momenta vanish for parity). D
Analytic solution of the compact model in the M „ Q
2 limit In particular
for the first few of them one gets a2 “ m12
g2 νp0q
a3 “ m13 ´ 12m1νp1q
8g4
a4 “ 5m14
g4
` m16
32g5 ` 5m1νp2q
2g6
´ 5m13νp1q
4g6
a5 “
m17
128g8 ` 7m14
16g6 ´ 35m12νp1q
4g6
´ 21m15νp1q
32g8
` 35m13νp2q
8g8
´ 7m1νp3q
2g8
(D.29) (D.29) JHEP06(2016)035 where the ak have been computed in [26] and read where the ak have been computed in [26] and read ak “ p´1qk´1
2k ´ 1 pγk ` x3 γk´1q
γk “
ÿ
p`q`r“k
˜
´1
2
p
¸ ˜
´1
2
q
¸ ˜
´1
2
r
¸
xp
1 xq
2 xr
4
(D.30) As the x’s are completely determined in terms of complete elliptic integrals, so are
the momenta. For a smooth distribution ρpxq “ ř c2 x2n this is an efficient way to fix the
coefficients cn recursively and compute a polynomial approximation to ρpxq that converges
quickly enough. But this turns out not to be always the case under study, at least not for
arbitrary values of the coupling constant, as ρpxq can develop cusps at its endpoints. The
first of equations (D.29) is simply the normalization of the eigenvalue distribution; we are
in particular interested in the second one because of the fact that the second momentum
is proportional to the derivative of the free energy. The expression we get is a slight
modification of its homologous in [26] due to the presence of the mass parameter νp1qplq “ m12
12 ´ 2m1K2plqθplq
“
5θplq
`
θplq ` l ´ 2
˘
` pl ´ 6ql ` 6q ` p2 ´ lqpl ´ 1q
‰
5π2“
θplq
`
3θplq ` 2l ´ 4
˘
´ l ` 1
‰
(D.31) (D.31) E
Analytic continuation and behaviour of the solution at the boundar Analytic continuation and behaviour of the solution at the boundary Interestingly, it turns out that there exist alternative descriptions of the analytic solution
of the master field equation (D.1). In the limit where the effective mass m is very small
compared to average eigenvalue separation, the kernel of (D.1) is the discretized version of
the Laplace operator acting on the Hilbert kernel LrHpxqs “ lim
ϵÑ0
1
ϵ2
ˆ
Hpxq ´ 1
2Hpx ´ ϵq ´ 1
2Hpx ` ϵq
˙
(E.1) (E.1) Setting m Ñ 0 we can take the continuous limit of L, so that the saddle point equation
can be written as Setting m Ñ 0 we can take the continuous limit of L, so that the saddle point equation
can be written as
ż µ
1
8
2 ż µ
´µ
dz ρpzq
1
px ´ zq3 “ ´ 8π2
m2λ
(E.2) (E.2) – 36 – – 36 – Physical values of Q, M imply that m is a real number, which in turn determines
boundary conditions to be ρp˘µq “ 0. Hence we can look for a solution of the kind ρRpzq “
a
µ2 ´ z2
ÿ
ně1
an z2n
(E.3) (E.3) To lowest order in this expansion, one can fix the first coefficient through the saddle
point equation, and subsequently determine µpλq requiring normalisation of the density
ş
ρdz “ 1 To lowest order in this expansion, one can fix the first coefficient through the saddle
point equation, and subsequently determine µpλq requiring normalisation of the density
ş
ρdz “ 1 a1 “
4π
3m2λ,
µRpλq “
c m
ζπp6 λq1{4
(E.4) (E.4) JHEP06(2016)035 where we have set back dimensionful quantities and ζ “ ?R1R2 was introduced in (3.1)
and keeps track of the compactification scale (or equivalently the energy scale). We then
have a representation of the solution that behaves near the boundaries according to Wigner
semi-circle law
2 where we have set back dimensionful quantities and ζ “ ?R1R2 was introduced in (3.1)
and keeps track of the compactification scale (or equivalently the energy scale). We then
have a representation of the solution that behaves near the boundaries according to Wigner
semi-circle law ρRpzq “ 8πz2
3µ2
R
b
µ2
R ´ z2
(E.5) (E.5) Let us further consider the analytic continuation to imaginary values of m. With a
slight abuse of notation we set m2 Ñ ´m2. Analytic continuation and behaviour of the solution at the boundary The relevant master field equation then admits
solutions with inverse square root behaviour near the boundary of the eigenvalue support Let us further consider the analytic continuation to imaginary values of m. With a
slight abuse of notation we set m2 Ñ ´m2. The relevant master field equation then admits
solutions with inverse square root behaviour near the boundary of the eigenvalue support ρIpzq “
1
a
µ2 ´ z2
ÿ
ně1
bn z2n
(E.6) (E.6) The lowest term in the polynomial clearly gives null contribution, to first non-trivial
order in this expansion one then has The lowest term in the polynomial clearly gives null contribution, to first non-trivial
order in this expansion one then has ρIpzq “
8z4
3πµ4
I
b
µ2
I ´ z2
µIpλq “
c m
ζπp2 λq1{4
(E.7) (E.7) Through analytic continuation the eigenvalue distribution changes drastically, but it is
not a surprising phenomenon. Indeed, changing the sign of m2 amounts to changing the sing
of the coupling constant and therefore this is a well known matrix model phase transition. There is a whole variety of questions that arise in this context, such as determining the role
of matrix models instantons to this phase transition and whether the transition itself can
have relevant effects in our original problem, but we will not discuss them any further herein. Open Access. This article is distributed under the terms of the Creative Commons
Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in
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Mediastinal Pseudocyst: Varied Presentations and Management—Experience from a Tertiary Referral Care Centre in India
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Hindawi
HPB Surgery
Volume 2017, Article ID 5247626, 7 pages
http://dx.doi.org/10.1155/2017/5247626 Hindawi
HPB Surgery
Volume 2017, Article ID 5247626, 7 pages
http://dx.doi.org/10.1155/2017/5247626 Hindawi
HPB Surgery
Volume 2017, Article ID 5247626, 7 pages
http://dx.doi.org/10.1155/2017/5247626 Durairaj Segamalai, Abdul Rehman Abdul Jameel, Naveen Kannan, Amudhan Anbalagan,
Benet Duraisamy, Prabhakaran Raju, and Kannan Devy Gounder
Institute of Surgical Gastroenterology, Madras Medical College, Chennai 600003, India Durairaj Segamalai, Abdul Rehman Abdul Jameel, Naveen Kannan, Amudhan Anbalagan,
Benet Duraisamy, Prabhakaran Raju, and Kannan Devy Gounder
Institute of Surgical Gastroenterology, Madras Medical College, Chennai 600003, India Correspondence should be addressed to Kannan Devy Gounder; malarkan08@gmail.com Received 22 September 2016; Revised 1 February 2017; Accepted 26 February 2017; Published 14 March 2017 Copyright © 2017 Durairaj Segamalai et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited. Pseudocysts are a recognised complication following acute or chronic pancreatitis. Usually located in peripancreatic areas, they
have also been reported to occur in atypical regions like liver, pelvis, spleen, and mediastinum. Mediastinal pseudocysts are a rare
entity and present with myriad of symptoms due to their unique location. They are a clinical challenge to diagnose and manage. In
this paper, we describe the clinical and radiological characteristics of mediastinal pseudocysts in 7 of our patients, as well as our
experience in managing these patients along with their clinical outcome. 3. Results Figure 2: CT chest coronal view showing mediastinal pseudocyst of
size 8 cm. The details of the patients are mentioned in Table 1. Of the
7 patients 6 were male and one was female with age ranging
from 17 to 40. Our analysis reveals that chronic pancreatitis
was present in 6/7 patients with mediastinal pseudocyst, with
ethanol being the most common etiological factor causing
chronic pancreatitis in 5/6 patients and it was idiopathic in
the other patient. Acute necrotising pancreatitis was observed
in one patient who had mediastinal pseudocyst. y
Almost all of the patients had abdominal pain in addi-
tion to symptoms attributed to mediastinal pseudocyst like
dyspnoea (𝑛= 3), dysphagia (𝑛= 2), chest pain (𝑛= 2),
and retrosternal discomfort (𝑛= 1). The mean size of the
mediastinal pseudocyst encountered in this series was 5.7 cm
(ranging from 3 cm to 8 cm) (Figures 1 and 2). All the patients
had abdominal pseudocyst. Two patients had dysphagia
due to compressive effects of pseudocyst on the esophagus
(Figure 3). One of the patients, who had dysphagia with
considerable weight loss (>15% in 3 months), was referred to
our unit as achalasia cardia on the basis of barium swallow
report and he was totally relieved of his symptom following
internal drainage (Figure 4). Two patients who presented
with dyspnoea had severe left ventricular dysfunction, due
to compressive effects on cardiac chambers (Figure 5), which
improved following treatment. Pleural effusion was observed
in five individuals. Figure 2: CT chest coronal view showing mediastinal pseudocyst of
size 8 cm. advocate internal drainage in the form of cystogastrostomy
or cystojejunostomy based on the anatomic proximity of the
pseudocyst to the stomach wall. In our series, we performed
cystogastrostomy as most of the pseudocysts were related to
posterior gastric wall. We encountered pleural effusion in five
patients but only two of them required insertion of intercostal
drainage tube, in view of symptoms. i
We summarize our proposed treatment algorithm in
Figure 6, which is based on our experience in managing
such cases. We have noted in our series that mediasti-
nal pseudocyst is invariably associated with peripancreatic
pseudocyst and management directed towards the pancre-
atic pseudocyst leads to resolution of mediastinal compo-
nent. Pancreatic ductal morphology and its communication
(Figure 7) with the pseudocyst as documented by MRCP
are the key parameters influencing treatment strategies. 1. Introduction This study is a retrospective analysis of patients diagnosed
to have mediastinal pseudocyst between Jan 2010 and March
2016 at our Institute. Our Institute is a high volume tertiary
referral care centre in India, where more than 200 pan-
creatic surgeries are performed annually for various benign
and malignant disorders. We reviewed clinical records and
imaging database and were able to identify 7 patients with
mediastinal pseudocyst. Thorough analysis regarding their
clinical symptomatology, etiology of pancreatitis, radiological
features, supplementary investigations, management strategy,
and their follow-up was done. Basic laboratory investigation
including serum amylase, lipase, and c-reactive protein was
done. All patients underwent oesophagogastroduodenoscopy
(OGD), ultrasound (USG) with portal Doppler, contrast
enhanced computed tomography (CT) scan of abdomen and
chest, and other investigations like echocardiogram, barium
swallow, and magnetic resonance cholangiopancreatography
(MRCP) as required. Patient presenting with ascites or
pleural effusion underwent aspiration and biochemical fluid
analysis. Patients who had dysphagia were graded as per
dysphagia score of Knyrim et al. [3]; grade 0 denoted the Pancreatic pseudocysts are common findings in patients
with acute or chronic pancreatitis, usually located in peri-
pancreatic areas. Mediastinal pseudocyst is rare and often
reported as case reports, exact incidence being unknown
[1]. Pancreatic ductal disruption due to inflammatory injury
leads to leakage of amylase rich pancreatic secretions along
the paths of least resistance. Posterior disruptions can lead to
thoracopancreatic fistulae while anterior disruptions produce
pancreatic ascites. Thoracopancreatic fistulas are divided into
four types based on the termination site of the fistula: pancre-
aticopleural, mediastinal pseudocyst, pancreaticobronchial,
and pancreaticopericardial. Mediastinal pseudocyst by way
of its unique location can present with myriad symptoms
like dysphagia, chest pain, or palpitations and in extreme
cases pericardial effusion, tamponade, and respiratory dis-
tress [2]. It can be a diagnostic and therapeutic challenge. High index of suspicion is often needed in diagnosing this
entity. Pancreatic ductal morphology and its communication
with the pseudocyst hold the key for successful manage-
ment. We present our experience in managing mediastinal
pseudocysts. 2 HPB Surgery Mediastinal pseudocyst
Pleural effusion
Figure 1: CT chest demonstrating mediastinal pseudocyst of size
3 cm, associated with left pleural effusion. ability to eat a normal diet; 1, the ability to eat some solid food;
2, the ability to eat semisolids only; 3, the ability to swallow
liquids only; and grade 4 referred to complete dysphagia. 1. Introduction Subsequent to the definitive intervention, the patients were
advised to attend the follow-up clinic at our institution after
3 months or earlier if symptomatic. All patients underwent
CT chest/abdomen to document the resolution of pseudocyst
and to study the disease activity after 3 months following the
primary intervention. Patients were advised to review with
us after 6 months thereafter, to document any complications. Follow-up of patients was for a mean of 13 months (range 10 to
18 months). We did not encounter any dropouts in our series. Figure 1: CT chest demonstrating mediastinal pseudocyst of size
3 cm, associated with left pleural effusion. 3. Results In
the absence of ductal communication, ultrasound guided
insertion of percutaneous catheter drains (PCDs), preferably
10–12 Fr, has been found to be helpful. From our expe-
rience, invariably multiple PCDs are required in patients
with necrotic or infected collections to ensure complete
resolution as they tend to block the catheters and are also
not adequately drained by a single catheter. Patients with
multiple ductal strictures or intraductal calculi will require a
Frey’s procedure to adequately drain the entire ductal system. For those with ductal communication with strictures, we Internal drainage could also be performed by endoscopic
approach, preferably with endoscopic ultrasound guidance
(EUS), if expertise is available. We did not employ endoscopic
approach in our series as endoscope could not be negotiated
beyond oesophagogastric junction in two patients, while two
patients did not require internal drainage and were managed
with PCDs. One of our patients had multiple peripancreatic
pseudocyst with walled-off pancreatic necrosis, for whom
we felt that surgical drainage would offer better results. Endotherapy could not be considered for the patient with
multiple ductal stricture associated with intraductal calculi. To summarize, internal drainage in the form of cystogastros-
tomy was performed in four patients, while Frey’s procedure
was done in one patient. Two patients were managed with
multiple PCDs and intercostal drainage.i Apart from the specific management, patients were gen-
erally managed with intravenous fluids, analgesics, antiemet-
ics, and antibiotics being initiated in those with infected 3 HPB Surgery Table 1: The table shows patient demographics, presentation, clinical and radiological finding, management, and follow-up. S. 3. Results Figure 4: Barium swallow (preop and postop) demonstrating the
esophageal dilatation due to mediastinal pseudocyst and resolution
of compressive effects following surgery. and fibrosis along the traditional peripancreatic spaces cre-
ates pathways of lesser resistance to mediastinum to form
thoracopancreatic fistulas [6]. Thoracopancreatic fistulas are
divided into four types based on the termination site of the
fistula: pancreaticopleural, mediastinal pseudocyst, pancre-
aticobronchial, and pancreaticopericardial [7]. In most cases,
the pseudocyst is located in the posterior mediastinum, with
entry to the mediastinum via the aortic or esophageal hiatus
[4]. Figure 4: Barium swallow (preop and postop) demonstrating the
esophageal dilatation due to mediastinal pseudocyst and resolution
of compressive effects following surgery. Most patients are alcoholics with a clinical history of
previous pancreatitis. The presentation is often confusing
because of the paucity of clues suggestive of pancreatic
disease and the preponderance of pulmonary signs and
symptoms. The most common presenting symptoms are chest
or abdominal pain and dyspnoea [8]. Due the mediastinal
location of the pseudocyst, dysphagia occurs because of
compression of the esophagus by the pseudocyst [6]. In
our study, two patients had dysphagia. Since the origin of
pseudocyst is a ductal disruption in the pancreas, invariably
most of our patients had abdominal pain and coexistent
abdominal pseudocysts. pseudocysts. Patients tolerating oral diet were encouraged to
do so with low-fat diet, while those with low or no intake
were put on nasojejunal feeding and total parenteral nutrition
instituted when nasojejunal tube could not be placed. Our
unit does not use somatostatin analogues in such patients due
to lack of strong evidence in literature. Interestingly, we noted increased incidence of vascular
complications in these patients. For the patient with pancre-
aticopleural fistula, reported with hemoptysis 2 weeks after
intercostal drainage, CT-angiogram showed multiple pseu-
doaneurysm involving inferior phrenic and lower intercostal
arteries which was eventually managed with angioembolisa-
tion with coils (Figure 8), while another patient with infected
pseudocyst developed splenic artery pseudoaneurysm in the
follow-up period and required angioembolisation (Figure 9). Mediastinal pseudocyst resulted in left brachiocephalic and
left internal jugular vein (IJV) thrombosis (Figure 10) in a
patient who was put on anticoagulant therapy for 6 months
and subsequently recanalised. p
y
Ultrasound is an easily available investigation to diagnose
peripancreatic pseudocyst, but it is less helpful in mediastinal
pseudocyst owing to its location. 3. Results number Age/sex
Etiology
Acute/chronic
Presenting
symptoms
Size of
mediastinal
pseudocyst
Presence of abdominal
pseudocyst
Associated complications
Management
Follow-up
1
40/M
Ethanol
Chronic
Grade 3 dysphagia1
Weight loss
Abdominal pain
5 cm
Yes
Communicating with
mediastinal pseudocyst
Severe OG2 junction
narrowing in barium swallow
Endoscopy could not
negotiate beyond
oesophagogastric junction
Open cystogastrostomy
Dysphagia relieved
Postop barium
swallow: normal
Gained weight
2
29/M
Ethanol
Acute
Dsypnoea
Pain abdomen
Hemoptysis, 2
weeks following
intercostal drainage
8 cm
Yes
8 × 8 cm infected
necrosis involving
body & tail of pancreas
Pancreaticopleural fistula
40% necrosis of pancreatic
body & tail
Echocardiogram: severe left
ventricular dysfunction EF3
37%
Inferior phrenic & intercostal
artery pseudoaneurysms
Infected necrosis was
managed with 2
percutaneous drainage
catheters inserted with
ultrasound guidance in
left subphrenic &
perinephric region
Intercostal drainage
Angioembolisation of
pseudoaneurysms
Follow-up
Complete resolution
of pseudocyst after 2
months
Echocardiogram: EF
70%
3
31/M
Ethanol
Chronic
Abdominal pain
Dsypnoea
4.5 cm
Yes
Infected pseudocyst 8
× 8 cm: head and body
of pancreas
Left pleural effusion
Pancreatic ascites
Three PCDs inserted in
left subphrenic, left
perinephric region and
pelvis
Left intercostal drainage
2 months later, he
developed splenic
artery
pseudoaneurysm:
angioembolisation
done
4
36/M
Ethanol
Chronic
Chest pain
Early satiety
Abdominal pain
3 cm
Yes
8 × 6 cm pseudocyst in
head of pancreas
Nil
Open cystogastrostomy
Complete resolution
of pseudocyst after 1
week
5
39/M
Ethanol
Chronic
Retrosternal
discomfort
Dsypnoea
Pain abdomen
8 cm
Yes
Multiple peripancreatic
pseudocyst
Left pleural
effusion/walled-off pancreatic
necrosis
Echocardiogram: severe left
ventricular dysfunction
EF 40%
Open cystogastrostomy,
open necrosectomy,
external drainage
Pseudocyst Resolved
EF improved to 64%
6
33/M
Ethanol
Chronic
Chest pain
Dysphagia
Abdominal pain
6.5 cm
Yes. Multiple peripancreatic
pseudocyst
Bilateral pleural effusion
Extrinsic compression over
esophagus from 34–38 cm
Open cystogastrostomy
Resolutions of
symptoms
7
17/F
Idiopathic
Chronic
Abdominal pain
Left sided neck
pain and edema
4 cm
Yes
Pseudocyst 2 × 2 cm
head of pancreas
Multiple parenchymal
and ductal calculi
Left pleural effusion
Left IJV4/brachiocephalic vein
thrombosis
Frey’s procedure
Thrombus
recanalised after 6
months of
anticoagulant therapy
1D
h
i
f K
i
t l HPB Surgery 4 Figure 3: CT chest showing mediastinal pseudocyst compressing
the esophagus. Figure 5: CT chest sagittal view demonstrating the compressive
effects on cardiac chambers. Figure 3: CT chest showing mediastinal pseudocyst compressing
the esophagus. Figure 5: CT chest sagittal view demonstrating the compressive
effects on cardiac chambers. 3. Results Computed tomography
(CT) is excellent in defining pancreatic abnormalities and
should be the first abdominal imaging study in suspected
cases [9]. CT can also comment on the connection between
the mediastinal cystic structures and the pancreas. MRI
can help in delineating the communication of mediastinal
pseudocysts with an abdominal pseudocyst; in addition
ductal morphology like disruption, communication with
pseudocyst, stricture, and dilatation are best defined by
magnetic resonance cholangiopancreatography (MRCP) [5]. Endoscopic ultrasound (EUS) is an important diagnostic
tool for the evaluation of mediastinal mass and cysts, and
it can help in planning the optimal therapy and allow EUS
guided aspiration and drainage of the cysts but is limited by
availability of equipment and expertise [10]. 4. Discussions Abdominal complications like pseudocyst and pancreatic
necrosis are recognised complications which occur as seque-
lae to acute or chronic pancreatitis [4]. Mediastinal pseudo-
cyst is a rare complication, usually detected on imaging stud-
ies performed for pancreatitis [5]. Presence of inflammation HPB Surgery 5 Assess: ductal communication
MRCP
Yes
Ductal morphology: multiple strictures/
intraductal calculi
Yes
Frey’s procedure
No
Internal drainage: cystogastrostomy/
EUS guided endotherapy
No
Percutaneous catheter drainage
Mediastinal pseudocyst
peripancreatic pseudocyst
+
Figure 6: Proposed treatment algorithm for management for symptomatic mediastinal pseudocyst. Percutaneous catheter drainage Figure 6: Proposed treatment algorithm for management for symptomatic mediastinal pseudocyst. Figure 7: MRI abdomen showing pseudocyst involving tail of
pancreas with mediastinal extension and MRCP showing ductal
communication of the pseudocyst. Figure 8: Chest X-ray demonstrating multiple coil embolization
done for pseudoaneurysm of left inferior phrenic artery and mul-
tiple intercostal arteries. Note the presence of percutaneous placed
catheter for drainage of peripancreatic necrosis. Figure 7: MRI abdomen showing pseudocyst involving tail of
pancreas with mediastinal extension and MRCP showing ductal
communication of the pseudocyst. Figure 8: Chest X-ray demonstrating multiple coil embolization
done for pseudoaneurysm of left inferior phrenic artery and mul-
tiple intercostal arteries. Note the presence of percutaneous placed
catheter for drainage of peripancreatic necrosis. The management of mediastinal pseudocysts depends
on the clinical symptomatology, underlying etiology, ductal
anatomy, size of the pseudocyst, and availability of exper-
tise. Spontaneous resolution of mediastinal pseudocyst with
conservative management is a rare event [11]. Endoscopic
procedures have significantly influenced the management of
mediastinal pseudocysts. EUS assisted endoscopic drainage
through either a transoesophageal [1, 12] or transgastric
approach has been described with immediate technical
success in 90–95% of patients and long term success in
85–90% patients [13]. Endoscopic retrograde cholangiopan-
creatogram (ERCP) has been employed for transpapil-
lary stenting of the pancreatic duct and few reports have
described successful resolution of mediastinal pseudocysts
with transpapillary stenting alone [12, 14]. ERCP carries
with it its own set of complications, including pancreatitis, haemorrhage, duodenal perforation, and cholangitis. Com-
plications of EUS guided cyst aspiration include perforation
of the oesophagus, infection, and stricture formation [12]. The general complication rate of endoscopic management
procedures for pancreatic pseudocysts is approximately 5%
and pancreatic pseudocysts recur in approximately 15% of
patients [15].t Surgical treatment has often been used for therapeutic
management of patients with mediastinal pseudocyst and
these can vary from pancreatic resections to external or
internal drainage. Disclosure Findings of this paper have been presented to Indian
Association of Surgical Gastroenterology (IASGCON 2016)
conference. 4. Discussions In our study the majority of the patients
were treated with surgical internal drainage like cystogas-
trostomy in four patients and Frey’s pancreaticojejunostomy HPB Surgery HPB Surgery 6 Figure 9: Contrast enhanced CT abdomen showing splenic artery
pseudoaneurysm. There have been multiple reports of mediastinal cysts
causing cardiac failure due to their compressive effects on
the cardiac chambers [21]. It is interesting to note that
two patients in our series had left ventricular failure with
decreased ejection fraction which recovered after resolution
of the cyst following treatment. Conflicts of Interest The authors declare that there are no conflicts of interest
regarding the publication of this paper. in one patient. Successful resolution of mediastinal pseu-
docysts with less invasive procedures, such as combined
laparoendoscopic or thoracoscopic approaches, has also been
reported [16]. Interventional radiological procedures are
often useful adjunct in management of complications follow-
ing pancreatitis and are also useful in managing mediastinal
pseudocyst. The same principle of percutaneous catheter
guided drainage (step-up approach) [16] for peripancreatic
collections/necrosis is applicable here as well. Drainage of
the abdominal pseudocyst/necrotic collections resulted in
resolution of mediastinal pseudocyst as observed in two of
our patients. Authors’ Contributions Durairaj Segamalai, Abdul Rehman Abdul Jameel, and
Naveen Kannan designed the report; Amudhan Anbalagan,
Benet Duraisamy, and Prabhakaran Raju collected patients
clinical data; Durairaj Segamalai, Abdul Rehman Abdul
Jameel, and Kannan Devy Gounder analysed the data and
wrote the paper. p
Vascular complication in the background of chronic pan-
creatitis is a rarely observed complication but is potentially
life threatening [17]. It could range from bleeding from vis-
ceral artery pseudoaneurysm to thrombosis of peripancreatic
veins as encountered in our series [18, 19]. We are probably
the first to report brachiocephalic vein thrombosis, due to
mediastinal pseudocyst. The development of thrombosis is
due to local, prothrombotic, inflammatory changes in the
vascular endothelium and by extrinsic compression due to
pseudocysts [20]. We would like to highlight the fact that
development of a complication following pancreatitis creates
an opportune environment for another complication. Additional Points Core Tip. Mediastinal pseudocysts are a rare entity, most of
the them published as case reports; we present a series of 7
cases. The highlighting point is the varied symptomatology
of the patients. The various management strategies that were
employed in these were tailored to the individual patients. Interesting complications and outcomes are also recorded. This paper might serve as guide to surgeons who encounter
such rare cases in their clinical practice. Figure 10: Contrast enhanced CT chest showing nonvisualization of
left internal jugular vein and brachiocephalic vein due to thrombosis
in a case of mediastinal pseudocyst. 5. Conclusion Mediastinal pseudocyst is a rare complication following acute
or chronic pancreatitis, which should be kept in mind in a
patient presenting with atypical symptoms. Thorough eval-
uation guides to the optimal treatment required. Traditional
surgical drainage which treats the underlying pancreatic dis-
ease, including ductal decompression or pseudocyst decom-
pression, is effective. Radiological interventions are a useful
adjunct to surgical management. EUS guided approach, when
feasible, is gaining more favour with growing expertise and
advancing technology in endoscopic adjuncts. Mediastinal
pseudocysts often require multiple expertise and should be
managed in centers with such expertise. Figure 9: Contrast enhanced CT abdomen showing splenic artery
pseudoaneurysm. Brachiocephalic
vein thrombosis
Figure 10: Contrast enhanced CT chest showing nonvisualization of
left internal jugular vein and brachiocephalic vein due to thrombosis
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Environmental Sustainability and Energy Efficiency in Historical Buildings: GeoFit Project Implementation in the Case Study of a Medieval Fortress in Perugia
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Building Simulation Conference proceedings
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Introduction Figure 2: View of the stable of the Sant’Apollinare after
renovations. While energy and cost efficiency solutions are widely
acknowledged in new constructions, a still deep
multidisciplinary interaction is required in case of
retrofitting of historical building with cultural heritage
value to be preserved. Such value typically represents a
huge constraint to face during the course of design and
construction phases, and therefore tailored low-impact
solutions have to be fitted according to case-specific
boundary conditions. The case study building was built in
the second half of the XIX century as a part of the
medieval complex of Sant’Apollinare fortress in Perugia,
Italy (Figure 1). Figure 2: View of the stable of the Sant’Apollinare after
renovations. The GBC Historic Building™ protocol addresses the
issue of the enhancement of the historical and cultural
value of the building heritage. This protocol is a tool able
to combine and enhance both the sustainability of the
construction process and the respect for the high cultural
value of the artefacts during the restoration process. The
combination of energy efficiency requirements and those
of the historical memory drives in particular the choice of
materials. Figure 1: View of the stable of the Sant’Apollinare
before renovations. Abstract Italian cities are mainly constituted by buildings
constructed until the mid-20th century by pre-industrial
construction techniques. A HVAC system for the energy
retrofit of historical buildings is evaluated when applied
in the case study of Sant’Apollinare. It consists of a
ground source heat pump a water tank for thermal energy
storage connected to a low-temperature radiant system
and air handling unit. The building thermal-energy
behavior, typically influenced by thermal inertia in
historical buildings, and the novel HVAC system
performance interactions are comparatively assessed
together with more traditional scenarios. Energy demand
decreases by about one third compared to the pre-retrofit
situation. In particular, the building has just won Gold Certification
given by GBC Historic Building™ protocol (Green
Building
Council
Italy,2016). Environmental
certifications of buildings represent an important tool for
monitoring the impacts of the whole construction process,
with the purpose being the development of an
environmental consciousness of both occupants and
construction stakeholders. Environmental sustainability and Energy Efficiency in Historical Buildings: GeoFit Project
Implementation in the Case Study of a medieval fortress in Perugia
Jessica Romanelli1, Matteo Di Grazia1, Cristina Piselli1,2, Anna Laura Pisello1,2, Franco Cotana1,2
1 CIRIAF - Interuniversity Research Centre, University of Perugia, Italy
2 Department of Engineering, University of Perugia, Italy
________________________________________________________________________________________________ _____________________ request by the cultural heritage authority, namely
“Soprintendenza ai Beni Culturali”, not to glue the cork
panels to the historical masonry respecting the principle
of reversibility, i.e. the principle according to which the
intervention must be removed without damaging the
original historical masonry preserved. The actual roof is
characterized by a wooden structure like the original
(Figure 2). Environmental sustainability and Energy Efficiency in Historical Buildings: GeoFit Project
Implementation in the Case Study of a medieval fortress in Perugia
Jessica Romanelli1, Matteo Di Grazia1, Cristina Piselli1,2, Anna Laura Pisello1,2, Franco Cotana1,2
1 CIRIAF - Interuniversity Research Centre, University of Perugia, Italy
2 Department of Engineering, University of Perugia, Italy
________________________________________________________________________________________________ Environmental sustainability and Energy Efficiency in Historical Buildings: GeoFit Project
Implementation in the Case Study of a medieval fortress in Perugia
Jessica Romanelli1, Matteo Di Grazia1, Cristina Piselli1,2, Anna Laura Pisello1,2, Franco Cotana1,2
1 CIRIAF - Interuniversity Research Centre, University of Perugia, Italy
2 Department of Engineering, University of Perugia, Italy
________________________________________________________________________________________________ Methods Material Thickness
[m]
Solar
Transmittance
Front Solar
Reflectance
Back Solar
Reflectance
Front Thermal
Emissivity
Back Thermal
Emissivity
Conductivity
[W/m*K]
42
0.006
0.600
0.170
0.220
0.84
0.1
0.9
3
0.006
0.775
0.071
0.071
0.84
0.84
0.9
Material
ThermalResistance
[m2*K / W]
Thickness
[m]
Conductivity
[W/m*K]
Density
[kg/m3]
Specific Heat
[J/kg*K]
BRICK 16 CM
0.19
0.16
0.84
1700
800
BRICK 8 CM
0.10
0.08
0.84
1700
800
CERAMIC FLOOR TILES 14MM
0.02
0.01
0.80
1700
850
CERAMIC TILES PIANELLE 30MM
0.04
0.03
0.80
1700
850
CONCRETE 1600 10CM
0.14
0.10
0.73
1600
1000
CONCRETE 2400 5CM
0.03
0.05
1.91
2400
1000
CORK PANEL 10CM
2.44
0.10
0.04
150
1670
CORK PANEL 4CM
0.98
0.04
0.04
150
1670
INSULATION RADIANT 4CM
1.74
0.04
0.02
30
1450
INSULATION XPS 10 CM
2.63
0.10
0.04
30
1450
LIMESTONE MORTAR 10MM
0.14
0.10
0.70
1600
840
LIMESTONE MORTAR 13MM
0.19
0.13
0.70
1600
840
LIMESTONE MORTAR 20MM
0.29
0.20
0.70
1600
840
LIMESTONE MORTAR 30MM
0.43
0.30
0.70
1600
840
LIMESTONE MORTAR 40MM
0.06
0.04
0.70
1600
840
OSB BOARD
0.09
0.01
0.13
650
1210
PAINTED OAK
0.18
0.04
0.19
700
2390
SCREED LEKA 12CM
1.33
0.12
0.09
330
1000
SCREED MR81 METAL 6CM
0.03
0.06
1.85
2000
1000
SCREED RENONE 5CM
0.04
0.05
1.30
2000
840
STONE 32CM
0.09
0.32
3.49
2880
840
STONE 39CM
0.11
0.39
3.49
2880
840
STONE 40 CM
0.11
0.40
3.49
2880
840
STONE 44CM
0.13
0.44
3.49
2880
840
STONE 62CM
0.18
0.62
3.49
2880
840
STONE 72CM
0.21
0.72
3.49
2880
840
STONE 74CM
0.21
0.74
3.49
2880
840
STONE 84CM
0.24
0.84
3.49
2880
840
WEBER CALCE TS
0.09
0.05
0.54
1500
1000
WEBER COTE ACTION
0.01
0.01
1.10
1900
1000
WEBER THERM
0.71
0.03
0.04
200
1000
WOOD FIBRE INSULATION 14CM
3.68
0.14
0.04
160
2100 Table 1: Thermophysical properties transparent
materials. Table 1: Thermophysical properties transparent
materials. Figure 3: Building information model. Figure 3: Building information model. After modelling the new HVAC system with a water to
water heat pump and ground heat exchangers we had
compared the results obtained in terms of cost-data
analysis and in terms of CO2 retained emissions. Methods Figure 1: View of the stable of the Sant’Apollinare
before renovations. In order to evaluate the achievable energy savings, both
in terms of economics and environmental impacts related
to the retrofit of the gas boiler with a geothermal system,
it was conducted a building energy performance
simulation in EnergyPlus. After the restoration, the building hosts the offices and
conference rooms of an International research center
dedicated to the development of innovative solutions and
technologies for the energy production from biomasses. The building thermal energy model was derived directly
from the related BIM (Figure 3) model made in the design
authoring platform Revit (Autodesk) while the HVAC
system was modelled directly into EnergyPlus simulation
engine. The data exchange via gbXML schema version
6.0.1 (gbXML 2017) allowed to reduce the amount of The external bearing walls, which are completely
preserved, are composed by a mix of local stone and
bricks and cork insulations. The historical aspect of the
building has determined in the renovation phase the ____________________________________
Proceedings of the 16th IBPSA Conference
Rome, Italy, Sept. 2-4, 2019 ____
286 https://doi.org/10.26868/25222708.2019.210323 time needed to redraw the building geometry into another
properly CAD software. emissivity type and the filling material of the cavity is
constituted by Argon with its relative properties present
in the calculation engine. The main thermophysical
properties of the opaque surface materials, shown in Table
2, are the thermal conductivity, the thickness, the density
and the specific heat. For
a
fair
comparison
between
the
simulated
performances and the existing building’s ones, it was
necessary to validate the model firstly based on the
thermal zone temperature and secondly on the gas
consumption for the entire building. The free-floating
validation aimed to verify the building envelope
properties and it was conducted in the month of August
when the HVAC system was off, whereas the
consumption-based validation was related to the operative
expenses associated with the HVAC System. Table 1: Thermophysical properties transparent
materials. Table 2: Thermophysical properties opaque materials. Methods Material Thickness
[m]
Solar
Transmittance
Front Solar
Reflectance
Back Solar
Reflectance
Front Thermal
Emissivity
Back Thermal
Emissivity
Conductivity
[W/m*K]
42
0.006
0.600
0.170
0.220
0.84
0.1
0.9
3
0.006
0.775
0.071
0.071
0.84
0.84
0.9
Material
ThermalResistance
[m2*K / W]
Thickness
[m]
Conductivity
[W/m*K]
Density
[kg/m3]
Specific Heat
[J/kg*K]
BRICK 16 CM
0.19
0.16
0.84
1700
800
BRICK 8 CM
0.10
0.08
0.84
1700
800
CERAMIC FLOOR TILES 14MM
0.02
0.01
0.80
1700
850
CERAMIC TILES PIANELLE 30MM
0.04
0.03
0.80
1700
850
CONCRETE 1600 10CM
0.14
0.10
0.73
1600
1000
CONCRETE 2400 5CM
0.03
0.05
1.91
2400
1000
CORK PANEL 10CM
2.44
0.10
0.04
150
1670
CORK PANEL 4CM
0.98
0.04
0.04
150
1670
INSULATION RADIANT 4CM
1.74
0.04
0.02
30
1450
INSULATION XPS 10 CM
2.63
0.10
0.04
30
1450
LIMESTONE MORTAR 10MM
0.14
0.10
0.70
1600
840
LIMESTONE MORTAR 13MM
0.19
0.13
0.70
1600
840
LIMESTONE MORTAR 20MM
0.29
0.20
0.70
1600
840
LIMESTONE MORTAR 30MM
0.43
0.30
0.70
1600
840
LIMESTONE MORTAR 40MM
0.06
0.04
0.70
1600
840
OSB BOARD
0.09
0.01
0.13
650
1210
PAINTED OAK
0.18
0.04
0.19
700
2390
SCREED LEKA 12CM
1.33
0.12
0.09
330
1000
SCREED MR81 METAL 6CM
0.03
0.06
1.85
2000
1000
SCREED RENONE 5CM
0.04
0.05
1.30
2000
840
STONE 32CM
0.09
0.32
3.49
2880
840
STONE 39CM
0.11
0.39
3.49
2880
840
STONE 40 CM
0.11
0.40
3.49
2880
840
STONE 44CM
0.13
0.44
3.49
2880
840
STONE 62CM
0.18
0.62
3.49
2880
840
STONE 72CM
0.21
0.72
3.49
2880
840
STONE 74CM
0.21
0.74
3.49
2880
840
STONE 84CM
0.24
0.84
3.49
2880
840
WEBER CALCE TS
0.09
0.05
0.54
1500
1000
WEBER COTE ACTION
0.01
0.01
1.10
1900
1000
WEBER THERM
0.71
0.03
0.04
200
1000
WOOD FIBRE INSULATION 14CM
3.68
0.14
0.04
160
2100 Table 1: Thermophysical properties transparent
materials. Table 2: Thermophysical properties opaque materials. Temperature Validation For validating the building thermal energy model and
verify the correctness of the thermophysical properties of
the building envelope, it was necessary to create a specific
climatic file for the month of August, since it was the only
month in which the external and internal environmental
data were available without the HVAC system being
operational, and also without the building occupancy. The
weather data for the construction of the climatic file were
obtained from an on-site weather station, where it was
possible to acquire both the dry bulb temperature of the
outside air and the relative humidity for the month of
August. Subsequently, the climatic file was built using
Elements software (Big Ladder Software). On the other
hand, the annual thermal energy simulations of the
building system were carried out using typical
meteorological year (TMY) weather data files. The distribution to the various unit terminals is carried out
by a small water storage tank maintained at setpoint
temperature by means of a plate heat exchanger between
the gas boiler and the tank itself. For the domestic hot
water, a2000 litres thermally insulated water tank has
been installed in the technical room of the basement but
since this study focused on the analysis of the space
heating requirement it was not taken into account. The entire space heating system was modelled via an air
loop and three water loops. As requested by EnergyPlus,
each system has been divided into two complementary
parts: one for the demand side and the other for the supply
side (EnergyPlus Plant Application Guide,2018). The
three water loops were arranged as follows: Then, regarding the thermal zones with available
monitored data, it was made a comparison between the
simulated temperatures and the internal temperatures
recorded by the monitoring system. The comparison
returned a good accuracy evaluated by the NMBE (1) and
RMSE (2) indices. Their analytical expression is
represented in the following equations: Hot Water 1 Loop: represents the heat generation part
of the system with the gas boiler on the supply side
and the plate heat exchanger on the demand
side(Figure 6). Hot Water 2 Loop: models the intermediate loop
between generation and distribution, where the plate
heat exchanger provides the heat necessary to mantain
the setpoint temperature in the water storage tank
(Figure 7). Building thermal energy model 2-4, 2019 ____
287 exchanger on the exhaust air and a water heating coil
which conditions the mixed air. The operating profile and internal heat gain due to the
presence of people and to the presence of electrical and
lighting equipment have been entered through schedules
that shows both the values in terms of magnitude and the
times at which these contributions are available. Air
infiltrations from outside have been included establishing
the ASHRAE method of leakages for square meters,
(ASHRAE, Handbook Fundamentals 2017). The time of
activation of these schedules follows the typical profile of
an office building occupation with a peak load by the end
of the morning and during mid-afternoon of the
weekdays. The heating temperature setpoint is 20°C in
each thermal zone with the exception of the unheated
zones. Figure 5: Temperature based validation (August). Figure 5: Temperature based validation (August) Figure 5: Temperature based validation (August) The thermal load of the building during the heating period
is mainly satisfied by the radiant floors in all the rooms,
except for the services where wall radiators are installed. The distribution to the various unit terminals is carried out
by a small water storage tank maintained at setpoint
temperature by means of a plate heat exchanger between
the gas boiler and the tank itself. For the domestic hot
water, a2000 litres thermally insulated water tank has
been installed in the technical room of the basement but
since this study focused on the analysis of the space
heating requirement it was not taken into account. The thermal load of the building during the heating period
is mainly satisfied by the radiant floors in all the rooms,
except for the services where wall radiators are installed. Temperature Validation 𝑁𝑀𝐵𝐸 =
∑(𝑉𝑎𝑐𝑡𝑢𝑎𝑙−𝑉𝑚𝑜𝑑𝑒𝑙𝑒𝑑)
(𝑁−1) ×𝑚𝑒𝑎𝑛(𝑉𝑎𝑐𝑡𝑢𝑎𝑙) (1)
𝐶𝑉(𝑅𝑀𝑆𝐸) =
√∑(𝑉𝑎𝑐𝑡𝑢𝑎𝑙−𝑉𝑚𝑜𝑑𝑒𝑙𝑒𝑑)2
(𝑁−1)
𝑚𝑒𝑎𝑛(𝑉𝑎𝑐𝑡𝑢𝑎𝑙)
(2) (1) Hot Water 3 Loop: represents the final part of the
system from the storage tank to the various unit
terminals such as radiators, radiant floors and heating
coil(Figure 8) (2) Where V states for values while actual and modelled state
respectively for monitored and simulated data. These
statistical indicators quantify the discrepancies between
the simulated outputs and the measured data (ASHRAE
Handbook Fundamentals,2017). A simulation model can
be considered calibrated if RMSE<30% and NMBE
<10% (ASHRAEGuideline,14-2014). The results of the
validation for the thermal zone most exposed to summer
weather conditions are reported in Figure 5. . Figure 6: Hot Water 1 Loop. . Figure 6: Hot Water 1 Loop. Building thermal energy model The Ex Stable building of Sant’Apollinare fortress has
been recently restored by seismic and energy renovations. The replacement of the windows with low-emissivity
glass and seamless external perimeter insulation with 10
cm thick cork panels have improved the thermal
performance of the building which, thanks to the good
thermal inertia possessed by the massive masonry, has
reduced the heating load and the overall annual energy
demand. An example of the views of the building energy model
after importing the gbXML file into SketchUp is shown
in Figure 4 (a), the final view of the process of assigning
constructions to the analytical surfaces is shown in (c) ,
while the boundary conditions are shown in (b) where
blue highlights the parts of the building envelope that are
exposed to outdoor conditions and therefore subject to
solar radiation and wind, while yellow highlights the parts
of the structure that are in direct contact with the ground,
in (d) the thermal zones are rendered by different colours. The building consists of two floors above ground and a
basement. The basement is used as a laboratory for the
analysis of biogas samples produced by the biodigester,
while the upper floors are intended for offices. The usable
area is about 240 m2 for a heated volume of 700 m3. It has
been divided into 17 thermal zones, with three unheated
rooms that are the technical room for the heating and
domestic hot water system (basement), the technical room
for the electrical and automation system (basement) and
the stairwell. Figure 4: Building energy model. (a)
(b)
(c)
(d) (a)
(b) (a) (b) The various materials making up the building envelope
were modelled as layers and then were aggregated into
construction layers applied to the analytical surfaces of
the model. For transparent surface materials the
thermophysical
properties
entered
are
thickness,
transmittance and reflectance to solar radiation,
emissivity and thermal conductivity, as well as values
such as transmittance in the visible, useful for calculating
the daylight factor (Table 1).The double pane replaced
during the energy requalification of the building is low- (b) (a) Figure 4: Building energy model. (c)
(d) (c) (d) (d) Figure 4: Building energy model. ____________________________________
Proceedings of the 16th IBPSA Conference
Rome, Italy, Sept. 2-4, 2019 ____________________________________
Proceedings of the 16th IBPSA Conference
Rome, Italy, Sept. HVAC system model The weekly activation and
system has been describe
schedules, similar to the
metabolic activity profiles,
system had to be on or off
night time. The air loop is similar to th
terminals, consisting of d
demand side and connecte
supply side, shown in Fig
loop, whose elements disp
technological equipment in
Figure 9: Air
Furthermore, in this loop th
been detailed by means of
of the economizer on the o
allows free-cooling durin
whenever temperature an
contributing in the thermal
The thermal load distribut
radiant floor and AHU in t
systems are present, is carr
to the radiant system. In th
load is satisfied by the rad
function of supply outdoor
The main HVAC compo
(EnergyPlus Engineering R
1
Boiler Hot Water:repre
Figure 7: Hot Water 2 Loop. ________________________________________________________________________ Figure 8: Hot Water 3 Loop. Fig
________________________________________ Figure 8: Hot Water 3 Loop. Figure 7: Hot Water 2 Loop. The setpoint temperature for the heat transfer fluid in the
system is 68°C, which is the temperature required by the
AHU heating coil and the wall radiators. The setpoint
temperature regarding the radiant floor is considerably
lower, reaching 38 °C. The setpoint temperature for the heat transfer fluid in the
system is 68°C, which is the temperature required by the
AHU heating coil and the wall radiators. The setpoint
temperature regarding the radiant floor is considerably
lower, reaching 38 °C. The setpoint temperature for the heat transfer fluid in the
system is 68°C, which is the temperature required by the
AHU heating coil and the wall radiators. The setpoint
temperature regarding the radiant floor is considerably
lower, reaching 38 °C. The weekly activation and operation profile of the water
system has been described by means of availability
schedules, similar to the occupation and occupants
metabolic activity profiles, which define when the plant
system had to be on or off and whenever cycle on during
night time. The air loop is similar to the hydraulic one. The air unit
terminals, consisting of diffusers, are arranged on the
demand side and connected to the thermal zones, while
supply side, shown in Figure 9, is composed by a half
loop, whose elements disposed in series represent the
technological equipment in the AHU. Figure 9: Air loop supply side. HVAC system model In the building, the air conditioning is carried out by a
partially recirculating air handling unit (AHU) with inlets
into the offices at the first and second floor and outlet into
the service rooms. In the AHU there is a cross-flow heat Figure 6: Hot Water 1 Loop. ____
288 ____________________________________
Proceedings of the 16th IBPSA Conference
Rome, Italy, Sept. 2-4, 2019 ____
288 The setpoint temperature for the heat transfer fluid
system is 68°C, which is the temperature required b
AHU heating coil and the wall radiators. The se
temperature regarding the radiant floor is consid
lower, reaching 38 °C. The weekly activation and operation profile of the
system has been described by means of availa
schedules, similar to the occupation and occu
metabolic activity profiles, which define when the
system had to be on or off and whenever cycle on d
night time. The air loop is similar to the hydraulic one. The a
terminals, consisting of diffusers, are arranged o
demand side and connected to the thermal zones,
supply side, shown in Figure 9, is composed by
loop, whose elements disposed in series represe
technological equipment in the AHU. Figure 9: Air loop supply side. Furthermore, in this loop the weekly operation profi
been detailed by means of schedules, while the pre
of the economizer on the outdoor air mixer exhaus
allows free-cooling during the intermediate se
whenever temperature and humidity are suitabl
contributing in the thermal load reduction. The thermal load distribution management betwee
radiant floor and AHU in the thermal zones, where
systems are present, is carried out by assigning a pr
to the radiant system. In this case almost all the h
load is satisfied by the radiant floor relegating the
function of supply outdoor fresh air to AHU. The main HVAC components and their descr
(EnergyPlus Engineering Reference,2018) are as fo
1. Boiler Hot Water:represents the gas boiler for
it is possible to insert the efficiency curves an
type of fuel used in this case is natural gas. 2. Heat Exchanger Fluid to Fluid:type of heat exch
that allows to model a general heat exchanger
fl id t
fl id
h
d
ffi i
Figure 7: Hot Water 2 Loop. ____________________________________________________________________________________________ Figure 7: Hot Water 2 Loop. ________________________________________________________________________________________________ The setpoint temperature fo
system is 68°C, which is th
AHU heating coil and the
temperature regarding the
lower, reaching 38 °C. Figure 9: Air loop supply side. Furthermore, in this loop the weekly operation profile has
been detailed by means of schedules, while the presence
of the economizer on the outdoor air mixer exhaust path
allows free-cooling during the intermediate seasons,
whenever temperature and humidity are suitable for
contributing in the thermal load reduction. The thermal load distribution management between the
radiant floor and AHU in the thermal zones, where both
systems are present, is carried out by assigning a priority
to the radiant system. In this case almost all the heating
load is satisfied by the radiant floor relegating the main
function of supply outdoor fresh air to AHU. The main HVAC components and their description
(EnergyPlus Engineering Reference,2018) are as follows: 1. Boiler Hot Water:represents the gas boiler for which
it is possible to insert the efficiency curves and the
type of fuel used in this case is natural gas. 2. Heat Exchanger Fluid to Fluid:type of heat exchanger
that allows to model a general heat exchanger from
fluid to fluid whose power and efficiency are
described respectively in equations (3) and (4) below. Figure 8: Hot Water 3 Loop. p
y
q
𝑄̇ = ε (ṁ cp)min(TSupLoop,in − TDmdLoop,in) (3) (3) ____________________________________
Proceedings of the 16th IBPSA Conference
Rome, Italy, Sept. 2-4, 2019 ____
289 Figure 11: Simulated zone air temperatures (February). Consumption validation
_______________________________________________ ____________ Figure 11: Simulated zone air temperatures (February). Consumption validation (4) 3. Water Heater Mixed: water storage function and
therefore with no capacity of the auxiliary heater. Because of the small volume it is not affected by
stratification phenomena (mixed type). The energy
balance equation of storage(5) is as follows. ϱVcp
dT
dt = qnet (5) 3. Water Heater Mixed: water storage function and
therefore with no capacity of the auxiliary heater. Because of the small volume it is not affected by
stratification phenomena (mixed type). The energy
balance equation of storage(5) is as follows. ϱVcp
dT
dt = qnet (5) ϱVcp
dT
dt = qnet (5) (5) 4. Low Temperature Radiant Variable Flow:used to
represent the radiant floor and inserted within a type
of internal source construction layer to calculate the
relative conduction transfer function (CTF) Figure 11: Simulated zone air temperatures (February). Consumption validation 5. Figure 9: Air loop supply side. Convective Baseboard Water: represents the radiators
present in the service rooms and divide the thermal
power emitted into radiant and convective The available gas consumption monitored data covered
the year following the activation of the system and the
newly occupation of the building after renovation. 6. Water Heating Coil:heating coil downstream in the
AHU with water controller to vary the flow of hot
water depending on thermal load. The consumption recorded in this period, shown in Figure
12, includes the commissioning and the initial start-up of
the HVAC system during December, in which the results
of the simulation model deviate significantly. 7. Air to Air Sensible Heat Exchanger: sensible heat
recovery ventilation with a design conditions
efficiency of 0.8. However, the building performance model behaves
acceptably with an RMSE of 3.2% over a monthly period
at hourly timestep. The NBME of 2.7% is within the
limits. Not having at least three consecutive years of
consumption monitored data does not permit to define this
step as an accurate validation but allows seeing the
consistency of the model simulated following the same
order of winter consumption magnitude. y
The results of the simulation for a thermal zone are shown
in Figure 10, during the month of February. The trend of
the indoor dry-bulb temperature, the radiant temperature
and the operating temperature are compared to the
outdoor dry bulb temperature during the month of
February on hourly timesteps. The zone temperature
follows the occupancy and activation profiles with ramps
and slope changes. In Figure 11, a widening of the
previous graph allows observing in detail the proximity
between the operating and radiant temperature with the air
temperature, due to both the thermal insulation of the
building and the presence of radiative surfaces at higher
temperatures. Moreover, during the hours of occupation,
the dry-bulb air temperature is higher than the radiant
temperature, due to internal heat, gains while it falls
below during the closing periods with a thermal drop of
about 1.5 K in two days. Figure 12: Consumption based validation. Figure 12: Consumption based validation. G Figure 10: Simulated zone and outdoor air temperatures
(February). Figure 10: Simulated zone and outdoor air temperatures
(February). Figure 12: Consumption based validation. Geothermal system model ____________________________________
Proceedings of the 16th IBPSA Conference
Rome, Italy, Sept. 2-4, 2019 Figure 13: Ground Source Loop. Kusuda and Achenebach (1965) developed the model
used for representing the ground thermal behaviour as
function of time and depth which is correlated to the
average annual soil surface temperature, the amplitude of
the soil temperature changes throughout the year or day
of minimum surface temperature and to the thermal
diffusivity of the ground. The vertical U-Tube heat
exchanger model is based on the work of Eskilson (1987)
which solves the problem of heat transmission for
borehole by means of a mixed numerical analytical
method, determining the response factors under constant
initial and boundary conditions over a long timestep. Yavutzturk and Spliter (1999) integrates this model in a
short time step considering also the thermal capacity of
the ground and grout, the thermal resistance of the pipe
and the fluid flow. In this work for determining the
thermal response of vertical U-tube HEXs was used as
predefined dataset of response factors, based on
abovementioned
properties
and
boreholes
field
configuration. The system configuration of the boreholes
field has great influences over the response factors which
must be determined before all simulations (Yavuzturk and
Spitler,2001). Figure 15: Source energy. Figure 15: Source energy. Figure 15: Source energy. Figure 16: Annual operating cost. Figure 16: Annual operating cost. Figure 16: Annual operating cost. Eventually, considering the current national energy mix,
the electricity from the grid is responsible for 0.44
kg/kWh of CO2 emissions while the CO2 produced by the
combustion of a m3 of natural gas is equal to 1,89 kg. The
saving in terms of CO2 replacing the gas boiler with a
GSHP is equal to 3032 kg CO2 (Figure 17). Eventually, considering the current national energy mix,
the electricity from the grid is responsible for 0.44
kg/kWh of CO2 emissions while the CO2 produced by the
combustion of a m3 of natural gas is equal to 1,89 kg. The
saving in terms of CO2 replacing the gas boiler with a
GSHP is equal to 3032 kg CO2 (Figure 17). Geothermal system model The latter, called parameter estimation-
based model, form a method between the black box and
the deterministic model (Jin and Spitler,2002). A
simplified model from basic thermodynamic heat and
mass balance equations (Fisher and Rees,2005) is applied
to each internal component of the heat pump and to find
the required parameter values using manufacturers
catalogue data by means of a multi-variable optimization
algorithm (Nelder and Mead, 1965). This method is
suitable for alternative compressor heat pump but the
evaluation of the parameters, when varying the type of
compressor of the heat pump, has a huge range of errors
in the parameter estimation-based model. As a result, the
mathematical model chosen for the heat pump of this
paper is based on the equation fit model. Figure 14: GSHP electrical energy demand In terms of source energy demand the comparison
between the gas boiler model and the water-to-water heat
pump model shows an energy demand for space heating
decreased by more than half, as shown in the figure 15. In terms of source energy demand the comparison
between the gas boiler model and the water-to-water heat
pump model shows an energy demand for space heating
decreased by more than half, as shown in the figure 15. In order to estimate the achievable economic savings,
according to the energy market prices, it was assigned an
average global cost of 0.22 €/kWh to the electricity and
an average global cost of 0.8 €/m3 for the gas consumption
(Figure 16). In order to estimate the achievable economic savings,
according to the energy market prices, it was assigned an
average global cost of 0.22 €/kWh to the electricity and
an average global cost of 0.8 €/m3 for the gas consumption
(Figure 16). Figure 13: Ground Source Loop. (
g
)
Figure 15: Source energy. Figure 16: Annual operating cost. Figure 15: Source energy. Geothermal system model In order to evaluate the savings that could be achieved
replacing the gas boiler with a ground source heat pump
(GSHP), the energy model was modified by inserting a
vapor compression heat pump with a thermal power of 15
kW and ground source heat exchangers (HEXs). A further
hydronic loop has been added to represent the
contribution of the two vertical U-tube HEXs with a depth
of 70 m and a diameter equal to 3 cm, as shown in Figure
13. Figure 10: Simulated zone and outdoor air temperatures
(February). The mathematical models that can be used within
EnergyPlus to simulate the operation of the GSHP consist
of two types: the curve-fit model and the parameter
estimation-based model (Jin,2002). The former, classified
as an equation fit component model by Hamilton and
Miller (1990), treats the system as black box by means of
four dimensionless curves useful to predict the ____
290 ____________________________________
Proceedings of the 16th IBPSA Conference
Rome, Italy, Sept. 2-4, 2019 ____________________________________
Proceedings of the 16th IBPSA Conference
Rome, Italy, Sept. 2-4, 2019 Figure 14: GSHP electrical energy demand. _______________________________________________ performance of the heat pump in heating and cooling
mode. The generalized least square method generates a set
of coefficients from the catalogue data which are used in
the model to simulate the behaviour of the heat pump
(Tang, 2005). The latter, called parameter estimation-
based model, form a method between the black box and
the deterministic model (Jin and Spitler,2002). A
simplified model from basic thermodynamic heat and
mass balance equations (Fisher and Rees,2005) is applied
to each internal component of the heat pump and to find
the required parameter values using manufacturers
catalogue data by means of a multi-variable optimization
algorithm (Nelder and Mead, 1965). This method is
suitable for alternative compressor heat pump but the
evaluation of the parameters, when varying the type of
compressor of the heat pump, has a huge range of errors
in the parameter estimation-based model. As a result, the
mathematical model chosen for the heat pump of this
paper is based on the equation fit model. performance of the heat pump in heating and cooling
mode. The generalized least square method generates a set
of coefficients from the catalogue data which are used in
the model to simulate the behaviour of the heat pump
(Tang, 2005). Future Developments Jin, H. (2002). Parameter Estimation Based Models of
Water Source Heat Pumps. Ph.D. Thesis, Department
of
Mechanical
and
Aerospace
Engineering,
Oklahoma State University,Stillwater, Oklahoma. A new hBIM (BIM for historic buildings) platform is
proposed, starting from a terrestrial laser scanning
combined with digital cameras and with the purpose to
consider
historical
building
priorities
and
energy/structural
retrofit
actions. Such
approach
demonstrated its efficacy and will be implemented into
this project with the purpose to act as demonstrator of
historical building retrofits, by combining energy
efficiency, building modern functionality, structural
safety and reliability, while not only preserving cultural
heritage but also taking advantage of its peculiarities for
installing new technologies with low visual impact
(geothermal and storage systems). Kusuda,
T. and
P.R. Achenbach(1965). Earth
Temperatures and Thermal Diffusivity at Selected
Stations in the United States. ASHRAE Transactions
71(1), 61–74. Nelder, J. A., and R. Mead. 1965. A Simplex Method for
Function Minimization.Computer Journal 7(1), 308-
313 Tang, C.C.,2005. Modeling Packaged Heat Pumps in a
Quasi-Steady
State
Energy
Simulation
Program.Master of Science. Oklahoma: Department
of Mechanical and Aerospace Engineering, Oklahoma
State University Very interesting future development consists of a ground
source heat pump implementation coupled to innovative
“earth baskets” heat exchangers, that will represent an
intermediate configuration between fully horizontal and
fully vertical systems, with the related variations in terms
of cost, drilling depth and total heat exchange potentiality. Yavuzturk, C. and J.D. Spitler (2001). Field Validation of
a Short Time-Step Model for Vertical Ground Loop
Heat Exchangers. ASHRAE Transactions 107(1),
617–625. Results The paper was aimed at investigating novel simulation
techniques and procedures specifically tailored for
historical buildings, where the implementation of retrofit
actions and renewables may be difficult because of
architectural constraints. The simulation of the GSHP model predicts an annual
energy demand equal to 2920 electrical kWh, whose
monthly distribution is visible in the Figure 14. ____
291 ____________________________________
Proceedings of the 16th IBPSA Conference
Rome, Italy, Sept. 2-4, 2019 ____
291 Figure 17: CO2 emission savings. EnergyPlus, Input Output Reference (2018), Version
9.0.US Department of Energy (DoE). EnergyPlus, Input Output Reference (2018), Version
9.0.US Department of Energy (DoE). EnergyPlus, Engineering Reference (2018), Version
9.0.Department of Energy (DoE). EnergyPlus, Plant Application Guide (2018), Version
9.0.Department of Energy (DoE). Eskilson, P. (1987). Thermal Analysis of Heat Extraction
Boreholes. Ph.D. Thesis, Department of Mathematical
Physics, University of Lund, Lund, Sweden Fisher ,D.E. and S.J. Rees (2005) Modeling ground source
heat pump systems in a building simulation program
(Energyplus). Proceedings from BS2005: Building
Simulation.. Montréal (CDN), 23-24 August 2005. Figure 17: CO2 emission savings. The building performance simulation following the
replacement of the gas boiler with a ground source heat
pump gives considerable benefits in terms of source
energy savings and CO2 offset while maintaining the
same comfort conditions for the occupants, the same
settings of temperature control and operation of the
HVAC system. In fact, in the retrofitted scenario the
source energy demand decreases by about one third
compared to the pre-retrofit situation while the CO2
savings reach three quarters of the total. The advantage of
the
geothermal
system
is
both
economic
and
environmental and is strictly related to the possibility of
use the ground as heat source. gbXML. http://www.gbxml.org/, version 6.01, January
2017. Green Building Council (GBC) Historic Building (HB)
Protocol, http://www.gbcitalia.org/historic-building. Hamilton, J.F., and J.L. Miller. 1990. A Simulation
Program for Modeling an AirConditioning System. ASHRAE Transactions 96(1), 213-221 Jin, H. and J.D. Spliter (2002). A Parameter Estimation
Based Model Of Water-to-Water Heat Pumps for Use
in
energy
Calculation
Programs. ASHRAE
Transactions 108(1), 3–17. Acknowledgements This project has received funding from the European
Union’s
Horizon
2020
research
and
innovation
programme under grant agreement No 792210 (GEOFIT) Yavuzturk, C. and J.D. Spitler (1999). A Short Time Step
Response Factor Model for Vertical Ground Loop
Heat Exchangers. ASHRAE Transactions 105(2),
475–485. References American Society of Heating, Refrigerating and Air-
Conditioning Engineers (ASHRAE). Measurement of
energy, demand, and water savings, (Guideline 14-2014). American Society of Heating, Refrigerating and Air-
Conditioning Engineers (ASHRAE). (2017). Handbook
Fundamentals. Atlanta (GA). ____
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Rome, Italy, Sept. 2-4, 2019
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https://openalex.org/W2061890460
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English
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Spot the Difference: Mimicry in a Coral Reef Fish
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PloS one
| 2,013
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cc-by
| 5,922
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Abstract The funders had no role in
and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E mail: monica gagliano@uwa edu au * E-mail: monica.gagliano@uwa.edu.au Eyespots of the coral reef damselfish Pomacentrus amboinensis have
been suggested to serve as a subordinate signal directed at
dominant males [11]. They consist of a large black spot
surrounded by a brilliant white ring found on the dorsal fin of
all juvenile individuals and, similar to the sub-adult plumage of
young birds, may be a form of status signalling (i.e. status signalling
hypothesis [17]) relaying an honest signal of social sub-ordinance
to adult territorial males. Because P. ambionensis forms social groups
of individuals of all ages, where multiple mature and immature
females are loosely centred around a dominant male [18], the
main advantage of being accepted within defended male territories
is known to be a reduced risk of predation (e.g. survival rates
within male territories are approximately 4 times higher than in
adjacent areas [19]). In addition, because males show little
aggression towards recruit-stage conspecifics, the advantages of
living in defended territories may extend to increased access and
availability of food and habitat resources, resulting from lower
densities of competitive or aggressive species. Being a protogynous
species, all P. amboinensis juveniles start out as females and
interestingly, their eyespots fade as the fish approaches sexual
maturation to disappear completely in the vast majority of mature
adult individuals, but intriguingly, not all. Monica Gagliano1,2*, Martial Depczynski3 1 Centre for Evolutionary Biology, School of Animal Biology, University of Western Australia, Crawley, Western Australia, Australia, 2 Centre for Microscopy,
Characterisation and Analysis, University of Western Australia, Crawley, Western Australia, Australia, 3 Australian Institute of Marine Science, The Oceans Institute,
University of Western Australia, Crawley, Western Australia, Australia Spot the Difference: Mimicry in a Coral Reef Fish Monica Gagliano1,2*, Martial Depczynski3 Abstract Eyespots on the body of many animals have long been assumed to confer protection against predators, but empirical
evidence has recently demonstrated that this may not always be the case and suggested that such markings may also serve
other purposes. Clearly, this raises the unresolved question of what functions do these markings have and do they
contribute to an individual’s evolutionary fitness in the wild. Here, we examined the occurrence of eyespots on the dorsal fin
of a coral reef damselfish (Pomacentrus amboinensis), where these markings are typical of the juvenile stage and fade away
as the fish approaches sexual maturation to then disappear completely in the vast majority of, but not all, adult individuals. By exploring differences in body shape among age and gender groups, we found that individuals retaining the eyespot into
adulthood are all sexually mature males, suggesting that these eyespots may be an adult deceptive signal. Interestingly, the
body shape of these individuals resembled more closely that of immature females than mature dominant males. These
results suggest that eyespots have multiple roles and their functional significance changes within the lifetime of an animal
from being a juvenile advertisement to a deceptive adult signal. Male removal experiments or colour manipulations may be
necessary to establish specific functions. Citation: Gagliano M, Depczynski M (2013) Spot the Difference: Mimicry in a Coral Reef Fish. PLoS ONE 8(2): e55938. doi:10.1371/journal.pone.0055938
Editor: Nicole M. Gerardo, Emory University, United States of America Citation: Gagliano M, Depczynski M (2013) Spot the Difference: Mimicry in a Coral Reef Fish. PLoS ONE 8(2): e55938. doi:10.1371/journal.pone.0055938 Editor: Nicole M. Gerardo, Emory University, United States of America Received August 9, 2012; Accepted January 4, 2013; Published February 13, 2013 Copyright: 2013 Gagliano, Depczynski. 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. ng: MG was supported by Research Fellowships from the University of Western Australia and the Australian Research Council. The fund
design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding: MG was supported by Research Fellowships from the University of Western Austr
study design, data collection and analysis, decision to publish, or preparation of the manus by Research Fellowships from the University of Western Australia and the Australian Research Council. Ethics Statement The study was conducted in accordance with the James Cook
University Animal Ethics guidelines (Permit Number: A-1254) and
under appropriate permits from the Great Barrier Reef Marine
Park Authority. The study was conducted during the austral summer at Lizard
Island (14u 389S, 145u 289E) on the northern Great Barrier Reef. During the breeding season, two divers used hand nets to
randomly collect a total of 113 P. amboinensis of various sizes and
stages of maturation from a contiguous reef. Each fish was placed
in an individually numbered transparent plastic bag and sexed in
situ by visual inspection of its genital papillae [28] by both divers to
assign it to 1 of 4 groups: [j] true juveniles (i.e. recently settled
individuals; less than 3 months old), [f] immature females (i.e. 3–6
months old juvenile), [F] mature females or [M] mature males. Each individual was then photographed in side view against
a measuring board with 1-cm gradations as a scale for subsequent
image calibration and immediately released. A small subsample of
25 individuals was retained for dissection in the laboratory to
assess the maturation state of their gonads (i.e. immature vs. developed testes/ovaries). This examination indicated that our
field scores were 96% correct in assigning individuals to a specific
group, hence making the visual inspection method used here
a reliable and ethically sound approach. Using the first 20 standardized harmonics (H2–H21), the body
shape of all photographed P. amboinensis were compared in relation
to their maturation status and the presence/absence of an eyespot. The hypothesis of no difference in body shape among the groups
was tested using multivariate analysis of variance (MANOVA),
followed by a canonical discriminant analysis (CDA) to examine
and display the patterns of difference identified by MANOVA. Vectors of the original harmonics were plotted to aid interpreta-
tion of differences among groups. The length of the vectors
described the relative importance of each harmonic in discrimi-
nating among groups. Each group was represented by 95%
confidence cloud around group centroids. Digital images for body shape analyses were taken and each
individual scored based on the presence/absence of a dorsal fin
eyespot. All images were calibrated using the scale on the
photograph itself and the dimensions of each fish quantified using
the image analysis program OPTIMAS 6.5. Fast Fourier analysis
was then used to describe the overall body shape of the fish as
previously done in similar studies [29]. Introduction Secondly, the outline of the
body excluding all fins and across the base of the caudal peduncle
was then traced to estimate perimeter (P), area (A mm2) and
rectangularity (R, where R = A/SL x DB1), an index calculating
how well the body shape fits into a rectangular shape. Lastly, the
silhouette of each fish was described by sampling over 128
equidistant points along the same body outline, extracting a series
of successive cosine waves, having phase angle and amplitude
components. Hence, the outline of the body was produced as an
aggregate of simple waveforms, where the amplitude of each
cosine wave defined a Fast Fourier shape descriptor (also called
‘harmonic’ [Hn]). By setting the zeroth descriptor (H0) to 0+0i, we
standardize all successive harmonics to account for differences in
the position of the body silhouette on the screen; we then divided
all successive harmonics by H1 to remove any confounding effect
of body size from the data. Because each descriptor produces
a simplified representation of the original shape and thus
reproduces only some aspects of it, multiple descriptors contrib-
uting to increasingly finer details of the original shape may be
necessary to entirely reconstruct the complete outline. According-
ly, the first six harmonics (H2–H6) are referred to as low-order
descriptors and determine the gross shape of the body such as its
elongation, triangularity and squareness, whereas successively
higher descriptors measure increasingly finer details of the body
outline. In this study, the number of harmonics to be used as shape
descriptors for each fish was set to the first 20 (excluding H0 and
H1) because the contribution of higher order harmonics (i.e. 22
and above) to the definition of the shape was negligible [29]. by sharing common perceived characteristics of those being
mimicked, clear cases of dishonest signalling, where certain
individuals mask their true identity by taking on, for example,
the appearance of females (e.g. female mimicry [24]) may be
generally difficult to detect. While the adaptive significance of
signals used in mimicry is often examined in terms of colouration
and colour patterns of individuals, behavior and body shape (or
a combination of them) can also have a signal function and be
mimicked by others [25]. Introduction The widespread occurrence and adaptive significance of
eyespots on the body of many animals have long intrigued
biologists. Current understanding strongly promotes the paradigm
that these conspicuous eye-resembling colour markings have
various
antipredatory
functions,
such
as
deterring
hunting
predators to initiate an attack by resembling the eyes of their
predator’s own enemies (i.e. intimidation hypothesis) or diverting
their attacks toward less vital body parts (i.e. deflective hypothesis
[1]). A range of studies have indeed provided good evidence for an
intimidatory and startling function of eyespots in insects such as
peacock butterflies [2] and eyed hawk moths [3], and even
artificial prey [4,5], showing that these eyespots can be highly
effective in scaring birds. On the other hand, studies of deflective
effects generally ascribed to peripheral eyespots located at the
posterior end of the body far from the animal’s head (e.g. butterflies [6]; fishes [7] and frogs [8]) have not found any
convincing support ([9–11] but see [12–13]). If these eyespots are
not aimed at deflecting attacking predators, then what function (if
any) do they really serve and does it contribute to an individual’s
evolutionary fitness? The function of eyespots in a non-predatory
role has been previously investigated, specifically in the context of
mate choice in the butterfly Bicyclus anynana [14–16]. Yet, other
than in systems with Lepidoptera and avian or lizard predators,
investigations of the alternative, non-predatory value of eyespots
are relatively rare. Empirical evidence supporting the idea that
these markings may have a different function and that they may be
important in reproduction has only recently been provided in
fishes [11]. For those adults that retain their dorsal eyespots, we hypoth-
esized that the presence of the eyespot may be preventing
dominant males with territories from distinguishing ‘‘bluffing’’
mature males in juvenile (female) clothing from truly sexually
immature female individuals in order to go unrecognized as
competing and fertile ‘‘sneaker’’ males (i.e. dual male reproductive
strategies [20–23]). Because cheats are supposed to go unnoticed February 2013 | Volume 8 | Issue 2 | e55938 1 February 2013 | Volume 8 | Issue 2 | e55938 PLOS ONE | www.plosone.org Eyespot Dishonest Signalling from the anterior base of dorsal fin to the anterior base of pelvic
fin, and depth of body 2 (DB2) measured across the body at the
anterior base of anal fin were taken. Materials and Methods All data were checked for and met the requirements of
normality
and
homogeneity
of
variance
before
performing
statistical analyses. One-way ANOVAs were used to compare
morphological measures (standardized by standard length) of
individuals at different stages of maturation and with/without an
eyespot on their dorsal fin. All differences among treatments were
identified using post-hoc Tukey’s [honest significant difference
(HSD)] tests. To quantify the precision of our measures, randomly
selected digital images of 10 individuals were re-calibrated twice
on separate occasions and re-measured. The overall errors
associated with obtaining measurements from the digital images
were very low [e.g. coefficient of variation (CV) values: SL, 2.91%;
BD1, 3.07%; BD2, 3.57%, P, 1.98%; A, 2.52%; R, 1.31%],
indicating that the morphological data were collected and
quantified reliably. Introduction In fishes, subtle variations in body shape
can reveal important ecological and behavioural differences [26],
and hence provide useful information for improving our un-
derstanding of animal signals and their relevance in the evolution
of animal social systems. Hence, the overall aim of this study was
to examine the possible functional role of the dorsal eyespots in P. amboinensis by exploring differences in body shape among age and
gender groups. Because mature adults are not sexually di-
chromatic (aside from the retention or disappearance of the
eyespot) and all individuals mature first as females and later
change sex to function as males (i.e. monandric protogynous
hermaphroditism [27]), this model species and system provide an
ideal opportunity for examining ontogenetic changes in eyespot
function within a socio-behavioural context. Specifically, we first
tested whether larger individuals retaining the eyespot are in fact
all sexually mature individuals, and exclusively males. We then
asked (1) whether the body shape of eye-spotted adults more
closely resembles that of juveniles and immature females (i.e. mimicry) than that of mature males; and if so, (2) whether such
variation in reproductive tactics is age-dependent (e.g. individuals
may be sneakers when young and become parentals when older). February 2013 | Volume 8 | Issue 2 | e55938 Ethics Statement Briefly, measurements
(mm) of standard length (SL), depth of body 1 (DB1) measured To provide further information on the age of P. amboinensis with
and without eyespots, we examined the information stored within
the otoliths of a total of 80 individuals. Transverse sections of
sagittal otoliths were obtained by mounting individual otoliths in
thermoplastic cement and the daily increments visible from these
sections used to estimate age [30]. February 2013 | Volume 8 | Issue 2 | e55938 2 PLOS ONE | www.plosone.org Eyespot Dishonest Signalling Results and Discussion
and mature females are
Figure 1. The damselfish P. amboinensis. (a) Sexually mature male without the eyespot and (b) se
juvenile eyespot on the dorsal fin [indicated by the white arrow]. doi:10.1371/journal.pone.0055938.g001 Figure 1. The damselfish P. amboinensis. (a) Sexually mature male without the eyespot and (b) sexually mature male still retaining the typically
uvenile eyespot on the dorsal fin [indicated by the white arrow]. doi:10.1371/journal.pone.0055938.g001 Figure 1. The damselfish P. amboinensis. (a) Sexually mature male without the eyespot and (b) sexually mature male still retaining the typically
juvenile eyespot on the dorsal fin [indicated by the white arrow]. doi:10.1371/journal.pone.0055938.g001 February 2013 | Volume 8 | Issue 2 | e55938 Results and Discussion and mature females are loosely centred around a dominant male
(lacking eyespot) and where growth rates and time to sexual
maturity of juveniles is under social control [18], it is intriguing to
find individuals that have matured into males while retaining
a juvenile trait, such as the eyespot. While most P. amboinensis exhibiting an eyespot on the dorsal fin
ranged from newly settled recruits to older (but still) immature
individuals, a significant 25% of all individuals with eyespots were
sexually mature males (Figure 1). Given that this species forms
social groups that contain individuals of all ages, where juvenile Using Fast Fourier shape analyses, we found significant
differences in the body dimensions and shape of P. amboinensis February 2013 | Volume 8 | Issue 2 | e55938 PLOS ONE | www.plosone.org February 2013 | Volume 8 | Issue 2 | e55938 3 Eyespot Dishonest Signalling depending on their maturation status and the presence/absence of
mature but eye-spotted males more closely resembled that of
Figure 2. Comparison of the body dimensions of P. amboinensis belonging to different age and gender groups without eyespot
(diamonds; N-values = 23 males & 36 females) and with eyespot (circles; N-values = 14 males, 16 females, 24 juveniles). Means 695% CI. Letters indicate significant differences based on post-hoc Tukey’s HSD tests (i.e. fish groups exhibiting no significant difference in a given trait are
indicated by the same letter). doi:10.1371/journal.pone.0055938.g002
Eyespot Dishonest Signalling Figure 2. Comparison of the body dimensions of P. amboinensis belonging to different age and gender groups without eyespot
(diamonds; N-values = 23 males & 36 females) and with eyespot (circles; N-values = 14 males, 16 females, 24 juveniles). Means 695% CI. Letters indicate significant differences based on post-hoc Tukey’s HSD tests (i.e. fish groups exhibiting no significant difference in a given trait are Figure 2. Comparison of the body dimensions of P. amboinensis belonging to different age and gender groups without eyespot
(diamonds; N-values = 23 males & 36 females) and with eyespot (circles; N-values = 14 males, 16 females, 24 juveniles). Means 695% CI. Letters indicate significant differences based on post-hoc Tukey’s HSD tests (i.e. fish groups exhibiting no significant difference in a given trait are
indicated by the same letter). doi:10 1371/journal pone 0055938 g002 mature but eye-spotted males more closely resembled that of
juveniles and immature females than that of mature males. February 2013 | Volume 8 | Issue 2 | e55938 Results and Discussion males and females with no eyespot along canonical variate 1 but
occupied a similar position to males with no eyespots along the
second variate (accounting for 25% of the overall variation). The
arrangement of the 5 groups along this second axis suggests that
those sexually mature individuals who have retained their dorsal
eyespot have a ‘‘male’’ body shape at the finer scale (i.e. higher It is clear that becoming a dominant male in a social system full
of mature females is reproductively advantageous. Yet within this
social setting, what is the advantage of maturing into a male
without acquiring a dominant social status, but instead retaining
a juvenile trait? Visually prominent and colourful ‘‘ornaments’’ are
generally costly and hence paraded only by individuals of superior
quality or condition as signals of mate quality in a female choice
context. For example, eyespots on butterfly wings have been
previously reported to play an important role in intraspecific
interactions, where females may exercise their preference for males
with larger/darker eyespots [14,31]. In P. amboinensis, however,
eyespots are very affordable [11] and hence easily maintained and
displayed by non-dominant males, strongly discounting the
possibility that their message is directed to females in order to
sway their mating preferences. Figure 4. Mean age in days of P. amboinensis belonging to
different age and gender groups without eyespot (diamonds; N-
values = 21 males & 36 females) and with eyespot (circles; N-
values = 9 males & 14 females). Error bars indicate 95% CI. Letters
indicate significant differences based on post-hoc Tukey’s HSD tests. doi:10.1371/journal.pone.0055938.g004 Together with their overall body morphology, the occurrence of
reproductively active males with eyespots supports the idea that
eyespots in animals may indeed have multiple roles. Although it
has been previously recognized that eyespots can have multiple
roles with at times conflicting functions, such as sexual selection
(and specifically female choice) and predator avoidance [32], this is
the first time that these markings are found to be important for
their deceptive function in the context of intrasexual (male-male)
competition. Moreover, these findings suggest that the functional
significance of these markings in P. amboinensis switches within
a lifetime from being a juvenile advertisement to a deceptive signal
of age and non-breeding status. Clearly, the hypothesis that the
eyespot is a deceptive signal would benefit from examining if males
with eyespots have large gonads compared to males without Figure 4. Results and Discussion We
found significant differences in the overall body shape of fish from
the different groups (MANOVA Wilks l, F96,299 = 1.66, p,0.001). Much of the variation among groups (52%) was due to coarse (i.e. low level harmonics H2 and H5) differences between the body
shape of adults (with no eyespots) and that of individuals with
eyespots, including mature males (canonical variate 1, Figure 3). Males with eyespots were located well away from both mature depending on their maturation status and the presence/absence of
an eyespot on the dorsal fin. Unsurprisingly, mature non-spotted
individuals were significantly bigger (Figure 2A–E) and more
rectangular than immature ones (Figure 2F), with mature males
being the largest and having the most rectangular body (one-way
ANOVA
for
SL,
F4,98 = 45.98,
p,0.001;
A,
F4,98 = 37.99,
p,0.001;
DB1,
F4,98 = 46.03,
p,0.001;
DB2,
F4,98 = 40.25,
p,0.001; one-way ANOVA, F4,98 = 5.58, p,0.001). It was
interesting, however, to find that the body shape of sexually depending on their maturation status and the presence/absence of
an eyespot on the dorsal fin. Unsurprisingly, mature non-spotted
individuals were significantly bigger (Figure 2A–E) and more
rectangular than immature ones (Figure 2F), with mature males
being the largest and having the most rectangular body (one-way
ANOVA
for
SL,
F4,98 = 45.98,
p,0.001;
A,
F4,98 = 37.99,
p,0.001;
DB1,
F4,98 = 46.03,
p,0.001;
DB2,
F4,98 = 40.25,
p,0.001; one-way ANOVA, F4,98 = 5.58, p,0.001). It was
interesting, however, to find that the body shape of sexually February 2013 | Volume 8 | Issue 2 | e55938 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 4 Eyespot Dishonest Signalling Figure 3. Comparison of the body shape of P. amboinensis belonging to different age and gender groups. Results of a canonical
discriminant analysis are displayed with treatment centroids and 95% confidence clouds plotted together with the direction and importance (as
indicated by the length of the vector) of trends in statistically significant shape descriptors. doi:10.1371/journal.pone.0055938.g003 Figure 3. Comparison of the body shape of P. amboinensis belonging to different age and gender groups. Results of a canonical
discriminant analysis are displayed with treatment centroids and 95% confidence clouds plotted together with the direction and importance (as
indicated by the length of the vector) of trends in statistically significant shape descriptors. doi:10.1371/journal.pone.0055938.g003 level harmonics, H8, H14, H16), albeit camouflaged in juvenile/
immature female shape. February 2013 | Volume 8 | Issue 2 | e55938 Results and Discussion Mean age in days of P. amboinensis belonging to
different age and gender groups without eyespot (diamonds; N-
values = 21 males & 36 females) and with eyespot (circles; N-
values = 9 males & 14 females). Error bars indicate 95% CI. Letters
indicate significant differences based on post-hoc Tukey’s HSD tests. doi:10.1371/journal.pone.0055938.g004 February 2013 | Volume 8 | Issue 2 | e55938 February 2013 | Volume 8 | Issue 2 | e55938 5 PLOS ONE | www.plosone.org Eyespot Dishonest Signalling Eyespot Dishonest Signalling eyespots and whether these spotted males are indeed successful in
extra pair or group spawning activities. reproduction instead of growth. In addition, mature eye-spotted
males reached maturation and changed sex at an earlier age (at
0.6 yr defined as the median age of the individuals in this group)
than any of the mature fish without eyespots (over 9 months old for
both sexes), reinforcing the idea that these individuals may be pre-
disposed towards becoming mimics, while the others delay
maturation in favour of growth to then become parentals [35]. In systems where male reproductive success is shaped by male-
male competition and aggression, individuals have been observed
to adopt alternative reproductive behaviours [33]. For example,
males may parasitize the territory of dominant male conspecifics
when they are competitively inferior, possibly due to their smaller
size. These parasitic males may try to steal fertilizations by rushing
into the nest guarded by their large, aggressive territorial
counterparts just at the moment of egg deposition (i.e. sneakers). However, when a display of typical male behaviour is associated
with a low probability of successful competitive outcomes (and
hence high fitness costs) these non-dominant males may adopt
a deceptive appearance or behaviour approach enabling them, for
example, to follow the female to the nest in the guise of juveniles
(i.e. juvenile mimics) to fertilize freshly laid eggs before the resident
male can do so. Alternatively, they may mimic the behaviour and
appearance of reproductive females (i.e. female mimics) in order to
gain access to reproductive females attracted to the nests of
dominant males. Either way, non-dominant individuals with
inherently smaller body sizes are likely to be more reproductively
successful by adopting one of these strategies when competing
against dominant males than simply accepting their fate as overtly
inferior males (i.e. making the best of a bad situation). Results and Discussion So, do conditions experienced early in life determine which
tactic an individual should choose? In some species, reproductive
behaviours are fixed through genetic pathways (i.e. genetic
polymorphism [36]). In others, processes operating prior to
spawning such as non-genetic parental effects, may pre-determine
which fish will later become a dominant male in a group [37]. Alternatively, it is equally possible that their early juvenile
environment and individual personalities (e.g. shy/bold and
aggressive/subordinate; [38]) shape their future reproductive life
history strategy [39]. For instance, individuals recruiting late in the
season that settle in a reef environment already populated by
slightly older, larger and bolder juveniles from previous cohorts,
may be more likely to experience aggressive behaviours and such
social interactions may promote the development of a subordinate
‘personality’ with carry-over effects on their adult life. Of course,
we acknowledge that these are currently speculative interpreta-
tions of our results. (
g
)
Many species exhibit age-dependent variation in their re-
productive tactics by sneaking when young and becoming
parentals when older [33] and hence, sneaking is generally
considered to be a conditional strategy based on body size. Mimicry, and specifically female mimicry on the other hand,
requires gonadal development and gamete production to be
decoupled from the female phenotype, and hence it may
underscore some genetically embedded behavioural pathways
[34]. In principle, mimicry would allow young males to more
easily bypass the territorial defences of older males and gain access
deceptively to territories and females. In the attempt to elucidate
which strategy best describes the study system, we aged a total of
80 P. amboinensis with and without eyespots. Interestingly, eye-
spotted males were not significantly younger than mature
individuals who have lost their eyespots (one-way ANOVA,
F3,75 = 5.41, p,0.002, where [F without eyespot = M without
eyespot] ? F with eyespot; M with eyespot = all groups; Figure 4),
suggesting that these individuals are not adopting an age-
dependent conditional sneaking strategy (i.e. sneaking when young
and waiting to become parentals when older). However, it could
be that sneaking is conditional with respect to some other trait
than age (e.g. size). Acknowledgments We thank staff at the Lizard Island Research Station for their assistance
and reviewers for their constructive comments. Results and Discussion Our results show that these individuals were
clearly older for a given body size (ANCOVA, F3,74 = 3.02,
p,0.05), indicating that they may invest more heavily in We now know that eyespots do not function as an anti-
predatory device in this species, but are instead honest signals of
juvenile status and hence interpreted by con-specifics as sub-
ordinate and non-threatening [11]. Here we have shown that
while most individuals lose their eyespots on maturation, some
retain them into adulthood, that this retention is gender-specific
(suggesting some reproductive advantage) and that it is further
enhanced by eye-spotted males having a closer resemblance to
females than the males they actually are. Clearly, the next logical step is to test a number of hypotheses
using experimental and manipulative approaches so that we can
fully appreciate and understand the range of functional roles
eyespots have in animals. Author Contributions Conceived and designed the experiments: MG MD. Performed the
experiments: MG MD. Analyzed the data: MG. Contributed reagents/
materials/analysis tools: MG MD. Wrote the paper: MG MD. Conceived and designed the experiments: MG MD. Performed the
experiments: MG MD. Analyzed the data: MG. Contributed reagents/
materials/analysis tools: MG MD. Wrote the paper: MG MD. References 8. Van Buskirk J, Aschwanden J, Buckelmuller I, Reolon S, Ruttiman S (2004)
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2. Vallin A, Jakobsson S, Lind J, Wiklund C (2005) Prey survival by predator
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Superresolution Imaging Identifies That Conventional Trafficking Pathways Are Not Essential for Endoplasmic Reticulum to Outer Mitochondrial Membrane Protein Transport
|
Scientific reports
| 2,017
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cc-by
| 12,959
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Transport Kyle Salka1, Shivaprasad Bhuvanendran1, Kassandra Wilson1, Petros Bozidis1,2, Mansi
Mehta1, Kristin Rainey3, Hiromi Sesaki4, George H. Patterson3, Jyoti K. Jaiswal1,5 & Anamaris
M. Colberg-Poley1,5,6 Most nuclear-encoded mitochondrial proteins traffic from the cytosol to mitochondria. Some of
these proteins localize at mitochondria-associated membranes (MAM), where mitochondria are
closely apposed with the endoplasmic reticulum (ER). We have previously shown that the human
cytomegalovirus signal-anchored protein known as viral mitochondria-localized inhibitor of apoptosis
(vMIA) traffics from the ER to mitochondria and clusters at the outer mitochondrial membrane (OMM). Here, we have examined the host pathways by which vMIA traffics from the ER to mitochondria and
clusters at the OMM. By disruption of phosphofurin acidic cluster sorting protein 2 (PACS-2), mitofusins
(Mfn1/2), and dynamin related protein 1 (Drp1), we find these conventional pathways for ER to the
mitochondria trafficking are dispensable for vMIA trafficking to OMM. Instead, mutations in vMIA that
change its hydrophobicity alter its trafficking to mitochondria. Superresolution imaging showed that
PACS-2- and Mfn-mediated membrane apposition or hydrophobic interactions alter vMIA’s ability to
organize in nanoscale clusters at the OMM. This shows that signal-anchored MAM proteins can make
use of hydrophobic interactions independently of conventional ER-mitochondria pathways to traffic
from the ER to mitochondria. Further, vMIA hydrophobic interactions and ER-mitochondria contacts
facilitate proper organization of vMIA on the OMM. Mitochondria consist of nearly a thousand proteins, and aside from the 13 proteins encoded by the mitochon-
drial genome, the rest are encoded by nuclear genes1. These proteins are synthesized in the cytosol and imported
into mitochondria using highly conserved translocation machinery2. Analysis of the mitochondrial proteome
has identified that a number of these proteins also localize in other organelles including over fifty proteins that
are classified as endoplasmic reticulum (ER) proteins3. Cellular proteins that traffic to mitochondria via the
ER include apoptosis inducing factor (AIF), acyl-CoA:diacylglycerol acyl-transferase 2 (DGAT2), and retinol 1Center for Genetic Medicine Research, Children’s National Health System, 111 Michigan Ave, NW, Washington,
DC, 20010, USA. 2Laboratory of Microbiology, Department of Medicine, School of Health Sciences, University
of Ioannina, Ioannina, 45500, Greece. 3Section on Biophotonics, National Institute of Biomedical Imaging
and Bioengineering, National Institutes of Health, Bethesda, MD, 20892, USA. 4Department of Cell Biology,
Johns Hopkins University School of Medicine Hunterian 111, 725 N. Wolfe Street, Baltimore, MD, 21205, USA. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 23 June 2016
Accepted: 20 December 2016
Published: xx xx xxxx Superresolution Imaging Identifies
That Conventional Trafficking
Pathways Are Not Essential for
Endoplasmic Reticulum to Outer
Mitochondrial Membrane Protein
Transport Received: 23 June 2016
Accepted: 20 December 2016
Published: xx xx xxxx Transport The second route for protein trafficking at the MAM involves
vesicular transport from ER to mitochondria, where membrane scission protein called dynamin related protein 1
(Drp1) facilitates transport of proteins from the ER to mitochondria4,10. Subpopulations of AIF and HIV-1 viral
protein Vpr are packaged and transported to mitochondria in vesicles. Knockdown of Drp1, ATPase family AAA
domain containing 3A (ATAD3A), or Mfn2 decreases AIF and HIV Vpr trafficking to mitochondria4,10. Drp1,
ATAD3A and Mfn2 are suggested to play distinct roles by facilitating budding, movement and fusion of the ves-
icles, respectively.fi y
Similar to the mitochondrial signal-anchored proteins, which traffic from the cytosol to the OMM, we found
that CMV vMIA is signal-anchored by an N-terminal single pass hydrophobic leader that serves as part of its
mitochondrial targeting signal (MTS)7,11. Rather than direct transport from the cytosol to the OMM, we pre-
viously showed that vMIA traffics sequentially from the ER to mitochondria through MAM contacts between
the two organelles7–9,11. vMIA signal sequence is not cleaved through its trafficking to OMM, identifying that
its transport does not involve transiting through the Golgi complex. However, the role of host proteins in vMIA
trafficking has not yet been studied. Notably, vMIA targeting is efficient: vMIA is able to retarget a cellular ER
protein, viperin, to mitochondria and the vMIA MTS can retarget the Tom20 hydrophobic leader to this ER to
mitochondrial trafficking11,27. Thus, vMIA offers a valuable tool to define the mechanism for ER to mitochondrial
trafficking of signal-anchored OMM proteins.ffi fi
g
g
p
While no consensus MAM/mitochondrial targeting signal is known, the factors affecting trafficking at the
MAM are now emerging11,22. Affinity for lipids has been implicated in MAM trafficking of proteins including
the lipid synthetic proteins. Targeting of Rdh10 to the MAM and mitochondria requires its N- and C-terminal
hydrophobic domains and Rdh10 can relocalize to lipid droplets6. Similarly, cellular retinol-binding protein type
1 localizes to the MAM and mitochondria and can relocalize to lipid droplets6,28. Palmitoylation enriches cal-
nexin and thioredoxin related transmembrane protein in the MAM29. Sigma 1 receptor (Sig-1R) localizes to the
MAM using cholesterol binding30,31. Similarly, the N-terminal MTS of vMIA contains an evolutionarily conserved
hydrophobic leader, with a consensus cholesterol binding domain (CBD) and multiple basic residues and proline
rich domain (PRD) downstream of the CBD7,11. Transport 5Department of Integrative Systems Biology, and of Pediatrics, George Washington University School of Medicine
and Health Sciences, Washington, DC, 20037, USA. 6Departments of Biochemistry & Molecular Medicine, and of
Microbiology, Immunology & Tropical Medicine, George Washington University School of Medicine and Health
Sciences, Washington, DC, 20037, USA. Correspondence and requests for materials should be addressed to J.K.J. (email: jkjaiswal@childrensnational.org) or A.M.C. (email: acolberg-poley@childrensnational.org) Scientific Reports | 7: 16 | DOI:10.1038/s41598-017-00039-5 1 www.nature.com/scientificreports/ dehydrogenase 10 (Rdh10), which traffic directly from the ER to the mitochondria4–6. Aside from cellular proteins,
pathogen-encoded proteins such as the human cytomegalovirus (CMV) encoded viral mitochondrial-localized
inhibitor of apoptosis (vMIA), hepatitis c virus (HCV) encoded N3/4A protease, and human immunodeficiency
virus 1 (HIV-1) encoded viral protein R (Vpr) also traffic from the ER to mitochondria7–12. (
)
p
( p )fi
There are two routes proposed for protein trafficking from the ER to mitochondria. The first is based
upon ER and the OMM proximity, where a bridge (tether) facilitates calcium (Ca2+) transfer through the
mitochondria-associated membrane (MAM) calcium signaling complex, which contains inositol 1,4,5 trisphos-
phate receptors (IP3Rs), cytosolic glucose response protein 75 (Grp75) and the outer mitochondrial membrane
(OMM)-localized voltage dependent anion channel (VDAC), and lipids between these compartments13–16. In yeast, MAM tethers, known as ER mitochondria encounter structure (ERMES) facilitate phospholipid
exchange17. ER-OMM contacts may facilitate transfer of proteins between these compartments. In mammalian
cells, several proteins including phosphofurin acidic cluster sorting protein 2 (PACS-2), Nogo (or reticulon 4)
and mitofusins (Mfn1/2) have been implicated in regulating ER-mitochondrial apposition14,18–21. PACS-2 is
required for proper distribution of the MAM-enriched protein calnexin22. It is currently debated whether mito-
fusins regulate ER-mitochondrial tethering and mitochondrial Ca2+ uptake in positive or negative manner. Although homotypic interactions between Mfn2 and heterotypic interaction with Mfn1 have been implicated
in decreasing ER-mitochondria tethering and functional coupling23,24, a recent study re-established the previous
report that Mfn2 is an ER-mitochondrial tether and its ablation reduces mitochondrial Ca2+ uptake without
altering the mitochondrial Ca2+ uniporter complex21,25. While the precise mechanism of action of mitofusins
in ER-mitochondria coupling is yet to be resolved, lack of Mfn1/2 has been shown to affect the distribution of
proteins at the OMM by altered MAM tethering26. Scientific Reports | 7: 16 | DOI:10.1038/s41598-017-00039-5 Transport We found that vMIA mutations that increase its MTS hydropathy
score retarget vMIA to the secretory apparatus and reduce its mitochondrial trafficking11. Mutations in the vMIA
CBD block its association with MAM lipid rafts, but do not affect its trafficking to the OMM32. ptffi
g
Similar to many other OMM proteins, including Tom20, Tom22, VDAC, and the associated cytosolic hexoki-
nase I33–35, vMIA also organizes in nanoscale clusters13,36. Using superresolution imaging by multifocal structured
illumination microscopy (MSIM), gated stimulated emission depletion (gSTED) and photoactivated localization
microscopy (PALM), we established that vMIA forms clusters of ~100–150 nm at the OMM of human cells13,36. Clustering has also been reported for inner mitochondrial membrane (IMM) proteins including mitochondrial
inner membrane or cristae organizing system (MICOS or MINOS), and cytochrome C oxidase subunit 2 of com-
plex IV35,37. These mitochondrial protein clusters are not static as they can change in response to the functional
requirements of mitochondria34, and following viral infection38. However, the cellular mechanism for facilitating
clustering of mitochondrial proteins and how this is dynamically regulated is poorly understood.fi g
p
y
y
g
p
y
In this study, we investigated the cellular mechanisms reported for ER to OMM protein trafficking and OMM
clustering of a membrane-anchored protein. To test the requirements of ER-mitochondria apposition and tether-
ing (bridge model) for trafficking, we used PACS-2 null and Mfn1/2 null mouse embryonic fibroblasts (MEFs)39,40
and PACS-2 knockdown human cells. To investigate the role of membrane scission (vesicle model) in traffick-
ing from ER to mitochondria, we used Drp1-null cells41. Additionally, by using vMIA MTS mutants, we have
assessed the role of hydrophobic and lipid interactions on mitochondrial localization of vMIA and its ability to
dimerize and form clusters at the OMM. Our results identify that vMIA uses an unconventional hydrophobic
interaction-mediated mechanism to traffic from the ER to the OMM and does not require Drp1-, PACS-2-, and
mitofusin-mediated pathways. Instead, PACS-2 and Mfn-mediated ER-mitochondria apposition regulates the
ability of vMIA to form clusters at the OMM. Scientific Reports | 7: 16 | DOI:10.1038/s41598-017-00039-5 2 www.nature.com/scientificreports/ Figure 1. vMIA traffics to and clusters at the OMM in mouse embryonic fibroblasts. (a) Human (HFF and
HeLa) and mouse (MEF) fibroblasts were transfected transiently (19 hours) to express vMIA-EYFP and then
immunolabeled for the OMM marker Tom20. The images show an optical slice obtained by deconvolution of
a confocal z-stack. Transport (b) A single optical plane from deconvolved z-stack of gSTED images showing clustered
distribution of vMIA-EYFP and Tom20 (immunostained) on the OMM of a HeLa cell transfected as above. Zoom shows the merged image marked by white box in the merged channel. (c) Normalized intensity profile of
the red and green pixels marked by the dotted line in the zoomed region in panel b. (d) Single optical plane from
deconvolved z-stack of gSTED images showing clustered distribution of vMIA-EYFP on the OMM in a WT
MEF. Zoom shows the region marked by white box. (e) A single plane from an MSIM z-stack images showing
clustered distribution of vMIA-EGFP on the OMM of a transfected MEF cell. Zoom of a single mitochondrion
(boxed) shows vMIA clustering and line marks the region used to plot the intensity profile in the panel d. (f)
Normalized intensity profile of the mitochondrion along the pixels marked by the solid line in the zoomed
region. (g) Plot showing vMIA cluster size (FWHM) in WT MEFs. The line in the box and red, cross mark
indicate the median (177.0) and mean (179.9 ± 3.7), respectively (n = 75). Figure 1. vMIA traffics to and clusters at the OMM in mouse embryonic fibroblasts. (a) Human (HFF and
HeLa) and mouse (MEF) fibroblasts were transfected transiently (19 hours) to express vMIA-EYFP and then
immunolabeled for the OMM marker Tom20. The images show an optical slice obtained by deconvolution of
a confocal z-stack. (b) A single optical plane from deconvolved z-stack of gSTED images showing clustered
distribution of vMIA-EYFP and Tom20 (immunostained) on the OMM of a HeLa cell transfected as above. Zoom shows the merged image marked by white box in the merged channel. (c) Normalized intensity profile of
the red and green pixels marked by the dotted line in the zoomed region in panel b. (d) Single optical plane from
deconvolved z-stack of gSTED images showing clustered distribution of vMIA-EYFP on the OMM in a WT
MEF. Zoom shows the region marked by white box. (e) A single plane from an MSIM z-stack images showing
clustered distribution of vMIA-EGFP on the OMM of a transfected MEF cell. Zoom of a single mitochondrion
(boxed) shows vMIA clustering and line marks the region used to plot the intensity profile in the panel d. Transport (f)
Normalized intensity profile of the mitochondrion along the pixels marked by the solid line in the zoomed
region. (g) Plot showing vMIA cluster size (FWHM) in WT MEFs. The line in the box and red, cross mark
indicate the median (177.0) and mean (179.9 ± 3.7), respectively (n = 75). Resultsfi (d) Polar plot presentation of the fluorescence lifetime of
the ROI marked by white circle in panel c. The plot coordinates for (b) and (d) are x = M cosφ and y = M sinφ,
where M is the modulation and φ is the phase delay. (e) The fluorescence lifetime comparison of cytosolic EGFP
(τ = 3.41 ± 0.01 ns, n = 140 ROIs from 14 cells) and vMIA-EGFP (τ = 3.10 ± 0.01 ns, n = 130 ROIs from 13 cells)
in WT MEFs. ****Represents p < 0.0001. Figure 2. Use of fluorescence lifetime imaging to monitor vMIA homodimerization in WT MEFs. vMIA-
EGFP was transiently expressed (as above) in WT MEFs, and live cells were imaged by using frequency
domain lifetime microscopy. (a) Fluorescent lifetime image of a WT MEF expressing cytosolic EGFP. The
pixels are pseudocolored based on their lifetime values as indicated by the heat map scale. (b) Fluorescence
lifetime polar plot representing the frequency characteristics of each pixel in the ROI marked by the white
circle in panel a. The colored squares represent a unique pixel lifetime and the red square marks the median
lifetime value of the ROI (c) Fluorescent lifetime image of a WT MEF expressing vMIA-EGFP. The pixels are
pseudocolored based on their lifetime as in panel b. (d) Polar plot presentation of the fluorescence lifetime of
the ROI marked by white circle in panel c. The plot coordinates for (b) and (d) are x = M cosφ and y = M sinφ,
where M is the modulation and φ is the phase delay. (e) The fluorescence lifetime comparison of cytosolic EGFP
(τ = 3.41 ± 0.01 ns, n = 140 ROIs from 14 cells) and vMIA-EGFP (τ = 3.10 ± 0.01 ns, n = 130 ROIs from 13 cells)
in WT MEFs. ****Represents p < 0.0001. vMIA forms homodimers in MEFs. vMIA is known to homodimerize during ER to mitochondrial traf-
ficking in human (HFFs and HeLa) cells42,43. To monitor this process in mouse cells, we imaged live cells using
fluorescent lifetime imaging microscopy (FLIM) (Fig. 2). As has been demonstrated before, homoFRET imaging
allows monitoring protein-protein proximity by change in fluorescence lifetime44. We thus imaged EGFP lifetime
in WT MEFs expressing cytosolic EGFP or vMIA-EGFP. This shows a homogenously greater pixel lifetimes in
cell expressing EGFP (Fig. Resultsfi Results
vMIA traffics to and clusters at the OMM in MEFs. vMIA traffics from the ER to OMM in human cells
including human primary fibroblasts (HFFs), U373 and HeLa cells7–9,11,13,36. By using confocal microscopy and
gSTED superresolution microscopy of vMIA with the OMM marker Tom20, we investigated the localization
of vMIA at the OMM in human and mouse cells. Confocal microscopy confirmed mitochondrial localization
of vMIA in all (human and mouse) cells tested (Fig. 1a). Pixel co-localization analysis in WT-MEFs identified
83.9 ± 0.5% co-localization between vMIA and Tom20 (Fig. S1), showing that, similar to the human primary
fibroblasts, vMIA traffics efficiently to the OMM in WT MEFs. ififi
y
Using gSTED superresolution imaging, we examined if the vMIA clusters that we previously described in the
human cells13,36 localize with Tom20 clusters. Using HeLa cells, we found that vMIA exists in clusters that can
include or exclude Tom20 clusters (Fig. 1b inset, and corresponding line intensity profile in 1c). Use of gSTED
imaging together with MSIM superresolution imaging of the wild type (WT) MEFs showed that vMIA also
formed clusters at the OMM in mouse cells (Fig. 1d–g). MSIM analysis of vMIA clustering at the OMM in MEFs
identified that the full width half maximum (FWHM) of vMIA clusters is 179.9 ± 3.7 nm (n = 75) (Fig. 1c,d). This
size is similar to 100–150 nm sized clusters we previously detected in the human cells13,36. Together, above results
identify that vMIA mitochondrial trafficking and organization in clusters at the OMM occurs similarly in human
and mouse cells. Scientific Reports | 7: 16 | DOI:10.1038/s41598-017-00039-5 3 www.nature.com/scientificreports/ Figure 2. Use of fluorescence lifetime imaging to monitor vMIA homodimerization in WT MEFs. vMIA-
EGFP was transiently expressed (as above) in WT MEFs, and live cells were imaged by using frequency
domain lifetime microscopy. (a) Fluorescent lifetime image of a WT MEF expressing cytosolic EGFP. The
pixels are pseudocolored based on their lifetime values as indicated by the heat map scale. (b) Fluorescence
lifetime polar plot representing the frequency characteristics of each pixel in the ROI marked by the white
circle in panel a. The colored squares represent a unique pixel lifetime and the red square marks the median
lifetime value of the ROI (c) Fluorescent lifetime image of a WT MEF expressing vMIA-EGFP. The pixels are
pseudocolored based on their lifetime as in panel b. Resultsfi 2a,b), but a more heterogeneous pixel lifetime values in a cell expressing vMIA-EGFP
(Fig. 2c,d). Quantification of lifetime of the pixels (e.g.: those marked by the circles in Fig. 2a,c) showed the
average lifetime of cytosolic EGFP was 3.41 ± 0.01 ns (10 regions per cell, n = 14 cells), which was reduced to
3.10 ± 0.01 ns for vMIA-EGFP (10 regions per cell, n = 13 cells, p < 0.0001) (Fig. 2e). This decrease in EGFP life-
time when tagged to vMIA demonstrates that, similar to human cells, vMIA efficiently homodimerizes during its
trafficking from ER to mitochondria in MEFs. The quantitative nature of FLIM also provides an assay to quantify
alterations in vMIA homodimerization on a single cell basis. We have used this ability in subsequent sections to
study the effects of various genetic changes in host cells and in vMIA itself on its ability to form homodimers. Mfn2 affects vMIA clustering at the OMM. Mfn1/Mfn2 are proposed to tether the ER to mitochon-
dria21,25, and interaction of vMIA with Mfn2 is important for its anti-apoptotic function45. Thus, we hypothesized
that mitofusin-vMIA interactions may regulate vMIA trafficking from ER to OMM. To test this, we first examined
vMIA and Mfn2 colocalization in MEFs (Fig. 3). In MEFs, Mfn2 localizes at mitochondrial junctions, causing
Mfn2-YFP (green) to localize with the OMM marker, Tom20-mCherry (red) (89 ± 3.4%) (Fig. 3a). However,
unlike Tom20, Mfn2 forms discreet puncta at the mitochondrial junction sites, causing only a small percentage Scientific Reports | 7: 16 | DOI:10.1038/s41598-017-00039-5 4 www.nature.com/scientificreports/ Figure 3. Expression of vMIA causes redistribution of Mfn2 on the OMM. MEFs transfected with Mfn
(pseudocolored green) and Tom20-mCherry (red), (a) without or (b) with vMIA-CFP (blue) were analy
by confocal microscopy. (a) In MEFs not expressing vMIA, Mfn2 shows punctate distribution along the
mitochondria; boxes mark the zoomed regions shown below. (b) Expression of vMIA causes Mfn2 to di
more uniformly along the mitochondria; boxes mark the zoomed regions below. (c) Plot showing quant
of Mfn2 colocalization with OMM marker Tom20 in MEFs expressing or not expressing vMIA-CFP. T
in the box represents the median value of cells not expressing (13%) or expressing vMIA (73.9%) (n > 1
***Represents p<0.001. Figure 3. Expression of vMIA causes redistribution of Mfn2 on the OMM. Resultsfi MEFs transfected with Mfn2-YFP
(pseudocolored green) and Tom20-mCherry (red), (a) without or (b) with vMIA-CFP (blue) were analyzed
by confocal microscopy. (a) In MEFs not expressing vMIA, Mfn2 shows punctate distribution along the
mitochondria; boxes mark the zoomed regions shown below. (b) Expression of vMIA causes Mfn2 to distribut
more uniformly along the mitochondria; boxes mark the zoomed regions below. (c) Plot showing quantificatio
of Mfn2 colocalization with OMM marker Tom20 in MEFs expressing or not expressing vMIA-CFP. The line
in the box represents the median value of cells not expressing (13%) or expressing vMIA (73.9%) (n > 16 cells)
***Represents p < 0.001. Figure 3. Expression of vMIA causes redistribution of Mfn2 on the OMM. MEFs transfected with Mfn2-YFP
(pseudocolored green) and Tom20-mCherry (red), (a) without or (b) with vMIA-CFP (blue) were analyzed
by confocal microscopy. (a) In MEFs not expressing vMIA, Mfn2 shows punctate distribution along the
mitochondria; boxes mark the zoomed regions shown below. (b) Expression of vMIA causes Mfn2 to distribute
more uniformly along the mitochondria; boxes mark the zoomed regions below. (c) Plot showing quantification
of Mfn2 colocalization with OMM marker Tom20 in MEFs expressing or not expressing vMIA-CFP. The line
in the box represents the median value of cells not expressing (13%) or expressing vMIA (73.9%) (n > 16 cells). ***Represents p < 0.001. Scientific Reports | 7: 16 | DOI:10.1038/s41598-017-00039-5 5 www.nature.com/scientificreports/ Figure 4. Role of mitofusins (Mfn1/2) on vMIA trafficking and clustering on mitochondria. (a) Mfn1/2-null
MEFs were transiently transfected to express vMIA-CFP (pseudocolored green) and Tom20-mCherry (red) and
imaged by confocal microscopy. The images show a single confocal plane from a deconvolved z-stack presented
as monochrome images of the individual channels, which are pseudocolored for the merged image, and the
boxed region marks the region zoomed in the inset. (b) The fluorescence lifetime comparison of cytosolic EGFP
(τ = 3.46 ± 0.015 ns, n = 150 regions from 15 cells) and vMIA-EGFP (τ = 3.10 ± 0.011 ns, n = 130 regions from
13 cells) in Mfn1/2-null MEFs. (c) A single plane from an MSIM z-stack images showing clustered distribution
of vMIA-EGFP on the OMM of an Mfn1/2-null MEF. A zoom of mitochondria shows vMIA clustering. (d) The
number of vMIA clusters/µm of the OMM in WT MEFs (n = 50) and Mfn1/2-null MEFs (n = 40) are shown. Resultsfi (e)
Normalized intensity profile along the solid line shown in the zoomed inset in panel c. (f) The FWHM of vMIA
clusters (n = 40) in Mfn1/2-null MEFs were measured and plotted. The line and red cross mark indicate the
median and mean, respectively. ****Represents p < 0.0001. Figure 4. Role of mitofusins (Mfn1/2) on vMIA trafficking and clustering on mitochondria. (a) Mfn1/2-null
MEFs were transiently transfected to express vMIA-CFP (pseudocolored green) and Tom20-mCherry (red) and
imaged by confocal microscopy. The images show a single confocal plane from a deconvolved z-stack presented
as monochrome images of the individual channels, which are pseudocolored for the merged image, and the
boxed region marks the region zoomed in the inset. (b) The fluorescence lifetime comparison of cytosolic EGFP
(τ = 3.46 ± 0.015 ns, n = 150 regions from 15 cells) and vMIA-EGFP (τ = 3.10 ± 0.011 ns, n = 130 regions from
13 cells) in Mfn1/2-null MEFs. (c) A single plane from an MSIM z-stack images showing clustered distribution
of vMIA-EGFP on the OMM of an Mfn1/2-null MEF. A zoom of mitochondria shows vMIA clustering. (d) The
number of vMIA clusters/µm of the OMM in WT MEFs (n = 50) and Mfn1/2-null MEFs (n = 40) are shown. (e)
Normalized intensity profile along the solid line shown in the zoomed inset in panel c. (f) The FWHM of vMIA
clusters (n = 40) in Mfn1/2-null MEFs were measured and plotted. The line and red cross mark indicate the
median and mean, respectively. ****Represents p < 0.0001. of Tom20 pixels (18 ± 4.1%) at the OMM to overlap with the Mfn2 pixels (Fig. 3c). Expression of vMIA in MEFs
caused redistribution of Mfn2 along the mitochondria (Fig. 3b). While the punctate distribution of Mfn2 was
still detectable, Mfn2 redistributed along the entire OMM such that colocalization of Tom20 pixels with Mfn2
increased from 18 ± 4.1% to 75 ± 5.1% (Fig. 3b,c). Mfn2 also colocalized well with vMIA (85 ± 3.4%; Fig. 3b). In view of the redistribution of Mfn2 upon vMIA expression, we investigated whether mitofusins affect vMIA
trafficking. The two mitofusin proteins (Mfn1 and Mfn2) have been shown to be partly redundant, as overexpres-
sion of either protein is able to rescue the loss of the other46. Resultsfi Shown are
single confocal planes from a deconvolved z-stack as monochrome images of individual channels, which are
pseudocolored for the merged image. The box marks the zoomed region. (b) A single confocal plane from a
deconvolved z-stack of confocal images of PACS-2-null MEFs transiently expressing vMIA-EGFP (green) and
Tom2-mCherry (red). The monochrome image of each fluorophore and the merged images are shown. The
box marks the zoomed area. (c) The fluorescence lifetime comparison of cytosolic EGFP (τ = 3.44 ± 0.009 ns,
n = 150 regions from 15 cells) and vMIA-EGFP (τ = 3.08 ± 0.009 ns, n = 120 regions from 12 cells) in
PACS-2-null MEFs. (d) Zoomed single optical plane of a mitochondrion from a MSIM z-stack showing
clustered distribution of vMIA-EGFP on the OMM of a transfected PACS-2-null MEF. (e) The percentage of
mitochondria (20 mitochondria/cell) with clustered vMIA along the OMM in WT MEFs (n = 5 cells) and
PACS-2-null MEFs (n = 6 cells). (f) Normalized pixel intensity of vMIA-EGFP along the line shown in the
zoomed d inset. (g) Box plot showing the FWHM distribution of vMIA clusters (n = 60 clusters) in WT MEFs
and PACS-2-null MEFs. The line and red, cross mark indicate the median and mean, respectively. (h) HeLa
(PSS-120) cells8 were lipofected with nonspecific (NS siRNA) or PACS-2 (PACS-2 siRNA) siRNAs and vector
expressing WT vMIA and harvested 48 h later as described18. PACS-2 knockdown was assessed using rabbit
anti-PACS-2 antiserum (1:500; gift from Dr. G. Thomas). Transfected cells were fractionated to obtain purified
microsomes and mitochondria. 10 μg of fractionated proteins were separated by SDS-PAGE and analyzed by
Western using anti-vMIA (DC35, 1:500)8 or mitochondrial (Grp75, 1:1000; Stressgen) marker. Monomeric and
dimeric vMIA are indicated by the grey and open arrows, respectively. *Represents p < 0.05, ****represents
p < 0.0001. The blots were cropped to enhance the conciseness of presentation. Full-length blots are presented
in Supplementary Figure S5. Figure 5. Role of PACS-2 in vMIA trafficking and clustering on the mitochondria. (a) WT MEFs transiently
expressing vMIA-mCherry (red) and PACS-2-EGFP (green) were imaged by confocal microscopy. Shown are
single confocal planes from a deconvolved z-stack as monochrome images of individual channels, which are
pseudocolored for the merged image. The box marks the zoomed region. (b) A single confocal plane from a
deconvolved z-stack of confocal images of PACS-2-null MEFs transiently expressing vMIA-EGFP (green) and
Tom2-mCherry (red). Resultsfi In view of this partial redundancy in the function of
the mitofusins, to test if Mfn2 is required for vMIA trafficking from the ER to OMM, we made use of Mfn1/2-null
MEFs (Fig. 4). Similar to the extensive colocalization of vMIA-CFP with Tom20-mCherry in WT MEFs
(83.9 ± 0.5%; Fig. S1), vMIA colocalized with Tom20-mCherry even in the Mfn1/2-null MEFs (85.8 ± 1.4%)
(Fig. 4a, Fig. S1). This shows that vMIA can traffic normally to mitochondria even in the absence of Mfn1 and
Mfn2, and show that mitofusins are not required for vMIA trafficking to the OMM. FLIM analysis of vMIA-EGFP
in Mfn1/2-null MEFs showed that vMIA-EGFP lifetime in Mfn1/2-null MEFs (τ = 3.10 ± 0.01 ns, n = 13) was
significantly lower than the lifetime of cytosolic EGFP (τ = 3.46 ± 0.02 ns, n = 15, p < 0.0001) (Fig. 4b), and was
similar to that of the WT-MEFs (Fig. 2). Thus, lack of mitofusins does not affect vMIA homodimerization. l
b
d h
ll l
13 36
d h
(
b ) ghf
Similar to our previous observation in primary HFFs and human cell lines13,36 and herein in MEFs (Fig. 1b,c),
superresolution imaging by MSIM showed that vMIA is organized in nanoclusters at the OMM even in the
Mfn1/2-null MEFs (Fig. 4c). Compared to the density of vMIA clusters at the OMM in WT MEFs (0.8 ± 0.05 clus-
ters/µm, n = 50 mitochondria), Mfn1/2-null MEFs showed a 38% increase in the number of clusters (1.1 ± 0.05
clusters/µm/mitochondria, n = 40 mitochondria; p = 0.0005) (Fig. 4d, Fig. S2). Using MSIM imaging, we meas-
ured if this increase in the number of vMIA clusters affected the vMIA cluster size. This showed that despite
the increased number of clusters/mitochondria, the vMIA cluster size was unaltered between the WT MEFs
(179.9 ± 3.7 nm, n = 75 clusters) and Mfn1/2-null MEFs (177.1 ± 3.7 nm, n = 60) (Fig. 4e,f, Fig. S2). We found
no difference in the percentages of mitochondria with clustered vMIA between the WT (91 ± 5.6%, n = 5) and Scientific Reports | 7: 16 | DOI:10.1038/s41598-017-00039-5 6 www.nature.com/scientificreports/ Figure 5. Role of PACS-2 in vMIA trafficking and clustering on the mitochondria. (a) WT MEFs transiently
expressing vMIA-mCherry (red) and PACS-2-EGFP (green) were imaged by confocal microscopy. Resultsfi The monochrome image of each fluorophore and the merged images are shown. The
box marks the zoomed area. (c) The fluorescence lifetime comparison of cytosolic EGFP (τ = 3.44 ± 0.009 ns,
n = 150 regions from 15 cells) and vMIA-EGFP (τ = 3.08 ± 0.009 ns, n = 120 regions from 12 cells) in
PACS-2-null MEFs. (d) Zoomed single optical plane of a mitochondrion from a MSIM z-stack showing
clustered distribution of vMIA-EGFP on the OMM of a transfected PACS-2-null MEF. (e) The percentage of
mitochondria (20 mitochondria/cell) with clustered vMIA along the OMM in WT MEFs (n = 5 cells) and
PACS-2-null MEFs (n = 6 cells). (f) Normalized pixel intensity of vMIA-EGFP along the line shown in the
zoomed d inset. (g) Box plot showing the FWHM distribution of vMIA clusters (n = 60 clusters) in WT MEFs
and PACS-2-null MEFs. The line and red, cross mark indicate the median and mean, respectively. (h) HeLa
(PSS-120) cells8 were lipofected with nonspecific (NS siRNA) or PACS-2 (PACS-2 siRNA) siRNAs and vector
expressing WT vMIA and harvested 48 h later as described18. PACS-2 knockdown was assessed using rabbit
anti-PACS-2 antiserum (1:500; gift from Dr. G. Thomas). Transfected cells were fractionated to obtain purified
microsomes and mitochondria. 10 μg of fractionated proteins were separated by SDS-PAGE and analyzed by
Western using anti-vMIA (DC35, 1:500)8 or mitochondrial (Grp75, 1:1000; Stressgen) marker. Monomeric and
dimeric vMIA are indicated by the grey and open arrows, respectively. *Represents p < 0.05, ****represents
p < 0.0001. The blots were cropped to enhance the conciseness of presentation. Full-length blots are presented
in Supplementary Figure S5. Mfn1/2-null MEFs (87.5 ± 3.2%, n = 4) (Fig. S2). Thus, mitofusins are dispensable for the ability of vMIA to traffic
to the OMM, and to homodimerize and form clusters at the OMM. However, the presence of mitofusins prevents
the formation of excessive vMIA clusters at the OMM. An alternative possibility is that the increase in density of
vMIA clusters at the OMM in Mfn1/2-null cells reflects the alteration of OMM protein distribution caused by the
absence of mitofusins26. PACS-2 alters vMIA clustering without affecting vMIA trafficking to the OMM. Another regula-
tor of ER mitochondrial apposition is PACS-218. Therefore, we examined if PACS-2-mediated ER-OMM appo-
sition is required for vMIA trafficking from the ER to OMM. We tested the ability of vMIA-EGFP to traffic to
the OMM in PACS-2-null MEFs. Resultsfi 5h). Using these cells transfected with vMIA and the biochemical
fractionation approach we have developed to monitor ER to mitochondrial trafficking, we examined the role
of PACS-2 in vMIA trafficking in human cells (Fig. 5h). Use of ER resident enzyme DPM1 and mitochondrial
protein Grp75 as markers for ER and mitochondria, respectively, we verified the identity of these two fractions. As expected we observed vMIA in ER and mitochondrial fractions (Fig. 5h, filled arrow) and also observed vMIA
dimers (Fig. 5h, open arrow). However, while vMIA monomers were also present in the ER and mitochondria
fractions of PACS-2 depleted cells, the dimeric form was only detected in the mitochondrial fraction of cells
treated with nonspecific siRNA but not with PACS-2 siRNA. These results independently validate our findings
by superresolution imaging of the PACS-2 null MEFs that PACS-2 is not required for vMIA trafficking to the
mitochondria, but is required for dimerization and proper organization of vMIA at the mitochondria. Use of
both Mfn1/2-null and of PACS-2 null cells offers no evidence to support the bridge model for vMIA protein
trafficking from ER to the mitochondria. Thus, we next tested the Drp1-mediated vesicular trafficking model for
the ER-mitochondrial protein transport. Drp1 does not regulate vMIA trafficking and its clustering at the OMM. Drp1 is known to asso-
ciate with the ER and facilitate its normal morphology20,47, but its role in regulating vMIA-induced change in
mitochondrial morphology has been debated48,49. However, Drp1 has been implicated in vesicular trafficking
of AIF and Vpr from the MAM to mitochondria4,10. To test if Drp1 may facilitate vesicle-mediated trafficking of
vMIA between ER and mitochondria, we used Drp1-null MEFs41. Using cells transfected with vMIA-CFP and
mCherry-Drp1, we observed these two markers to colocalize in HFFs (mean, 65.9 ± 4.2%, n = 13 cells) and in WT
MEFs (Fig. 6a, Figs S1 and S3). In Drp1-null MEFs, we observed efficient trafficking of vMIA to the mitochondria
as shown by extensive colocalization of vMIA-CFP with Tom20-YFP (82.5 ± 2.8%, n = 11 cells) (Fig. 6b), which
is similar to the WT MEFs (83.9 ± 0.5%) (Fig. S1). Thus, vMIA can traffic efficiently to mitochondria even in the
absence of Drp1-mediated vesicular trafficking between ER and mitochondria. FLIM analysis of vMIA-EGFP
showed that the lack of Drp1 did not affect the ability of vMIA to homodimerize (Fig. Resultsfi Transfection of control WT MEFs with vMIA-mCherry and PACS-2-EGFP
showed that vMIA-labeled (red) mitochondria are interspersed between PACS-2-labeled (green) ER (Fig. 5a). vMIA-mCherry and PACS-EGFP distributed similarly in HFFs (Fig. S3). Similar to the WT MEF, vMIA-EGFP Scientific Reports | 7: 16 | DOI:10.1038/s41598-017-00039-5 7 www.nature.com/scientificreports/ and Tom20-mCherry colocalized extensively in the PACS-2 null MEFs (88.4 ± 1.0%, n = 11 cells) (Figs 1a and 5b,
Fig. S1). However, vMIA distributed more diffusely along the OMM in PACS-2-null MEFs, which increased colo-
calization of vMIA with Tom20 in PACS-2 null cells to 88.4 ± 0.8% (n = 11 cells) as compared to 83.9 ± 0.5% colo-
calization in the WT MEFs (p = 0.0006). This shows that vMIA traffics efficiently to mitochondria in PACS-2-null
MEFs. FLIM analysis of vMIA-EGFP in PACS-2 null MEFs showed vMIA-EGFP lifetime of 3.08 ± 0.01 ns (n = 12
cells) in PACS-2-null MEFs, is similar to that in the WT MEF (τ = 3.10 ± 0.01) and is significantly lower than the
corresponding PACS-2-null cells expressing cytosolic EGFP (τ = 3.44 ± 0.01 ns, n = 15 cells, p < 0.0001) (Fig. 5c). p
g
p
g y
(
p
) ( g
)
By MSIM imaging of vMIA-EGFP in PACS-2-null MEFs, we found that while mitochondria with clustered
vMIA can be detected (Fig. 5d), the percentage of such mitochondria with clustered vMIA (60.8 ± 7.5%, n = 6
cells) was lower than in WT MEFs (91.0 ± 5.7%, n = 5 cells, p = 0.04) (Fig. 5e, Fig. S2). These results show that
vMIA’s ability to form clusters at the OMM is reduced by the reduced apposition between ER and OMM due to
the lack of PACS-2. Concomitantly, the size (FWHM) of vMIA clusters was significantly larger in PACS-2-null
MEFs (190.9 ± 3.7 nm, n = 60 mitochondria) as compared to the WT MEFs (179.9 ± 3.7 nm, n = 75 mitochon-
dria, p = 0.02) (Fig. 5f,g, Fig. S2). However, the number of vMIA clusters/mitochondria was not different between
the PACS-2-null (0.8 ± 0.05 clusters/µm) and WT MEFs (0.8 ± 0.06 clusters/µm) (Fig. S2). Together, above results
with Mfn-null and PACS-2-null MEFs validate the importance of ER-OMM tethering on proper distribution and
clustering of vMIA at the OMM. g
To further verify the effects of PACS-2 on organization of vMIA at mitochondria, we used siRNA to
knockdown PACS-2 in human (HeLa) cells (Fig. Resultsfi 6c) - vMIA-EGFP lifetime
(τ = 3.04 ± 0.03 ns, n = 13 cells) was significantly lower than the cytosolic EGFP (τ = 3.40 ± 0.01 ns, n = 12 cells,
p < 0.0001), and no different from that of vMIA-EGFP lifetime in WT MEFs (Fig. 2e). f
MSIM imaging of vMIA-EGFP demonstrated the ability of vMIA to form clusters at the OMM in Drp1 null
cells (Fig. 6d–f). These vMIA-EGFP clusters in Drp1-null cells were similar in size (172.1 ± 2.8 nm; n = 100 mito-
chondria) to those observed in WT MEFs (179.9 ± 3.7 nm; n = 75 mitochondria) (Fig. 6e,f, Fig. S2). Compared
to the WT MEFs, lack of Drp1 affected neither the number of vMIA clusters/µm (0.8 clusters/µm/mitochondria)
nor the percentage of mitochondria with clustered vMIA (100 ± 0%) (Fig. S2). Thus, by all the measures used,
we found that lack of Drp1-mediated vesicle trafficking did not affect the ability of vMIA to traffic from ER to
mitochondria, dimerize, or properly cluster at the OMM. Lipid and hydrophobic interactions reduce vMIA dimerization and clustering at the OMM. Above results identify that protein-mediated ER-OMM tethering regulates the ability of vMIA to form clusters at
the OMM, but does not affect ER to OMM protein trafficking. Through the use of two vMIA mutants that have
leader sequence with higher hydrophobicity - high hydrophobicity A (HHA) and B (HHB) mutants we have
previously identified that hydrophobic interactions of vMIA affect its trafficking to the mitochondria11. Between
the vMIA hydrophobicity mutants, HHA and HHB, it is the HHB mutant that has larger hydropathy score of its
N-terminal leader (residues 1–22)11. In contrast, the vMIA cholesterol binding domain II (CBDII) mutant has
a mutant cholesterol binding domain (residues 14–23), which reduces its association with the MAM lipid rafts,
without affecting trafficking to the OMM32. Using these two vMIA mutants with defects in lipid binding proper-
ties and divergent trafficking fates, we assessed if altered lipid interactions affect vMIA’s ability to homodimerize
and form clusters. Using FLIM, we assessed the ability of vMIA and its mutants to homodimerize. Compared to the WT
vMIA-EGFP (τ = 3.10 ± 0.01 ns), lifetimes of both the mutant vMIA-EGFP were increased - vMIA-CBDII-EGFP
(τ = 3.14 ± 0.01 ns; p = 0.003) and vMIA-HHB-EGFP (τ = 3.2 ± 0.01 ns; p < 0.0001) (Fig. 7g). Resultsfi Using MSIM
imaging, we readily detected clustering of WT vMIA and of vMIA CBDII at the OMM (Fig. 7a and b, Fig. S4). In contrast, vMIA HHB mutant localized away from the mitochondria, on the ER and did not form clusters 8 Scientific Reports | 7: 16 | DOI:10.1038/s41598-017-00039-5 www.nature.com/scientificreports/ Figure 6. Role of Drp1 on vMIA trafficking and clustering on the mitochondria. (a) HFFs were lipofected
with vectors expressing vMIA-CFP (pseudocolored green) and mCherry-Drp1 (red)57. Cells were imaged by
confocal microscopy and single slice of the deconvolved images is shown as monochrome and pseudocolored
images as above. (b) Confocal image of a Drp1-null MEF transiently expressing vMIA-CFP (pseudocolored
green) and Tom20-YFP (pseudocolored red). Monochrome images show the individual channels and the
merged image demonstrates vMIA colocalization with Tom20-YFP. A zoom of the boxed region of interest
is shown. (c) The fluorescence lifetime comparison of cytosolic EGFP (τ = 3.40 ± 0.013 ns, n = 120 regions
from 12 cells) and vMIA-EGFP (τ = 3.04 ± 0.026 ns, n = 130 regions from 13 cells) in Drp1-null MEFs. (d) A
single plane from an MSIM z-stack images showing clustered distribution of vMIA-EGFP on the OMM of a
transfected Drp1-null MEF. A zoom of a single mitochondrion shows vMIA clustering. (e) Normalized pixel
intensities of a line shown along the mitochondrion in inset of panel d. (f) Box plot showing the distribution of
vMIA clusters (n = 40 cells) in WT MEFs and Drp1-null MEFs. ****Indicates p < 0.0001. Figure 6. Role of Drp1 on vMIA trafficking and clustering on the mitochondria. (a) HFFs were lipofected
with vectors expressing vMIA-CFP (pseudocolored green) and mCherry-Drp1 (red)57. Cells were imaged by
confocal microscopy and single slice of the deconvolved images is shown as monochrome and pseudocolored
images as above. (b) Confocal image of a Drp1-null MEF transiently expressing vMIA-CFP (pseudocolored
green) and Tom20-YFP (pseudocolored red). Monochrome images show the individual channels and the
merged image demonstrates vMIA colocalization with Tom20-YFP. A zoom of the boxed region of interest
is shown. (c) The fluorescence lifetime comparison of cytosolic EGFP (τ = 3.40 ± 0.013 ns, n = 120 regions
from 12 cells) and vMIA-EGFP (τ = 3.04 ± 0.026 ns, n = 130 regions from 13 cells) in Drp1-null MEFs. (d) A
single plane from an MSIM z-stack images showing clustered distribution of vMIA-EGFP on the OMM of a
transfected Drp1-null MEF. Resultsfi A zoom of a single mitochondrion shows vMIA clustering. (e) Normalized pixel
intensities of a line shown along the mitochondrion in inset of panel d. (f) Box plot showing the distribution of
vMIA clusters (n = 40 cells) in WT MEFs and Drp1-null MEFs. ****Indicates p < 0.0001. (Fig. 7c). Further, compared to WT vMIA, which formed clusters on 88.0 ± 3.7% (n = 5 cells) mitochondria,
vMIA CBDII mutant formed clusters in fewer mitochondria (71.7 ± 4.8%, n = 6 cells; p = 0.03) (Fig. 7d). Despite
the reduced homodimerization and reduction in the number of mitochondria with vMIA-CBDII clusters, neither
the vMIA-CBDII cluster sizes (Fig. 7e) nor the cluster density per mitochondria (Fig. 7f) were different from WT
vMIA. Together, these results show that lipid and other hydrophobic interactions facilitate ER-OMM trafficking
and the nanoscale organization of vMIA at the OMM. Discussionh Shown are the fluorescence lifetime comparisons of vMIA-EGFP
(τ = 3.10 ± 0.009 ns), vMIA-CBDII-EGFP (τ = 3.14 ± 0.007 ns) and vMIA-HHB-EGFP (τ = 3.2 ± 0.008 ns). *Represents p < 0.03, **represents p < 0.003, ****represents p = 0.0001. Figure 7. Analyses of vMIA HHB and CBDII mutant clustering by MSIM and FLIM. HeLa cells were Figure 7. Analyses of vMIA HHB and CBDII mutant clustering by MSIM and FLIM. HeLa cells were
transiently transfected with vectors expressing (a) vMIA-EGFP, (b) vMIA-CBDII-EGFP or (c) vMIA-HHB-
EGFP (green) as previously described36. The cells were co-transfected with only a mitochondrial marker (Mito-
BFP, blue) (a,b), or with Mito-BFP and an ER marker (ER-RFP, red) (c). Cells were fixed and a single plane from
an MSIM z-stack image showing distribution of vMIA-EGFP and mitochondrial and ER marker is shown. A
zoom of regions marked by the white boxes in each image is shown below. (d) The percentages of mitochondria
with clustered vMIA in HeLa cells expressing vMIA-EGFP (88.0 ± 3.7%, n = 5 cells) or vMIA-CBDII-EGFP
(71.7 ± 4.8%, n = 6 cells). (d) The size of vMIA (mean 169.18, min: 110, median 167, max: 233, n = 50) and
vMIA-CBDII (mean 164.55, min: 111, median 159, max: 230, n = 60) clusters were determined. p = 0.4024. The
lines and red crosses indicate the medians and means, respectively. (f) The number of vMIA clusters in HeLa
cells expressing vMIA-EGFP (mean 0.638, min: 0.194, median 0.562, max: 1.73, n = 50) or vMIA-CBDII-EGFP
(mean = 0.563, min: 0.153, median 0.526, max: 1.312, n = 60) were determined (p = 0.3118). The line and
red, cross mark indicate the median and mean, respectively. (g) vMIA-HHB and vMIA-CBDII are defective
in dimerization as measured by FLIM. Shown are the fluorescence lifetime comparisons of vMIA-EGFP
(τ = 3.10 ± 0.009 ns), vMIA-CBDII-EGFP (τ = 3.14 ± 0.007 ns) and vMIA-HHB-EGFP (τ = 3.2 ± 0.008 ns). *Represents p < 0.03, **represents p < 0.003, ****represents p = 0.0001. transiently transfected with vectors expressing (a) vMIA-EGFP, (b) vMIA-CBDII-EGFP or (c) vMIA-HHB-
EGFP (green) as previously described36. The cells were co-transfected with only a mitochondrial marker (Mito-
BFP, blue) (a,b), or with Mito-BFP and an ER marker (ER-RFP, red) (c). Cells were fixed and a single plane from
an MSIM z-stack image showing distribution of vMIA-EGFP and mitochondrial and ER marker is shown. Discussionh Through the use of multiple available MEF knockout cell lines, we have performed a comprehensive analysis of
ER-mitochondrial trafficking and OMM organization of signal-anchored viral protein vMIA. Our approach of
using a combination of mouse (MEFs) and human (HeLa cells and HFFs) cells to study vMIA trafficking is based
upon the previous findings from multiple laboratories that vMIA traffics efficiently and similarly in these CMV
permissive and non-permissive cells7–9,11,13,27,32,36,43,50,51. Furthermore, most of vMIA functions (including mito-f chondrial fragmentation, actin rearrangement, host cell protein retargeting to mitochondria, metabolic effects,
inhibition of ATP synthesis, and inhibition of antiviral signaling) are unaffected by the cell being permissive or
not to CMV growth27,43,51. Our results herein show that Mfn1, Mfn2, and PACS-2-mediated ER-mitochondria
tethering is not required for ER-mitochondria trafficking. Instead, we show that these proteins facilitate cluster-
ing of proteins at the OMM. Further, we find that lack of Drp1 does not affect ER to mitochondrial trafficking Scientific Reports | 7: 16 | DOI:10.1038/s41598-017-00039-5 9 www.nature.com/scientificreports/ Figure 7. Analyses of vMIA HHB and CBDII mutant clustering by MSIM and FLIM. HeLa cells were
transiently transfected with vectors expressing (a) vMIA-EGFP, (b) vMIA-CBDII-EGFP or (c) vMIA-HHB-
EGFP (green) as previously described36. The cells were co-transfected with only a mitochondrial marker (Mito-
BFP, blue) (a,b), or with Mito-BFP and an ER marker (ER-RFP, red) (c). Cells were fixed and a single plane from
an MSIM z-stack image showing distribution of vMIA-EGFP and mitochondrial and ER marker is shown. A
zoom of regions marked by the white boxes in each image is shown below. (d) The percentages of mitochondria
with clustered vMIA in HeLa cells expressing vMIA-EGFP (88.0 ± 3.7%, n = 5 cells) or vMIA-CBDII-EGFP
(71.7 ± 4.8%, n = 6 cells). (d) The size of vMIA (mean 169.18, min: 110, median 167, max: 233, n = 50) and
vMIA-CBDII (mean 164.55, min: 111, median 159, max: 230, n = 60) clusters were determined. p = 0.4024. The
lines and red crosses indicate the medians and means, respectively. (f) The number of vMIA clusters in HeLa
cells expressing vMIA-EGFP (mean 0.638, min: 0.194, median 0.562, max: 1.73, n = 50) or vMIA-CBDII-EGFP
(mean = 0.563, min: 0.153, median 0.526, max: 1.312, n = 60) were determined (p = 0.3118). The line and
red, cross mark indicate the median and mean, respectively. (g) vMIA-HHB and vMIA-CBDII are defective
in dimerization as measured by FLIM. Discussionh A
zoom of regions marked by the white boxes in each image is shown below. (d) The percentages of mitochondria
with clustered vMIA in HeLa cells expressing vMIA-EGFP (88.0 ± 3.7%, n = 5 cells) or vMIA-CBDII-EGFP
(71.7 ± 4.8%, n = 6 cells). (d) The size of vMIA (mean 169.18, min: 110, median 167, max: 233, n = 50) and
vMIA-CBDII (mean 164.55, min: 111, median 159, max: 230, n = 60) clusters were determined. p = 0.4024. The
lines and red crosses indicate the medians and means, respectively. (f) The number of vMIA clusters in HeLa
cells expressing vMIA-EGFP (mean 0.638, min: 0.194, median 0.562, max: 1.73, n = 50) or vMIA-CBDII-EGFP
(mean = 0.563, min: 0.153, median 0.526, max: 1.312, n = 60) were determined (p = 0.3118). The line and
red, cross mark indicate the median and mean, respectively. (g) vMIA-HHB and vMIA-CBDII are defective
in dimerization as measured by FLIM. Shown are the fluorescence lifetime comparisons of vMIA-EGFP
(τ = 3.10 ± 0.009 ns), vMIA-CBDII-EGFP (τ = 3.14 ± 0.007 ns) and vMIA-HHB-EGFP (τ = 3.2 ± 0.008 ns). *Represents p < 0.03, **represents p < 0.003, ****represents p = 0.0001. or OMM clustering of vMIA. While Drp1 has been implicated in vesicle mediated trafficking between ER and
mitochondria4,10, it is conceivable that lack of Drp1 resulting in elongated mitochondria may also impact on
ER-mitochondria tethering. However, our observation that Drp1 deletion did not affect vMIA trafficking or clus-
tering at the mitochondria suggests a potential role of Drp1 in mediating ER-mitochondria tethering is also
not relevant for vMIA trafficking or clustering at the OMM. Mfn1/2 and PACS-2 have been shown to maintain
ER-mitochondria apposition18,21. pp
vMIA interacts with Mfn245, while PACS-2 is known to alter subcellular distribution of the cellular MAM
protein calnexin22. Despite these results suggesting a potential involvement of Mfn1/2 and PACS-2 in vMIA traf-
ficking from the ER to mitochondria, we find this is not the case. We thus envision that vMIA may traffic to mito-
chondria by multiple, parallel pathways for trafficking from the ER to mitochondria, such that knock out of any
one pathway does not inhibit its trafficking but affects its optimal organization at the OMM. Alternatively, vMIA
may traffic by a pathway that is independent of ER-mitochondrial tethering by PACS-2 and Mfn2 and of vesicle
formation by Drp1. Discussionh Such a pathway may use lipid affinity or a cytosolic lipid transport protein to translocate
without vesicular or bridging requirements. It was recently found that phosphatidylserine (PS) transport from the
ER to mitochondria does not require proteinaceous linkers between the organelles but a cytosolic protein, VAT-1,
which has affinity for PS52. In favor of such a mode of trafficking, we found that vMIA mutant that has increased
hydropathy fails to traffic to the OMM11. y
p
yfi
vMIA’s ability to translocate to the lipid-synthetic rich MAM subdomains potentially enables it to usurp lipid
trafficking pathways. Association with lipids is known to enable some proteins to target the MAM. For example,
Sig-1R protein utilizes cholesterol affinity to associate with the MAM31. Palmitoylation enriches calnexin and
TMX in the MAM29. Indeed, lipid association appears to enable vMIA trafficking from the MAM to mitochon-
dria. We found that the vMIA HHA and HHB mutants, which have increased hydrophobicity of the N-terminal Scientific Reports | 7: 16 | DOI:10.1038/s41598-017-00039-5 10 www.nature.com/scientificreports/ leader, are retargeted to the secretory apparatus and are less efficient in mitochondrial trafficking11. These results
suggest that vMIA’s targeted association with lipids in the ER/MAM subdomain, in part, underlies its ability to
translocate to the OMM. In addition, other vMIA MTS elements, including modification of its 21SY motif and
downstream proline rich domain (33PLPP), regulate its trafficking to the OMM11. Nonetheless and in contrast
to Sig-1R, vMIA targeting to the MAM and mitochondria is not mediated by cholesterol binding. vMIA CBD
mutants, defective in cholesterol binding, traffic efficiently from the MAM (but not to the MAM lipid rafts) to
mitochondria32. Once in the MAM lipid synthetic enriched sub-domain, we suggest that vMIA could use a cyto-
solic lipid carrier to translocate to the OMM as has been shown for PS trafficking from the outer to inner leaflet of
the mitochondrial membrane, by the cytosolic protein VAT-152. y
y
p
Following trafficking to the mitochondria, vMIA forms nanometric clusters in the OMM13,36. This organ-
ization of vMIA clusters at the OMM is similar to that of multiple mitochondrial proteins33,35,37. However, the
mechanism underlying clustering of these proteins has not been addressed. Our results show that Drp1 does not
affect the ability of vMIA to homodimerize, but PACS-2 and mitofusins are needed for proper distribution and
clustering of vMIA at the OMM. Methods
C ll
l Cell culture and transfection. HFFs (Viromed Laboratories) were grown in Dulbecco’s Modified Eagle’s
medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U/ml of penicillin, 100 µg/ml of strepto-
mycin and 2 mM of L-glutamine at 37 °C and 5% CO2. WT MEFs, Mfn-null MEFs (Mfn1 −/−, Mfn2 −/−)40, and
PACS-2-null (PACS-2 −/−) MEFs39 were cultured in DMEM supplemented with 10% FCS, 100 U/ml of penicil-
lin and 100 µg/ml of streptomycin; whereas, Drp1-null MEFs (Drp1 −/−)41 were cultured in Iscove’s Modified
Dulbecco’s Medium (IMDM) supplemented with 10% FCS, 100 U/ml of penicillin and 100 µg/ml streptomycin. HFFs and MEFs were plated on 18 mm (for confocal microscopy) or 25 mm (for MSIM) cover slips and trans-
fected one day later using Lipofectamine 2000 (Life Technologies) as previously described36. Plasmids encoding
vMIA-EGFP55, vMIA-CFP11, mouse Tom20 (Tom20)-YFP56, PACS-2-EGFP18, mCherry-Drp157, Mfn2-YFP48, Cell culture and transfection. HFFs (Viromed Laboratories) were grown in Dulbecco’s Modified Eagle’s
medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U/ml of penicillin, 100 µg/ml of strepto-
mycin and 2 mM of L-glutamine at 37 °C and 5% CO2. WT MEFs, Mfn-null MEFs (Mfn1 −/−, Mfn2 −/−)40, and
PACS-2-null (PACS-2 −/−) MEFs39 were cultured in DMEM supplemented with 10% FCS, 100 U/ml of penicil-
lin and 100 µg/ml of streptomycin; whereas, Drp1-null MEFs (Drp1 −/−)41 were cultured in Iscove’s Modified
Dulbecco’s Medium (IMDM) supplemented with 10% FCS, 100U/ml of penicillin and 100µg/ml streptomycin. HFFs and MEFs were plated on 18 mm (for confocal microscopy) or 25 mm (for MSIM) cover slips and trans-
fected one day later using Lipofectamine 2000 (Life Technologies) as previously described36. Plasmids encoding
vMIA-EGFP55, vMIA-CFP11, mouse Tom20 (Tom20)-YFP56, PACS-2-EGFP18, mCherry-Drp157, Mfn2-YFP48,
Tom20-mCherry, or vMIA-mCherry were used for transfection. Monomeric EGFP expression vector (Clontech
Laboratories, Madison WI) was used as a control. Tom20-mCherry expression plasmid was generated by sub-cloning of the mouse Tom20 cDNA upstream
of the mCherry open reading frame in mCherry N-1 vector (Clontech Laboratories). The vMIA open reading
frame was analogously sub-cloned into pmCherry N-1. The plasmid sequence was confirmed using a commercial
sequencing company (Macrogen). PACS-2 siRNAs. PACS-2 siRNAs18 were used as described to reduce PACS-2 expression and destabi-
lize ER-mitochondria contacts. A nonspecific random siRNA (5′-CGUUUGCGGUGUUUAUGGCtt-3′;
5′-GCCAUAAACACCGCAAACGtt-3′) was used as a control. BLASTN searches with the nonspecific siRNA
detected no significant similarity in the human genome database (A. Colberg-Poley, unpublished results). Discussionh The density of vMIA clusters at the OMM appears to be negatively regulated by
Mfn1/2 such that in their absence, more vMIA clusters are localized in the OMM. This may result from vMIA’s
ability to interact with Mfn1/245. Herein, we documented that vMIA redistributes Mfn2 at the OMM. This inter-
action with and redistribution of Mfn2 may exclude interactions with cellular proteins present in vMIA clusters
and thereby reduce vMIA clustering in presence of Mfn1/2. In contrast, the absence of PACS-2 reduces the per-
centage of mitochondria with clustered vMIA. As PACS-2 regulates apposition of the ER and OMM, its presence
may enhance the number of MAM contact sites which may, in turn, affect vMIA distribution and clustering in
OMM subdomains close to the MAM tethering sites. g
Emerging evidence suggests that mitochondrial lipids can modulate the organization and distribution of some
mitochondrial nanoscale complexes - low ergosterol content of yeast mitochondrial membranes for targeting of
some OMM proteins with C-terminal anchors53. Consistent with this, we find that the cholesterol binding mutant
of vMIA (CBD II) reduced the number of mitochondria with clustered vMIA. Moreover, in addition to sterol, the
mitochondria specific lipid cardiolipin has been shown to be required for the localization of yeast Mic27/Mic10/
Mic12 sub-complex at cristae junctions in the inner mitochondrial membrane54. These roles of lipids suggest the
possibility that the lipid composition of the MAM and of the OMM facilitate ER to mitochondrial trafficking of
vMIA through association with its leader sequence. Our work highlights the need to study the role of lipids in
ER-mitochondria targeting and OMM organization of proteins. Methods
C ll
l The image analysis was carried out using
Olympus Fluoview version 4.0, MetaMorph Premier and Fiji ImageJ software. For image deconvolution CellSens
Dimension software version 1.12 (Olympus Life Science) was used. Constrained iterative 3-D deconvolution
module with the advanced maximum likelihood estimation algorithm (ADVMLE) was employed. The plots used
to identify clustering on the deconvolved images were made using MetaMorph Premier (7.7.0) software, which
was provided by Molecular Devices, LLC. For pixel colocalization analysis the individual channels of the confocal
images were separated and the average intensity of pixels within the selection was calculated. The resulting value
of 0.9 X average intensity was used to threshold the channel. Integrated morphometry analysis (IMA) module of
the MetaMorph Premier 7.0 software was used to identify objects in the image larger than 0.14 µm2 to generate
the mask for use in colocalization analysis. Gated Stimulated Emission Depletion Microscopy (GSTED). STED imaging was done using the
Leica TCS SP8 equipped with a White Light Laser, two depletion lasers and hybrid detectors. A HC PL APO CS2
100x/1.40 Oil objective was used to acquire 12-bit images with pixel size less than 25 nm. The Tom20-Alexa 647
was excited at 633 nm and depleted with 775 nm laser. The emission was collected between 640 nm to 750 nm with
a time gating of 0.3 ns to 6.0 ns. Sequential stack for vMIA-YFP, using a 514 nm excitation and 660 nm depletion,
was acquired from 520 nm to 610 nm and time-gated between 1.5 ns and 6.0 ns. Z-stacks with a step size of 170 nm
or less was acquired and deconvolved using the Huygens Professional software (Scientific Volume Imaging). MSIM Sample Preparation and Imaging. At 16 to 24 hours post-transfection, cells were fixed using 4%
paraformaldehyde (PFA) (Electron Microscopy Sciences) in PBS, pre-warmed to 37 °C, for 15 minutes at room
temperature. After fixation cells were washed with PBS and coverslips were mounted with ProLong Diamond
Antifade Reagent (Life Technologies) onto slides and left overnight to dry at room temperature. For MSIM imag-
ing, tetraspecs (Thermo Fisher) were added onto 25 mm coverslips that contained cells with multiple fluorophores
prior to mounting onto another 25 mm coverslip with ProLong Diamond Antifade Reagent and left to dry over-
night at room temperature. g
p
The MSIM microscope used for these experiments is made from an Olympus IX-71 widefield microscope
as previously described36. Methods
C ll
l Briefly, critical modifications made to provide an increase its resolution capabilities
include placing a microlens array in the illumination pathway to provide a two-dimensional pattern of excita-
tion spots within the focus of the objective lens. This pattern was translated in approximately 1 pixel size steps
over the field of view while collecting an image at each position. Images were taken in z-stacks with 200 images
taken per plane for each fluorophore used. MSIM processing was performed using code written in Python and
provided by the Hari Shroff lab at the NIH59. After processing, the resulting images were a widefield image, and
deconvolution of the widefield, and the MSIM image. MSIM images taken were 512 × 512 pixels; however, during
processing, images were scaled up to 1024 × 1024 pixels. Any further processing of the MSIM images was done in
ImageJ (NIH). Light sources used to excite fluorophores within cells were a 50 mW 488 laser to illuminate green
fluorophores and a 50 mW 561 Sapphire laser to illuminate red fluorophores. All settings for the microscope were
controlled using Micro-Manager. A 100 × 1.4 NA Nikon oil immersion objective was used for imaging in these
experiments. FLIM. The frequency domain FLIM analysis was performed with live cells roughly 20 hours after transfec-
tion. An Olympus BX81 inverted microscope was used to acquire widefield FLIM images. All the images were
acquired using a UApoN 100 X/1.49 oil objective. A 488 nm laser (Intelligent Imaging Innovations) modulated at
50 MHz was used to excite the fluorophores in the samples. The fluorescence lifetime signal was measured using
the II18MD modulated image intensifier (Lambert Instruments). The modulation was set to 0.23 and the inten-
sification was set to 2,500. A CoolSNAP EZ camera (Photometrics) was used to acquire phase-shifted images. Rhodamine 101 (Sigma-Aldrich) dissolved in water, with a lifetime of 4.32 ns, was used to calibrate the system. It was also used to check the stability of the system over the course of the experiment. Images from 10–15 fields
of view were collected for each sample type. At least 10 regions of interest (ROI) were marked randomly on each
cell. The fluorescence lifetime from these ROIs were analyzed. The image acquisition and analysis was done using
Slidebook version 6 software from Intelligent Imaging Innovations. The lifetime of individual pixels was calcu-
lated using the polar plot representation for the frequency-domain fluorescence lifetime data60. Methods
C ll
l Briefly,
HeLa cells expressing EGFP tagged human phosphatidylserine synthase 1, named PSS-120 cells8, were transfected
with 7 μg of vMIA expression vector and 100 nM of PACS-2 siRNAs (Dharmacon) or 100 nM of nonspecific siR-
NAs (Ambion). Fractionation of mitochondria and microsomes. Mitochondria and microsomes were isolated from
transfected PSS-120 cells as described8,58. Briefly, cells were washed twice in PBS and pelleted by centrifuga-
tion, resuspended and lysed using a motor-driven Potter-Elvehjem homogenizer. The supernatant from pel-
leted homogenates was then centrifuged at 10,300 × g to separate microsomal (microsomes and cytosol) from
crude mitochondrial (MAM and mitochondria) fraction. Microsomes were pelleted by ultracentrifugation
at 100,000 × g from the crude microsomal fraction (supernatant). Mitochondria fractions were separated in
self-generating Percoll gradients. Isolated fractions were diluted fivefold in sucrose homogenization medium and
subjected separately to a centrifugation at 6,300 × g for 10 min at 4 °C. The pellet of the mitochondria centrifu-
gation was used as the purified mitochondria in our analysis. All of the fractions were resuspended in sucrose
homogenization medium and analyzed by Western blot analysis. Confocal microscopy and analysis. Confocal images were acquired on Olympus FV 1000 and Leica TCS
P8 microscopes. On the Leica TCS SP8, a HC PL APO CS2 100x/1.40 Oil objective was used to acquire images Scientific Reports | 7: 16 | DOI:10.1038/s41598-017-00039-5 11 www.nature.com/scientificreports/ 514 nm and 633 nm laser lines from a White Light Laser was used to excite YFP and Alexa 647 fluorophores. The emission was collected on hybrid detectors with the AOBS set to 520–610 nm and 640–750 nm respectively. These images were deconvolved using the Huygens software (Scientific Volume Imaging). On the Olympus FV
1000, UPlanSApo 100 X/1.40 NA oil objective was used to acquire 1024 × 1024 pixels image, where each pixel
was 62 nm in size. A step size of 410 nm was used for the z-stacks. For images that were to be deconvolved, step
size of 110 nm was used for acquiring the z-stacks. 440 nm, 488 nm, 515 nm and 559 nm lasers were used to excite
CFP, GFP, YFP and mCherry fluorophores, respectively. For triple color (CFP-YFP-mCherry) imaging 405 nm
laser was used to excite CFP and emission collected with (425–475 nm filter), which prevented any excitation or
emission bleedthrough and optimal excitation of YFP and mCherry. www.nature.com/scientificreports/ www.nature.com/scientificreports/ References Trafficking of UL37 proteins into mitochondrion-associated
membranes during permissive human cytomegalovirus infection. J Virol 84, 7898–7903 (2010). doi:JVI.00885-10. g p
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Mediated ER Mitochondria Interaction PLoS One 7 e33657 doi:10 1371/journal pone 0033657 (2012) 10. Huang, C. Y., Chiang, S. F., Lin, T. Y., Chiou, S. H. & Chow, K. C. HIV-1 Vpr Triggers Mitochondrial Destruction by Impairing Mfn2-
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This work was funded by the NSF grant (MCB 1244509) to A.C.-P. and J.K.J., and by the Intramural Research
Program of the National Institutes of Health, including the National Institute of Biomedical Imaging and
Bioengineering, to G.H.P. The CRI microscopy imaging core was supported by the NIH grant P30HD40677. We
thank Dr. Gary Thomas for the gift of the PACS-2 null MEFs, parental MEFs and PACS-2-YFP plasmid and anti-
PACS-2 antiserum, Dr. David Chan for the Mfn1/2 null MEFs and parental MEFs and Dr. Richard Youle for the
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wrote the paper. K.S. performed most of the experiments and was helped by G.H.P. and K.R. for superresolution
imaging and data analysis. S.B. performed the FLIM, confocal, and gSTED experiments. K.W. carried out
experiments in Fig. 3. P.B. performed the PACS-2 siRNA and fractionation experiments. H.S. generated Drp1
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26185–26192, doi:10.1074/jbc.M503062200 (2005). Scientific Reports | 7: 16 | DOI:10.1038/s41598-017-00039-5 13 www.nature.com/scientificreports/ Additional Information Supplementary information accompanies this paper at doi:10.1038/s41598-017-00039-5 Competing financial interests: The authors declare no competing financial interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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English
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Brain structural abnormalities in migraine patients: an observational study
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© 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
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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: Migraine is a common neurological disorder characterized by a complex physiopathology. We
assessed brain morphologic differences in migraine and the possible pathogenetic mechanism underlying this
disease. Methods: We analyzed brain morphologic images of migraine patients, 14 with aura (MwA) [the mean (SD) age
was 42.36 (2.95) years (range, 37–47)] and 14 without aura (MwoA) [the mean (SD) age was 43.5 (3.25) years (range,
39–50)] during episodic attack compared with health subjects balanced (HS) [the mean (SD) age was 42.5 (5.17)
years (range, 34–51)]. All subjects underwent a Magnetic Resonance Imaging (MRI) examination with a scanner
operating at 3.0 T and voxel based morphometry (VBM) approach was used to examine the gray matter volume
(GMV). The statistical analysis to compare clinicl characteristics was performed using unpaired t-test an one-way
Anova. Results: Total cerebral GMV showed a significant difference between MwA and HS (p = 0.02), and between
MwoA and HS (p = 0.003). In addition, not significative differences were found between MwA and MwoA groups
(p = 0.17). We found three clusters of regions which showed significant GMV reduction in MwA compared with
MwoA. MwA subjects showed a less of GMV in 4 clusters if compared with HS, and MwoA subjects showed a less
of GMV in 3 clusters if compared with HS. We observed that MwA and MwoA patients had a significant reduction
of GMV in the frontal and temporal lobe and the cerebellum, if compared to HS. The bilateral fusiform gyrus and
the cingulate gyrus were increase in MwoA patients compared with HS. Conclusion: Our findings could provide a approach to understand possible differences in the pathogenesis of two
type of migraine. Keywords: Aura, Magnetic resonance imaging, Migraine, No Aura, Voxel-based Morphometry interest (ROI) [2]. VBM is largely used because it is rela-
tively easy to use [3]. The Journal of Headache
and Pain The Journal of Headache
and Pain (2020) 21:39 Bonanno et al. The Journal of Headache and Pain (2020) 21
https://doi.org/10.1186/s10194-020-01109-2 Open Access Brain morphologic abnormalities in
migraine patients: an observational study Lilla Bonanno, Viviana Lo Buono*, Simona De Salvo, Claudio Ruvolo, Viviana Torre, Placido Bramanti,
Silvia Marino and Francesco Corallo a Bonanno, Viviana Lo Buono*, Simona De Salvo, Claudio Ruvolo, Viviana Torre, Placido Braman
via Marino and Francesco Corallo Introduction Magnetic resonance imaging (MRI) is an instrumental
method which provides a non-invasive observation of
brain changes. Voxel-based morphometry (VBM) is an
automated technique using to analize the MRI images
[1] and to identify changes in brain anatomy. It is char-
acterized by high regional specificity and it does no re-
quire preventively the definition of a particular region of A recent VBM study, in which regional volumes based
voxel-wise on were compared between patients with mi-
graine and controls, detected structural differences in
brain tissue composition of migraine patients [4]. Mi-
graine is a common neurological disorder, characterized
by recurrent unilateral pain attacks associated with nau-
sea and other neurovegetative symptoms of moderate or
severe intensity, whose physiopathology is complex [5]. Up to one third of migraineurs with aura have visual
symptoms followed by motor somatosensory symptoms * Correspondence: viv.lobuono@gmail.com
IRCCS Centro Neurolesi “Bonino-Pulejo”, S.S. 113, Via Palermo, C. da Casazza,
98124 Messina, Italy * Correspondence: viv.lobuono@gmail.com
IRCCS Centro Neurolesi “Bonino-Pulejo”, S.S. 113, Via Palermo, C. da Casazza,
98124 Messina, Italy Voxel-based Morphometry method We used VBM approach to examine the gray matter. The VBM is based on 3 basic preprocessing steps follow-
ing by statistical analysis. These steps are: tissue classifi-
cation, spatial normalization and spatial smoothing [2]. The local tissue morphology was maintained perform-
ing a modulation/correction for volume changes on seg-
mented brain images. The latter were also levelled off
with an isotropic 12 mm FWHM Gaussian kernel. After-
wards, the last step consisted in the esteem of global
GM and WM volumes and total intracranial volume
(TIV) by using segmented images in native space. Bonanno et al. The Journal of Headache and Pain (2020) 21:39 Bonanno et al. The Journal of Headache and Pain Page 2 of 6 number of slices = 173, field of view 240 mm] was
acquired. during attacks [6]. Literature data reported an associ-
ation between migraine and morphologic brain alter-
ations [7]. The aim of this study is to applie VBM
approach in migraineurs patients with aura (MwA) and
without aura (MwoA) in order to analyze their brain
morphologic differences and to evaluate a possible
pathogenetic mechanism underlying these two types of
migraine. Methods
Subjects Twenty-eight
migraine
patients
(14
MwA
and
14
MwoA), according to International Headache Society
criteria (Headache Classification Committee of the Inter-
national Headache, 2013), and 14 sex and age matched
healthy subjects (HS) were recruited. MwA group com-
posed of 14 female patients with aura attacks of visual
area. MwoA group composed of 14 female patients with-
out aura attacks. Demographic and clinical characteris-
tics showed in Table 1. The protocol was approved by
the Local Ethics Committee of IRCCS Centro Neurolesi
Bonino-Pulejo of Messina (Italy) and conducted accord-
ing to the Declaration of Helsinki. Informed consent was
obtained from all participants included in the study. The
exclusion criteria were: 1) other types of headache; 2)
vascular disease or trauma; 3) history of major psychi-
atric disorders; 4) presence of metabolic disorders; 5)
aura greater than 70 min. Results All participants completed the study. The total cerebral
GMV showed a significant difference between MwA and
HS (p = 0.02), and between MwoA and HS (p = 0.003). In addition, not significative differences were found be-
tween MwA and MwoA groups (p = 0.17) (Table 1). We
corrected the analysis for multiple comparisons (p < 0.05
family-wise error corrected), and we detected the regions
with significant GM changes between MwA and MwoA
groups. At p < 0.05, we found 3 clusters of regions that
showed significant GMV reductions in MwA if com-
pared with MwoA and more GMV in the right frontal
lobe (Fig. 1 and Table 2). MwA group showed less GMV
in 4 clusters (right cerebellum, left postcentral and pre-
central gyrus, right inferior frontal gyrus, left Broadman
area 20–22 and left lingual gyrus), and more GMV in
right superior parietal gyrus and left thalamus (Fig. 1
and Table 2) if compared to HS. Finally, MwoA subjects
showed less GMV in 3 clusters (bilateral cerebellum, left
cerebellum crus I, left superior/medial and right infer-
ior/middle frontal gyrus, right superior frontal gyrus, left
fusiform gyrus, left Broadman area 20, right parahippo-
campal gyrus and insula) and more GMV in right thal-
amus (Fig. 1 and Table 2) if compared to HS. In this study, we applied the VBM approach to MwA
and MwoA patients and HS. We observed that MwA
and MwoA subjects had a significant reduction of GMV
compared to HS in cerebellum, and frontal and temporal
lobe. Our previous study [6] analyzed the resting state
findings in the same patient sample and we found an
hyperactivity increase of cortical activity in bilateral fusi-
form and cingulate gyrus of MwoA subjects compared
with controls. In this study, the VBM approach showed
a reduction in the volumes of same cerebral areas. Al-
though the volume of bilateral fusiform and cingulate
gyrus is descreased, the hyperactivity of cortical activity
could be ascribed to the fusiform gyrus that seems to be
hyperactive in migraineurs for it involvment in the cog-
nitive pain treatment, while the cingulate gyrus is in-
volved in the transformation process of migraine from
“an episodic” to “a chronic brain disorder” [14]. Fusiform
gyrus is involved in nociception/antinociception and
neurocognitive aspects of pain processing. Data processing and analysis patients
14
14
14
Age
43.5±3.25
42.36±2.95
42.5±5.17
0.70±
Education
12.96±3.49
13.45±3.28
13.46±3.75
0.91±
Disease Duration (year)
6.78±3.66
5.21±1.31
–
0.14¥
Attacks per year
22.75±10.03
29.83±11.9
–
0.10¥
Frequency of attacks (mounth)
1.89±1.18
2.48±1.40
–
0.24¥
Duration of headache attacks (hours)
2.5±1.2
3.3±1.9
–
0.19¥
VBM analysis
Grey Matter
793.83±47.10
814.73±29.10
850.98±46.11
0.003*±
*p < 0.05
¥unpaired t-test
±one-way Anova
SD Standard Deviation, HAM-A Hamilton Rating Scale for Anxiety, BDI-II Beck Depression Inventory Table 1 Socio-demographic and clinical characteristics of three groups (% frequency) one way Anova
SD Standard Deviation, HAM-A Hamilton Rating Scale for Anxiety, BDI-II Beck Depression Inventory Page 3 of 6 Page 3 of 6 Page 3 of 6 Bonanno et al. The Journal of Headache and Pain (2020) 21:39 Bonanno et al. The Journal of Headache and Pain to fit the GM tissue segments modulated and normal-
ized for each subject. to fit the GM tissue segments modulated and normal-
ized for each subject. connectivity in pain network involved in migraine patho-
physiology [6, 9, 10]. Abnormalities have been reported
in multiple brain areas as evidenced by VBM. It is an on-
going matter of debate whether the changes are cause or
consequence of migraine, but in many VBM studies the
changes correlated with disease duration argues in favor
of the latter. The exact underlying mechanisms, leading
to alterations in grey matter density as evidenced by
VBM, are not clear. These alterations may reflect modi-
fications of dendritic complexity or changes in the num-
bers of synapses or simply in water content. These
changes may be an index of the disorder, its progression
or an effective therapy. Migraine patients showed signifi-
cant GM abnormalities of several brain regions involved
in central pain processing [11, 12]. In particular, VBM
data established that the GMV was decreased in the an-
terior cingulate cortex, insula, amygdala, parietal opercu-
lum, middle and inferior frontal gyrus [11]. In addition,
regions with less grey matter density are located in bilat-
eral insula, motor premotor, prefrontal, cingulate cortex,
right posterior parietal cortex and orbitofrontal cortex
[13]. Results In idiopathic
or primary headaches, including migraine, tension-type
headaches, and cluster headaches, the accepted view is
that these conditions are due to abnormal brain function
that occurs with normal brain structure. A decrease in
GMV suggest that the central reorganization processes
in chronic pain syndromes may involve degeneration of
anti-nociceptive brain areas. Statistical analysis y
The VBM analysis was carried out using the CAT12
toolbox in MatLab (www.mathworks.com). The first step
consisted to perform a 2-sample t-test, with age, attacks
per year, frequency of attack, duration of headache at-
tacks and TIV as covariates, to compare the GMV be-
tween patients and HCs. Statistical parametric maps
were generated after family-wise error (FWE) correction
for multiple voxel-wise comparison. These maps were
created using an initial threshold p < 0.001 and estimated
at peak statistical significance level for p < 0.05. The ex-
tent threshold was set at 100 voxels. Data processing and analysis Data processing and analysis
Image data processing was performed with SPM12
(www.fil.ion.ucl.ac.uk). We considered GM tissue to cal-
culate the GM tissue volume (GMV) and TIV in the na-
tive space. Subsequently, we used the affine registration
algorithm to record all the native-space tissue segments
to the standard Montreal Neurological Institute template
(included in SPM12). The use of the exponentiated lie
algebra toolbox (DARTEL) to all participants’ GM and
WM was necessary to refine the inter-subject registra-
tion via the application of the diffeomorphic anatomical
registration. In the last step of DARTEL, we used a non-
linear approach to modulate the GM tissues, in order to
compare the relative GMV tailored for individual brain
size. In addition, we performed the spatial normalization
[8] to estimate the Jacobian determinant that was used
to modulate the voxel values in the tissue maps. More-
over, an assessment of the GM tissue homogeneity was
needed. For this reason, we performed a quality check
with a CAT12 toolbox after preprocessing pipeline. Fi-
nally, a Gaussian filter with 8 mm of FWHM was used The patients were in treatment with analgesics (18/
28), triptans (4/28) and combination of analgesics (2/28). We did not find cognitive impairment in our patients. All patient underwent a MRI examination with a scan-
ner operating at 3.0 T (Achieva, Philips Healthcare, Best,
The Netherlands), by using a 32-channel SENSE head
coil. MRI scans were performed in the interictal phase at
least 3 days after migraine attack. For each subject, T1
[TR = 8 ms,
TE = 4 ms,
slice
thickness/gap = 1/0 mm, Table 1 Socio-demographic and clinical characteristics of three groups (% frequency)
Migraine without aura (Mean±SD)
Migraine with aura (Mean±SD)
Health Control (Mean±SD)
p
N. Discussion Neuroimaging developments have provided highly sensi-
tive and non invasive approaches to investigate the
neural mechanisms of brain alterations associated with
several disorders. Although advances in migraine re-
search contributed to improve the disease understand-
ing, the use of advanced MRI techniques allowed the
accurate investigation of migraine patients. Migraine is
not only relate to pain occurring intermittently or con-
stantly, but a process that over time affects the brain act-
ing on a predisposed brain (genetic) and modifining it
the
function
or
morphology. Several
fMRI
studies
revealed
abnormalities
of
resting
state
functional In addition, the cerebellum of migraineurs and con-
trols differs structurally. In a study of Mehnert [15] the
GMV and the neuronal activity, in response to trigemi-
nal pain, increased in posterior part of the cerebellum
(crus). Migraine patients had also a connectivity decrease
in the thalamus and higher cortical areas, suggesting a Page 4 of 6 (2020) 21:39 Bonanno et al. The Journal of Headache and Pain (2020) 21:39 Fig. 1 Gray matter volume (GMV) changes. A) GMV of migraine with aura compared with migraine without aura. B) GMV of migraine with aura
compared with health control. C) GMV of migraine without aura compared with health control. Statistical parametric maps show gray matter
volume alterations with a threshold of P < 0.001 uncorrected superimposed on a standard T1 image. The color bar reflects t values (red/yellow =
increased volume, blue = decreased volume). MwA = Migraine with Aura; MwoA = Migraine without Aura; HS = Heath Subjects Fig. 1 Gray matter volume (GMV) changes. A) GMV of migraine with aura compared with migraine without aura. B) GMV of migraine with aura
compared with health control. C) GMV of migraine without aura compared with health control. Statistical parametric maps show gray matter
volume alterations with a threshold of P < 0.001 uncorrected superimposed on a standard T1 image. The color bar reflects t values (red/yellow =
increased volume, blue = decreased volume). MwA = Migraine with Aura; MwoA = Migraine without Aura; HS = Heath Subjects that found a less volume of insula bilaterally, motor/pre-
motor, prefrontal and cingulate cortex, right posterior
parietal cortex, and orbitofrontal cortex. Moreover, Jin
et al. [9] showed a less GMV in several brain regions in-
volved in pain processing, such as left medial prefrontal
cortex, cingulate, right occipital lobe, cerebellum, and
brainstem. Discussion The Journal of Headache and Pain (2020) 21:39 Page 5 of 6 Table 2 Significant regions with less or more grey matter volume in migraine patients compared with controls
Location
Coordinates (mm)
Peak Z
Corrected p
x
y
z
MwA vs MwoA
L Inferior Temporal
−52
−34
−27
−3.76
0.04
R Frontal lobe
33
18
40
3.83
0.04
R Cerebellum
16
−82
−20
−3.51
0.04
MwA vs HS
R Cerebellum
2
−64
−57
−5.14
0.01
L Postcentral gyrus
−26
30
74
−4.87
0.03
R inferior frontal gyrus
39
34
9
−3.51
0.01
L Brodmann area 22
−58
8
−2
−4.30
0.04
L lingual gyrus
−15
−46
−4
−3.52
0.03
R superior parietal gyrus
15
−45
63
4.12
0.02
L Thalamus
−14
−15
10
3.67
0.01
MwoA vs HS
2003Cerebellum
9
−60
−54
−7.03
0.03
L Cerebellum crus 1
−45
−64
−24
−4.27
0.02
L superior/medial frontal gyrus
−10
38
39
−4.54
0.004
R Inferior/middle frontal gyrus
40
30
−6
−4.74
0.01
R superior frontal gyrus
32
57
26
−4.29
0.001
L fusiform gyrus
−22
−62
−15
−4.31
0.001
L Brodmann area 20
−34
−20
−33
−5.25
0.02
R parahippocampal gyrus
24
−52
−3
−5.14
0.04
R Insula
40
30
−6
−4.74
0.03
R Thalamus
15
−18
3
4.33
0.04
Legend: MwA Migraine with Aura, MwoA Migraine without Aura, HS Heath Subjects, R Right, L Left, BA Brodmann’s area ns with less or more grey matter volume in migraine patients compared with controls Legend: MwA Migraine with Aura, MwoA Migraine without Aura, HS Heath Subjects, R Right, L Left, BA Brodmann’s area migraine is a heterogeneous disorder, whereby it is diffi-
cult to obtain a phenotypically homogeneous group. microvascular volume) as well as more complex pro-
cesses as neurodegeneration. Therefore, there are several
possible explanations for the observed abnormalities in
our patients. Variations in gray matter may result from
repeated ischaemia caused by blood flow both during
migraine attacks and in the interictal phase. In contrast,
the reduction of gray matter may be a consequence of
migraine specific neurotoxic mechanisms. It has been
hypothesized that migraine is associated with a state of
neuronal hyperexcitability, involving over-activity of the
aminoacid exciters glutamate and aspartate. VBM ana-
lysis shown that migraineurs present a significant reduc-
tion in the gray matter of different brain areas involving
to the pain activation network [6]. Discussion Although we investigated a small sample of patients,
our results could provide a new instrumental approach
useful to understand the pathogenesis of MwA and
MwoA. Conclusion LB: a substantial contribution to the concept and design of the work;
analysis and interpretation of data; drafted the article. VLB: acquisition and
interpretation of data and drafted the article. FC: acquisition and
interpretation and revised it critically for important intellectual content. SDS:
manuscript revision. CR: acquisition of data. VT: acquisition of data. PB:
revised it critically for important intellectual content. SM: revised it critically
for important intellectual content and approved the version to the
published. The VBM approach is an important and useful tool to
assess brain morphologic changes in neurological disor-
ders, such as migraine. The different results reported by
aforementioned studies could be attributed, in part, to
the use of different MRI scanners (3 T vs. 1.5 T) [23, 24]. In fact, different scanners may led to the different ap-
proaches for GM segmentation and to detect morpho-
logic
abnormalities
of
different
types. In
addition, Funding
Not applicable. Abbreviations Abbreviations
MwA: Migraine with aura; MwoA: Migraine without aura; HS: Health subjects;
MRI: Magnetic Resonance Imaging; GMV: Gray matter volume;
NBV: Normalized brain volume; ROI: Region of interest; TIV: Total intracranial
volume; FWE: Family-wise error Discussion The results obtained affirm that migraine pa-
tients have a less GMV in the precentral gyrus as well as
in the post-central gyrus and temporal lobe. less inhibitory involvement of migraine cerebellum on
trigeminal nociception. The frontal cortex is the area as-
sociated with cerebral abnormalities in migraine patients
[16, 17]. Previous studies suggested that the medial pre-
frontal cortex could be involved in mediating the attenu-
ation
of
pain
perception
by
a
cognitive
control
mechanisms [18, 19], associated with pain modulation
[20, 21]. Schmitz et al. [22] reported that migraine pa-
tients had a less gray matter density in the medial pre-
frontal cortex correlated significantly with a slower
response time to the set-shifting task. In literature, the possible mechanisms underlying the
reduction of grey matter in migraine are currently un-
known. The observed decrease in grey matter may re-
flect tissue shrinkage (changes in extracellular space and In coherence with previous VBM findings, [11, 12, 17]
our results corroborate with the study of Kim et al. [4], Bonanno et al. References 1. Whitwell JL, Jack CR Jr, Boeve B (2009) F., et al. voxel-based morphometry
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approved by the Local Ethics Committee according to Declaration of
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ICNIRP threshold limit (for workers) exceeded? Int J Environ Res Public
Health 15:1298 Competing interests
The authors declare no competing interests. The authors declare no competing interests. Received: 12 February 2020 Accepted: 13 April 2020 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
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https://openalex.org/W2580494565
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https://www.frontiersin.org/articles/10.3389/fcimb.2016.00204/pdf
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English
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Comparative Genomics of the Zoonotic Pathogen Ehrlichia chaffeensis Reveals Candidate Type IV Effectors and Putative Host Cell Targets
|
Frontiers in cellular and infection microbiology
| 2,017
|
cc-by
| 11,207
|
ORIGINAL RESEARCH
published: 25 January 2017
doi: 10.3389/fcimb.2016.00204 Comparative Genomics of the
Zoonotic Pathogen Ehrlichia
chaffeensis Reveals Candidate Type
IV Effectors and Putative Host Cell
Targets Christophe Noroy 1, 2, 3 and Damien F. Meyer 1, 2* 1 CIRAD, UMR ASTRE, Guadeloupe, France, 2 INRA, UMR 1309 ASTRE, Montpellier, France, 3 Université des Antilles,
Guadeloupe, France 1 CIRAD, UMR ASTRE, Guadeloupe, France, 2 INRA, UMR 1309 ASTRE, Montpellier, France, 3 Université des Antilles,
Guadeloupe, France During infection, some intracellular pathogenic bacteria use a dedicated multiprotein
complex known as the type IV secretion system to deliver type IV effector (T4E) proteins
inside the host cell. These T4Es allow the bacteria to evade host defenses and to subvert
host cell processes to their own advantage. Ehrlichia chaffeensis is a tick-transmitted
obligate intracellular pathogenic bacterium, which causes human monocytic ehrlichiosis. Using comparative whole genome analysis, we identified the relationship between
eight available E. chaffeensis genomes isolated from humans and show that these
genomes are highly conserved. We identified the candidate core type IV effectome of
E. chaffeensis and some conserved intracellular adaptive strategies. We assigned the
West Paces strain to genetic group II and predicted the repertoires of T4Es encoded by
E. chaffeensis genomes, as well as some putative host cell targets. We demonstrated
that predicted T4Es are preferentially distributed in gene sparse regions of the genome. In addition to the identification of the two known type IV effectors of Anaplasmataceae,
we identified two novel candidates T4Es, ECHLIB_RS02720 and ECHLIB_RS04640,
which are not present in all E. chaffeensis strains and could explain some variations in
inter-strain virulence. We also identified another novel candidate T4E, ECHLIB_RS02720,
a hypothetical protein exhibiting EPIYA, and NLS domains as well as a classical type
IV secretion signal, suggesting an important role inside the host cell. Overall, our
results agree with current knowledge of Ehrlichia molecular pathogenesis, and reveal
novel candidate T4Es that require experimental validation. This work demonstrates that
comparative effectomics enables identification of important host pathways targeted by
the bacterial pathogen. Our study, which focuses on the type IV effector repertoires
among several strains of E. chaffeensis species, is an original approach and provides
rational putative targets for the design of alternative therapeutics against intracellular
pathogens. The collection of putative effectors of E. chaffeensis described in our paper
could serve as a roadmap for future studies of the function and evolution of effectors. Keywords: type IV effectors, Ehrlichia chaffeensis, comparative genomics, host-pathogen interactions, genome
plasticity Edited by:
Rey Carabeo,
Washington State University, USA Reviewed by:
Jere W. McBride,
University of Texas Medical Branch,
USA
Matteo Bonazzi,
Centre National de la Recherche
Scientifique, France *Correspondence:
Damien F. Meyer
damien.meyer@cirad.fr Received: 16 June 2016
Accepted: 21 December 2016
Published: 25 January 2017 INTRODUCTION isolates (Paddock and Childs, 2003). The DNA of TRP32 genes
amplified from cultured isolates of E. chaffeensis, or from ticks, or
from samples of patients’ blood infected with this pathogen, has
shown two to six repeats (summarized in Table 1). TRP120gene
plays an important role in E. chaffeensis infection as it is a
type I secretion system effector which is sumoylated on lysine
residues and mediates interactions with host protein targets
such as actin and myosin cytoskeleton components (Myo10)
or GGA1 involved in vesicular trafficking (Wakeel et al., 2009)
(Table 1). Ehrlichia chaffeensis is an intracellular rickettsial pathogen
transmitted by Amblyomma americanum ticks, which is the
etiologic agent of human monocytic ehrlichiosis (HME) (Dumler
et al., 1993). This pathogen also causes disease in several other
vertebrates, including dogs and deer (Paddock and Childs, 2003). The white-tailed deer is the reservoir host for E. chaffeensis, while
humans, dogs and other vertebrate hosts, such as coyotes and
goats, are regarded as incidental hosts (Paddock and Childs,
2003). This bacterium is able to replicate within two hosts, a
mammalian host and a tick vector, and is capable of orchestrating
highly sophisticated strategies to persist and infect their natural
hosts (Rikihisa, 2010). Thus, studying E. chaffeensis provide
a wealth of information about bacterial adaptation to various
environments. (
)
Like other mammalian pathogenic bacteria, E. chaffeensis uses
specific molecular mechanisms to evade host immune responses
and to modulate host cell processes to its own advantage. Among
these pathogenicity determinants, the type IV secretion system
(T4SS) is a specialized protein complex involved in the injection
of type IV effector (T4E) proteins into eukaryotic cells in order
to subvert host cell processes during infection (Cascales and
Christie, 2003). Rapid progress has been made toward identifying
the proteins that form different parts of the T4SS, the translocated
effectors and how these effectors subvert eukaryotic cellular
processes during infection (Voth et al., 2012). However, to date,
only two T4Es have been identified in the Anaplasmataceae
family and shown to be critical for pathogenicity. After being
injected in the host cells, AnkA (Anaplasma phagocytophilum),
is tyrosine-phosphorylated in the cytoplasm at EPIYA motifs
and binds to SHP-1 phosphatase (Lin et al., 2007; Garcia-
Garcia et al., 2009). INTRODUCTION AnkA is then translocated to the nucleus
of the infected cell and interacts with gene promoter regions,
leading to the downregulation of the CYBB and other key host
defense genes (IJdo et al., 2007). In E. chaffeensis, the only
known T4E is ECH_0825, homologous to A. phagocytophilum
Ats-1 (Liu et al., 2012). This effector is translocated to host
mitochondria where it restrains ROS and apoptosis for more
efficient infection. E. chaffeensis has a biphasic developmental cycle involving
two morphologically distinct forms (Zhang et al., 2007). The
infectious extracellular forms (dense core cells) first attach to the
surface of host target cells before entering by endocytosis. Inside
the host cells, the bacteria differentiate into reticulate cells within
a membrane-bound vacuole where they create a safe niche for
survival and replication by binary fission to form large colonies,
called morulae. After a few days, the bacteria redifferentiate into
infectious forms to be released outside the cell and start a new
cycle of infection (Zhang et al., 2007). In E. chaffeensis, the genome sequences of eight human
isolates with variable pathogenicity, are available (Table 1). The
first strain was discovered in 1991 in a 21-year old man and
was named Arkansas for its geographic origin (Dawson et al.,
1991). The most recently identified strain, called West Paces,
was found in Tennessee in 2000 (Cheng et al., 2003). The
other strains, Heartland, Jax, Liberty, Osceola, Saint Vincent, and
Wakulla have also been isolated in humans (Table 1) and show
different pathogenesis. In severe combined immunodeficiency
(SCID) mice, Miura et al. observed differences in virulence in
three of the strains, the Arkansas strain causing mild, the Liberty
strain causing acute severe pathogenesis, and the Wakulla strain
causing acute lethal pathogenesis (Miura and Rikihisa, 2007). The
eight strains of E. chaffeensis used in this study were separated
into three genetic groups based on the sequence polymorphisms
of the p28 outer membrane protein genes (Yu et al., 1999). The
Arkansas and Osceola strains were classified in group I, the
Heartland, Saint Vincent, and Wakulla strains in group II, and
the Jax and Liberty strains in group III. The West Paces strain
had not yet been isolated when the genetic groups were defined. Other genetic classifications were based on genes encoding
immunoreactive proteins. Citation: January 2017 | Volume 6 | Article 204 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org Comparative genomics of Ehrlichia chaffeensis Noroy and Meyer Frontiers in Cellular and Infection Microbiology | www.frontiersin.org Analysis of Type IV Effectome Distribution
According to Local Gene Density To visualize in a single representation the distance between each
gene and its closest neighbors on the five prime and three prime
borders, we sorted genes into two-dimensional bins defined by
the length of their 5′ and 3′ flanking intergenic regions (hereafter
denoted 5′ and 3′ FIRs) (Raffaele et al., 2010). The gene density
distribution is represented in R by a heat map. We used the
median length of FIRs to distinguish between gene-dense regions
(GDRs); in-between regions (IBRs); and gene-sparse regions
(GSRs). Putative type IV effectors identified by S4TE software
were plotted on this graph according to their 5′ and 3′ FIRs
(Figure 2A). The distribution of putative T4Es in each region was
calculated for each strain (Figure 2B). Phylogenetic Reconstruction and Genomic
Plasticity Analysis Complete genome sequences of E. chaffeensis strains were
obtained
from
the
National
Center
for
Biotechnology
Information (NCBI) database (ftp://ftp.ncbi.nih.gov/genomes/
Bacteria/). Eight complete genomes were used in this study. Orthologous groups of all E. chaffeensis genes were identified
using the PanOCT program (Fouts et al., 2012) with the following
parameters: E-value 10−5, percent identity ≥30, and length of
match ≥65. For phylogenetic reconstruction, whole-genome nucleotide
sequences of the eight E. chaffeensis strains were aligned
using the progressiveMauve algorithm (Darling et al., 2010,
http://gel.ahabs.wisc.edu/mauve/). FastTree
was
used
with
default parameters to build the unrooted tree (Price et al., 2009). Mauve software was also used to characterize the genomic
rearrangements between the eight genomes of E. chaffeensis by
showing LCBs. In order to accurately align conserved regions in
the genomes, the progressiveMauve algorithm was parameterized
with a match seed weight of 15 and a minimum LCB score of 70. The seed size parameter sets the minimum weight of the seed
pattern used to generate local multiple alignments (matches)
during the first pass of anchoring the alignment. The LCB weight
sets the minimum number of matching nucleotides identified in
a collinear region in order for the region to be considered a true
homology rather than a random similarity (Darling et al., 2010). The Ehrlichia chaffeensis Genomes Are
Highly Conserved In order to establish a whole genome-based phylogeny of these
eight E. chaffeensis strains, we used the Mauve progressive
alignment and FastTree to build the tree. Our results are in
agreement with those of previous studies, with the eight strains
being separated into three genetic groups. The Arkansas and
Osceola strains were assigned to group I, and the Wakulla,
Saint Vincent, West Paces, and Heartland strains were assigned
to group II. We also assigned the West Paces strain to group
II due to its phylogenetically close proximity to the Heartland
strain (Figure 1A). The Jax and Liberty strains were assigned
to group III (Figure 1A). With an average size of 1.2 Mb, the
genomic features of the eight strains used in this study are
similar. The GC (guanine-cytosine) content was seen to be
highly homogenous (30.1%) and genome sequences relatively
well-conserved (Figure 1B). The number of genes ranges from
871 to 883. Whole genome alignments revealed seven LCBs
with some inversions and with rearrangements in the genomes
with respect to one another (Figure 1B). In the Arkansas strain,
we found a rearrangement between three LCBs with green and
orange blocks switched with yellow LCB. The strains Arkansas,
Osceola, Heartland, and West Paces showed an inversion of
blue and red LCBs compared to other genomes. The Saint
Vincent and Wakulla strains showed inversion of one small
LCB (purple, Figure 1B). The structural variation among these
genomes suggests a low degree of inter-species genome plasticity
for E. chaffeensis. Prediction of E. chaffeensis Type IV
Effectomes The repertoires of T4Es were predicted using a S4TE algorithm
with default parameters (Meyer et al., 2013) . S4TE 1.4 predicts
and ranks candidate T4Es by using a combination of 11
independent modules to explore 14 characteristics of type
IV effectors. One module searches for consensus motifs in
promoter regions; three modules search for the five features
of the type IV secretion signal (C-terminal basicity, C-terminal
charges, C-terminal hydrophobicity, overall hydrophilicity, and
E-blocks); six modules search for several domains (eukaryotic–
like domains, the DUF domain, EPIYA motifs, the nuclear
localization signal, the mitochondrial localization signal, the
prenylation domain, coiled-coil domains); and one module
searches for homology with known T4Es (Meyer et al., 2013) INTRODUCTION The gene encoding tandem-repeat
proteins (TRP) 32 (formerly VLPT, the variable length PCR target
gene) contains the region specifying three to six nearly identical,
highly hydrophilic 90–amino acid tandem repeats (Sumner et al.,
1999). Similarly, in TRP120 (formerly gp120), there are two to
four imperfect, direct, tandem 80 bp repeats (Sumner et al.,
1999). The number of repeats varies depending on the isolate,
resulting in variations in size in the encoded protein. The TRP32
gene shows great inter-strain diversity and is characterized by
a series of direct tandem repeats whose number varies among Our laboratory developed a searching algorithm for type IV
effector proteins (S4TE), which identifies candidate T4Es in
genome sequences based on a combinatorial approach with 14
different parameters (Meyer et al., 2013). To better understand the evolution and pathogenicity of
E. chaffeensis, we analyzed the eight available E. chaffeensis
genomes of distinct geographical origin and of varying virulence
isolated from humans (Table 1). We identified the relationship
between E. chaffeensis strains using comparative whole genome
analysis based on phylogenetic analysis, alignment of locally
collinear blocks (LCB), and analysis of shared and specific
genetic content. We provide evidence that the West Paces
strain belongs to genetic group II and that E. chaffeensis is
a highly conserved species. We describe likely virulence traits
(candidate type IV effectors) encoded by their genomes and some
putative host cell targets. Most notably some strains lack one or
two candidate T4Es, but show conserved intracellular adaptive
strategies. Our results show that using our S4TE software and
approach even for strains which are really close at the
intraspecies level, enables the prediction of candidate type
IV effectors that could be relevant for the study of bacterial
pathogenesis. January 2017 | Volume 6 | Article 204 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 2 Comparative genomics of Ehrlichia chaffeensis Noroy and Meyer MATERIALS AND METHODS predicted using the CELLO2GO algorithm (Yu et al., 2014). S4TE
1.4 results were used to predict the location of T4Es (Meyer et al.,
2013) Prediction of E. chaffeensis Type IV
Effectors and Host Protein–Protein
Interaction Networks Protein-protein interactions between human genomes and
predicted type IV effectome of E. chaffeensis were predicted using
the Host-Pathogen Interaction Database (HPIDB) (Kumar and
Nanduri, 2010) with the identity and percentage query coverage
set at 30%. Based on the homology approach, the HPIDB
predicts protein-protein interactions from a plentiful template
of eukaryotic-prokaryotic inter-species interactions available
among 68 hosts and 602 pathogens. Subcellular locations of the
host proteins interacting with putative T4Es of E. chaffeensis were We then analyzed the pan-genome of E. chaffeensis. We used
PanOCT software to cluster the ortholog and compared the core
and accessory genomes of the eight strains of E. chaffeensis. January 2017 | Volume 6 | Article 204 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 3 Comparative genomics of Ehrlichia chaffeensis Noroy and Meyer TABLE 1 | Main biological and genetic characteristics of the eight Ehrlichia chaffeensis strains analyzed. Strain
Arkansas
Heartland
Jax
Liberty
Osceola
St. Vincent
Wakulla
West Paces
Year
1991
1999
1997
1998
1997
1996
1997
2000
Origin
Arkansas
Nebraska
Florida
Florida
Florida
Georgia
Florida
Tennessee
Source
21-year old male
Human
51-year old Woman
Human
Human
52-year old, HIV+
Human
Human
Human, clinical and
laboratory
observations
Fever, headache,
pharyngitis,
nausea, vomiting,
and dehydration. Cervical
lymphadenopathy,
splenomegaly,
thrombocytopenia,
leukopenia with left
shift, elevations in
serum aspartate
transaminase
concentration. HME, no clinical
description available. Fever, non-productive
cough, nausea,
vomiting, and diarrhea,
profoundly lethargic. Thrombocytopenia,
leukopenia, elevations
in serum aspartate
transaminase
concentration,
doxycycline therapy,
cerebrospinal fluid
mononuclear
pleocytosis, pulmonary
oedema, hypotension,
and anuria. The patient
died in hospital on
day 6. Acute HME, no clinical
description available. Acute HME, no clinical
description available. Fever, headache,
myalgia, nausea, and
vomiting, orthostatic
hypotension,
thrombocytopenia,
leukopenia with left
shift, elevations in
serum aspartate
transaminase
concentration,
doxycycline therapy. Lobar pneumonia and
acute renal failure. The
patient died. Acute HME, no
clinical
description
available. Acute HME, no clinical
description available. SCID mice
pathogenesis
Mild, chronic
UN
UN
Acute, severe
UN
UN
Acute, lethal
UN
Genetic Group/
TRP32/TRP120
repeats
I/4/4
II/3/3
III/4/4
III/4/4
I/4/3
II/3/3
II/6/4
II/3/3
Literature
Dawson et al.,
1991
Sumner et al., 1999
Paddock et al., 1997
Sumner et al., 1999
Sumner et al., 1999
Paddock et al., 1997
Sumner et al.,
1999
Cheng et al., 2003
Human isolates of E. January 2017 | Volume 6 | Article 204 Prediction of E. chaffeensis Type IV
Effectors and Host Protein–Protein
Interaction Networks chaffeensis were classified in three genetic groups according to the 28-kDa major outer membrane gene cluster, the number of TRP32 repeats (variable-length PCR target gene, NCBI accession version
# WP_011452439.1) and the number of TRP120 repeats (120-kDa immunodominant surface protein, NCBI accession version # WP_011452362.1). Data for pathogenesis in SCID mice for E. chaffeensis Arkansas, Liberty and
Wakulla strains were obtained from Miura and Rikihisa (2007) and indicated. UN, unknown. Frontiers in Cellular and Infection Microbiology | www frontiersin org
4 January 2017 | Volume 6 | Article 204 Comparative genomics of Ehrlichia chaffeensis Noroy and Meyer FIGURE 1 | Comparative genomics of 8 Ehrlichia chaffeensis strains. (A) Phylogenetic tree of 8 E. chaffeensis strains. FastTree based on the Mauve alignment
of the whole genomes of 8 E. chaffeensis strains. The node values indicate the local support values of the Shimodaira-Hasegawa test. The number outside the tree
shows the genetic group of each strain, the West Paces strain was assigned to genetic group II due to the high level of conservation with the Heartland strain. (B)
Alignments of 8 E. chaffeensis genomes generated using Mauve software (Darling et al., 2010) (http://gel.ahabs.wisc.edu/mauve/). Locally collinear blocks (LCBs),
shown as rounded rectangles, represent regions with no rearrangement of homologous sequences across genomes. The forward or reverse orientation of the LCBs is
indicated by their position, respectively above or below the line. Lines between the genomes trace orthologous LCBs. Using default parameters resulting in a minimum
LCB weight of 70, there are 7 LCBs across all the genomes. The LCB weight defines the minimum number of matching nucleotides in a collinear region for it to be
considered homologous across genomes and not the result of a spurious match. Regions outside LCBs were too divergent in at least one genome to be aligned
successfully. Inside each LCB, vertical bars represent the similarity profile of the genome sequence. The height of each bar corresponds to the average level of
conservation in that region of the genome sequence. (C) Shared and specific gene content between 8 E. chaffeensis strains. Each colored petal represents a different
E. chaffeensis genome. The number in the center of the diagram represents the number of orthologous genes shared by all the genomes, thus defining the FIGURE 1 | Comparative genomics of 8 Ehrlichia chaffeensis strains. (A) Phylogenetic tree of 8 E. chaffeensis strains. Prediction of E. chaffeensis Type IV
Effectors and Host Protein–Protein
Interaction Networks FastTree based on the Mauve alignment
of the whole genomes of 8 E. chaffeensis strains. The node values indicate the local support values of the Shimodaira-Hasegawa test. The number outside the tree
shows the genetic group of each strain, the West Paces strain was assigned to genetic group II due to the high level of conservation with the Heartland strain. (B)
Alignments of 8 E. chaffeensis genomes generated using Mauve software (Darling et al., 2010) (http://gel.ahabs.wisc.edu/mauve/). Locally collinear blocks (LCBs),
shown as rounded rectangles, represent regions with no rearrangement of homologous sequences across genomes. The forward or reverse orientation of the LCBs is
indicated by their position, respectively above or below the line. Lines between the genomes trace orthologous LCBs. Using default parameters resulting in a minimum
LCB weight of 70, there are 7 LCBs across all the genomes. The LCB weight defines the minimum number of matching nucleotides in a collinear region for it to be
considered homologous across genomes and not the result of a spurious match. Regions outside LCBs were too divergent in at least one genome to be aligned
successfully. Inside each LCB, vertical bars represent the similarity profile of the genome sequence. The height of each bar corresponds to the average level of
conservation in that region of the genome sequence. (C) Shared and specific gene content between 8 E. chaffeensis strains. Each colored petal represents a different
E. chaffeensis genome. The number in the center of the diagram represents the number of orthologous genes shared by all the genomes, thus defining the
E. chaffeensis core genome. The number inside each individual petal corresponds to the number of genes that are absent from the core genome, and the numbers in
brackets correspond to the number of genes specific to this strain. The number outside each petal shows the genetic group of each strain. January 2017 | Volume 6 | Article 204 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 5 Comparative genomics of Ehrlichia chaffeensis Noroy and Meyer TABLE 2 | Putative type IV effectors (T4Es) identified by the S4TE algorithm. January 2017 | Volume 6 | Article 204 Prediction of E. chaffeensis Type IV
Effectors and Host Protein–Protein
Interaction Networks Ehrlichia chaffeensis strains
NCBI protein names
Arkansas
Liberty
Wakulla
West Paces
S4TE Score
Promoter motif
Homology
Euk-like domain
EPIYA
NLS
MLS
Prenylation
Coiled coils
Cter basicity
Cter charges
Cter basicity
Cter charges
Cterhydrophobicity
Eblock
Location
ECH_RS02870
ECHLIB_RS01940
ECHWAK_RS01950
ECHWP_RS02750
Hypothetical protein
229
1
1
1
0
1
1
0
0
0
1
0
0
1
0
GSRs
ECH_RS03425
ECHLIB_RS01385
ECHWAK_RS01390
ECHWP RS03295
Hypothetical protein
164
1
1
0
0
0
0
0
0
0
0
1
0
0
0
GDRs
ECH_RS04335
ECHLIB_RS00490
ECHWAK_RS00485
ECHWP_RS00485
Gamma carbonic anhydrase
family protein
151
1
1
0
0
0
0
0
0
0
1
0
0
0
1
GSRs
ECH_RS02750
ECHLIB_RS02060
ECHWAK_R502070
ECHWP R502630
Hypothetical protein
141
1
0
1
0
1
1
0
0
0
1
0
0
0
0
GDRs
ECH_RS02620
ECHLIB_RS02190
ECHWAK_RS02200
ECHWP_RS02500
Alpha/beta hydrolase
139
1
1
0
0
0
1
0
0
0
1
1
0
0
0
GSRs
ECH_RS03745
ECHLIB_RS01065
ECHWAK_RS01070
ECHWP RS03615
AI-2E family transporter
122
1
1
0
0
0
0
0
0
0
1
0
0
0
0
GSRs
ECH_RS00450
ECHLIB_RS04345
ECHWAK_RS04360
ECHWP_RS04320
Hypothetica l protein
118
1
0
0
1
0
1
0
0
0
1
0
0
0
1
GSRs
ECH_RS01210
ECHLIB_RS03585
ECHWAK_RS03600
ECHWP RS01105
DNA ligase (NAD(+)) LlgA
117
0
0
1
0
1
1
0
0
0
1
0
0
1
0
BRs
ECH RS04225
ECHLIB RS00595
ECHWAK RS00600
ECHWP RS00590
Hypothetica l protein
115
1
0
0
0
1
1
0
0
0
1
1
0
1
0
GSRs
ECHLIB_RS02720
Hypothetical protein
114
0
0
0
0
1
1
0
0
0
1
0
0
1
1
GDRs
ECH_RS02365
ECHLIB_RS02435
ECHWAK_RS02455
ECHWP_RS02250
Translation initiation factor IF-2
114
0
0
0
0
1
1
0
0
0
0
1
0
1
1
GDRs
ECH_RS03205
ECHLIB_RS01605
ECHWAK_RS01615
ECHWP RS03075
Diguanylate cyclase response
regulator0
109
0
1
0
0
0
1
0
0
0
1
0
0
0
0
IBRs
ECH_RS03195
ECHLIB_RS01615
ECHWAK_RS01625
ECHWP_RS03065
NAD-glutamate dehydrogenase
108
1
0
0
0
1
0
0
1
0
1
0
1
1
0
IBRs
ECH_RS02495
ECHLIB_RS02315
ECHWAK_R502330
ECHWP R502375
Peptide chain release factor 1
105
1
0
0
0
0
0
0
0
0
1
0
0
1
1
IBRs
ECH_RS04685
ECHLIB_RS04640
ECHWAK_RS04650
Hypothetica l protein
103
0
0
0
1
1
1
0
0
0
1
1
1
1
0
GSRs
ECH_RS01570
ECHLIB_RS03225
ECHWAK_RS03240
ECHWP RS01465
Hypothetical protein
101
0
0
0
0
1
1
0
0
0
1
1
1
1
0
GDRs
ECH_RS04230
ECHLIB_RS00590
ECHWAK_RS00595
ECHWP_RS00585
Hypothetica l protein
99
1
0
0
0
1
1
0
0
0
0
0
0
1
0
GSRs
ECH RS02945
ECHLIB RS01860
ECHWAK RS01875
ECHWP RS02825
Transcriptional regulator
98
0
1
0
0
0
0
0
0
0
1
0
0
0
0
IBRs
ECH_RS02385
ECHLIB_RS02415
ECHWAK_RS02435
ECHWP_RS02270
Hypothetica l protein
98
1
0
0
0
1
0
0
0
0
1
0
0
1
0
GSRs
ECH_RS03860
ECHLIB_RS00950
ECHWAK_RS00955
ECHWP RS03725
Hypothetical protein
97
1
0
0
0
0
1
0
0
0
1
0
1
1
0
IBRs
ECH_RS04650
ECHLIB_RS00175
ECHWAK_RS00175
ECHWP_RS00175
Protein translocase subunit SecA 93
1
0
0
0
0
1
0
0
0
1
1
0
1
0
GSRs
ECH_RS02080
ECHLIB_R502725
ECHWAK_R502740
ECHWP R501965
Hypothetical protein
93
0
0
0
0
1
0
0
0
0
0
0
0
0
1
GDRs
ECH_RS02075
ECHLIB_RS02730
ECHWAK_RS02745
ECHWP_RS01960
Conjugal transfer protein Trbl
92
0
0
0
0
0
1
0
0
0
1
0
0
1
1
GDRs
ECH_RS03040
ECHLIB_RS01765
ECHWAK_R501780
ECHWP R502910
Peptidylprolyl isomerase
91
0
0
0
0
1
1
0
0
0
1
1
0
1
0
GSRs
ECH_RS02255
ECHLIB_RS02545
ECHWAK_RS02565
ECHWP_RS02140
165 rRNA (uracii(1498)-N(3))-
methyltransferase
88
0
1
0
0
0
0
0
0
0
0
0
0
0
0
GDRs
ECH_RS03605
ECHLIB_RS01205
ECHWAK_R501210
ECHWP R503475
Hypothetical protein
87
1
0
1
0
0
1
0
0
0
1
0
1
0
0
IBRs
ECH RS03515
ECHLIB RS01295
ECHWAK RS01300
ECHWP RS03385
Hypothetical protein
87
1
0
0
0
0
1
0
0
0
1
0
0
1
0
GSRs
ECH_RS02340
ECHLIB_RS02460
ECHWAK_RS02480
ECHWP RS02225
Hypothetical protein
87
1
0
0
0
0
1
0
0
0
1
0
0
1
0
IBRs
ECH_RS01565
ECHLIB_RS03230
ECHWAK_RS03245
ECHWP_RS01460
Exodeoxyribonuclease V subunit
beta
87
1
0
0
0
0
1
0
0
0
1
0
0
1
0
IBRs January 2017 | Volume 6 | Article 204 6 Comparative genomics of Ehrlichia chaffeensis Noroy and Meyer TABLE 2 | Continued
Ehrlichia chaffeensis strains
NCBI protein names
Arkansas
Liberty
Wakulla
West Paces
S4TE Score
Promoter motif
Homology
Euk-like domain
EPIYA
NLS
MLS
Prenylation
Coiled coils
Cter basicity
Cter charges
Cter basicity
Cter charges
Cterhydrophobicity
Eblock
Location
ECH_RS01140
ECHLIB_RS03660
ECHWAK_RS03675
ECHWP RS01035
Hypothetical protein
87
1
0
0
0
0
1
0
0
0
1
0
0
1
0
GSRs
ECH_RS03890
ECHLIB_RS00920
ECHWAK_RS00925
ECHWP_RS03755
DNA-directed RNA polymerase
subunit beta
85
0
0
0
0
1
1
0
0
0
1
0
0
1
0
GDRs
ECH_RS00205
ECHLIB_RS04580
ECHWAK_RS04590
ECHWP RS04555
Type IV secretion system protein
VirD4
85
0
0
0
0
1
1
0
0
0
1
0
0
1
0
IBRs
ECH_RS03630
ECHLIB_RS01180
ECHWAK_RS01185
ECHWP_RS03500
DNA processing protein DprA
84
1
0
0
0
0
1
0
0
0
1
0
1
0
1
GSRs
ECH_RS03440
ECHLIB_RS01370
ECHWAK_RS01375
ECHWP RS03310
Phage capsid protein
82
1
0
0
0
0
0
0
0
0
1
1
0
1
0
GSRs
ECH_RS03530
ECHLIB_RS01280
ECHWAK_RS01285
ECHWP_RS03400
Hypothetica l protein
81
0
0
0
0
0
0
0
0
0
1
0
0
1
1
IBRs
∼RS01950
ECHLIB_RS02855
ECHWAK_RS02870
ECHWP RS01835
Molecular chaperone DnaK
81
0
0
0
0
0
0
0
0
0
1
0
0
1
1
IBRs
ECH_RS03610
ECHLIB_RS01200
ECHWAK_RS01205
ECHWP_RS03480
Hypothetica l protein
80
0
0
0
0
1
0
0
0
0
1
1
0
1
0
GSRs
ECH_RS03260
ECHLIB_RS01550
ECHWAK_RS01560
ECHWP RS03130
abc-ATPase UvrA
77
1
0
0
0
0
1
0
0
0
0
0
0
1
0
GSRs
ECH_RS03895
ECHLIB_RS00915
ECHWAK_RS00920
ECHWP_RS03760
505 ribosomal protein L7 /L12
74
1
0
0
0
0
1
0
0
0
1
0
0
0
1
GDRs
ECH_RS02525
ECHLIB_RS02285
ECHWAK_RS02300
ECHWP RS02405
Glutamate-tRNA ligase
74
1
0
0
0
0
1
0
0
0
1
0
0
0
1
GSRs
ECH RS00785
ECHLIB_RS04010
ECHWAK_RS04025
ECHWP_RS03985
Hypothetica l protein
74
0
0
0
0
1
0
0
0
0
1
0
0
1
0
GSRs
’Eei(RS00330
ECHLIB_RS04455
ECHWAK_RS04465
ECHWP RS04430
1-acyl-sn-glycerol-3-phosphate
acyltransferase
74
1
0
0
0
0
1
0
0
0
1
0
0
0
1
GSRs
ECH_RS00255
ECHLIB_RS04530
ECHWAK_RS04540
ECHWP_RS04505
Hypothetical protein
74
1
0
0
0
0
1
0
0
0
1
0
0
0
1
BRs
ECH_RS03415
ECHLIB_RS01395
ECHWAK_RS01400
ECHWP RS03285
NAD+ synthetase
73
1
0
0
0
1
1
0
0
0
1
1
0
0
0
GSRs
ECH_RS02490
ECHLIB_RS02320
ECHWAK_RS02335
ECHWP_RS02370
GTP-binding protein
73
1
0
0
0
0
0
0
0
0
1
0
1
0
1
IBRs
ECH_RS00505
ECHLIB_RS04290
ECHWAK_RS04305
ECHWP_RS04265
Citrate (Si)-synthase
73
1
0
0
0
0
0
0
0
0
0
1
0
1
1
GDRs
ECH RS03555
ECHLIB RS01255
ECHWAK RS01260
ECHWP RS03425
Hypothetica l protein
72
0
0
0
0
0
0
0
1
0
0
1
1
0
1
GSRs
This table shows the candidate T4Es identified by S4TE software in four E. January 2017 | Volume 6 | Article 204 Prediction of E. chaffeensis Type IV
Effectors and Host Protein–Protein
Interaction Networks chaffeensis strains. The Liberty strain is used as a reference to sort predicted effectors, and the homolog candidate effectors are ranked by S4TE scores. Each
T4E is defined by the gene ID, Name, and S4TE features. January 2017 | Volume 6 | Article 204 7 Comparative genomics of Ehrlichia chaffeensis Noroy and Meyer FIGURE 2 | Distribution of Ehrlichia chaffeensis effectomes according to local gene density. (A) Distribution of E. chaffeensis str. Arkansas genes according
to the length of their flanking intergenic regions (FIRs). All E. chaffeensis genes were sorted into 2-dimensional bins according to the length of their 5′ (y-axis) and 3′
(x-axis) FIRs. The number of genes in the bins is represented by a color-coded density graph. Genes whose FIRs are both longer than the median FIR length were
considered as gene-sparse region (GSR) genes. Genes whose FIRs are both below the median value were considered as gene-dense region (GDR) genes. In-between region (IBR) genes are genes with a long 5′FIR and short 3′FIR, or inversely. Candidate effectors predicted using the S4TE algorithm were s plotted on this
distribution according to their own 3′ and 5′ FIRs. A color is assigned to each of the three following groups: Red to GDRs, orange to IBRs, and blue to GSRs. (B)
Distribution of genes in GDRs, IBRs, and GSRs of E. chaffeensis strains. The proportion of the genome and the effectome that occurs in GDRs (red), IBRs (orange),
and in GSRs (blue) is indicated. FIGURE 2 | Distribution of Ehrlichia chaffeensis effectomes according to local gene density. (A) Distribution of E. chaffeensis str. Arkansas genes according
to the length of their flanking intergenic regions (FIRs). All E. chaffeensis genes were sorted into 2-dimensional bins according to the length of their 5′ (y-axis) and 3′
(x-axis) FIRs. The number of genes in the bins is represented by a color-coded density graph. Genes whose FIRs are both longer than the median FIR length were
considered as gene-sparse region (GSR) genes. Genes whose FIRs are both below the median value were considered as gene-dense region (GDR) genes. In-between region (IBR) genes are genes with a long 5′FIR and short 3′FIR, or inversely. Candidate effectors predicted using the S4TE algorithm were s plotted on this
distribution according to their own 3′ and 5′ FIRs. Prediction of E. chaffeensis Type IV
Effectors and Host Protein–Protein
Interaction Networks A color is assigned to each of the three following groups: Red to GDRs, orange to IBRs, and blue to GSRs. (B)
Distribution of genes in GDRs, IBRs, and GSRs of E. chaffeensis strains. The proportion of the genome and the effectome that occurs in GDRs (red), IBRs (orange),
and in GSRs (blue) is indicated. causes acute pathogenesis in immunodeficient mice (Miura and
Rikihisa, 2007). (Figure 1C). The E. chaffeensis core-genome contained 853
orthologous genes, corresponding to ∼96% of the pan-genome
and indicating that the E. chaffeensis accessory genome is narrow. Thus, four percent of E. chaffeensis genes are not in the core
genome and only a few genes are specific to four out of these
eight strains. The Arkansas strain harbored eight specific genes,
the Osceola strain two specific genes and Liberty and Jax strains
only one specific gene (Figure 1C). Frontiers in Cellular and Infection Microbiology | www.frontiersin.org Prediction of Type IV Effectors for
E. chaffeensis Identifies the Core Type IV
Effectome among Four Human Isolates g
We used the S4TE algorithm to predict and compare the type
IV effector repertoires in four human isolates (Arkansas, Liberty,
Wakulla, and West Paces) of E. chaffeensis in order to determine
how these repertoires differed between strains with respect to the
presence or absence of whole candidate T4Es. We identified a
conserved repertoire of 45 candidate T4Es, defining the core type
IV effectome of E. chaffeensis. To test if the genome plasticity and effector repertoires
can
explain
the
differential
intra-species
pathogenesis
of
E. chaffeensis, we decided to focus our study on four different
representative strains. When data were available, we chose strains
belonging to different genetic groups showing variations in
virulence. From genetic group I, we chose the Arkansas strain,
which is the most widely studied and best-described strain in the
literature. This strain shows mild virulence in immunodeficient
mice (Miura and Rikihisa, 2007). From genetic group II, we chose
the West Paces and Wakulla strains, the latter causing acute
lethal pathogenesis in SCID mice (Miura and Rikihisa, 2007). Finally, from genetic group III, we chose the Liberty strain, which Based on orthology analysis, we found few differences
in T4E content between the four selected E. chaffeensis
isolates. E. chaffeensis str. Liberty was the only strain to
own all 47 predicted T4Es (Table 2). One candidate T4E,
ECHLIB_RS02720, is specific to E. chaffeensis str. Liberty,
whereas ECHLIB_RS04640 was only absent in E. chaffeensis str. January 2017 | Volume 6 | Article 204 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 8 Comparative genomics of Ehrlichia chaffeensis Noroy and Meyer IGURE 3 | Protein-protein interaction network between the E. chaffeensis str. Liberty effectome and the human genome. A sub-cellular location was
redicted with the S4TE algorithm (http://sate.cirad.fr) for Ehrlichia candidate effectors (left) and with CELLO2GO software (http://cello.life.nctu.edu.tw/cello2go/) for
uman proteins (right). Blue and red circles represent predicted T4Es located in the cytoplasm and in the nucleus of the host cell, respectively. Blue, red, pink, green, FIGURE 3 | Protein-protein interaction network between the E. chaffeensis str. Liberty effectome and the human genome. A sub-cellular location was
predicted with the S4TE algorithm (http://sate.cirad.fr) for Ehrlichia candidate effectors (left) and with CELLO2GO software (http://cello.life.nctu.edu.tw/cello2go/) for
human proteins (right). Blue and red circles represent predicted T4Es located in the cytoplasm and in the nucleus of the host cell, respectively. Prediction of the Host-Pathogen
Protein–Protein Interaction Network We predicted the interactions of E. chaffeensis T4Es with human
proteome and identified 57 protein-protein interactions with the
involvement of 13 putative T4Es of E. chaffeensis str. Liberty
(which harbors all predicted T4Es) and 56 human proteins
(Figure 3). The
targeted
host
proteins
are
located
in
cellular
compartments
relevant
to
the
pathogenesis
mechanisms. The predicted cellular localizations of human interacting
proteins were confirmed in cytoplasm, nucleus, extracellular,
mitochondrial, plasma membrane, endoplasmic reticulum, and
cytoskeleton (Figure 3). As described above, we predicted the
subcellular localization in human host cell of E. chaffeensis
T4Es using the S4TE algorithm (Table 2, Table S1). Out of the
13 predicted T4Es of E. chaffeensis that interact with human
proteins, eight (∼60%) harbor at least one nuclear location signal
(NLS). Interestingly, most of these proteins had putative human
targets located in the nucleus (Figure 3). Concerning other features related to the type IV secretion
signal,
17%
of
the
predicted
T4Es
showed
C-terminal
hydrophobicity, 68% showed global hydropathy < −200
(on the Kyte-Doolittle scale), 21.3% had a C-ter charge ≥2 and
89.4% had at least three alkaline amino acids in C-terminal 25
amino acids. Thus, the putative targets of the ABC-ATPase UvrA
(ECHLIB_RS01550) are involved in different processes including
innate immunity, response to stress, the cell cycle, cell signaling,
and cell death (Table S2). Another candidate nuclear effector
(ECHLIB_RS02285) interacts with 11 human proteins, most
of which are involved in metabolic processes such as amino
acid synthesis (IDHC), carbohydrate metabolic process (DPM1
and IDHC), lipid metabolism (DPM1), and nitrogen compound
metabolism (ZN627, IDHC, DPM1, WBS22, CNTRL, and
PUF60) (Table S2). Prediction of Type IV Effectors for
E. chaffeensis Identifies the Core Type IV
Effectome among Four Human Isolates Blue, red, pink, green,
purple, yellow, and turquoise hexagons represent the different locations of targeted human proteins in the host cell. Hexagons harbor several colors when CELLO2GO
predicts several probable subcellular locations. January 2017 | Volume 6 | Article 204 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 9 Comparative genomics of Ehrlichia chaffeensis Noroy and Meyer West Paces. All the other predicted T4Es (94% of predicted
effectors) are common to the four strains, revealing the low
diversity of effector repertoires in E. chaffeensis species. We did
not discover any relation between the presence or absence of an
effector and the variations in virulence exhibited by the Wakulla,
Liberty, and Arkansas strains. The gene dense region (GDR, genes with 5′ FIR < 202 bp and
3′ FIR < 223 bp) contains 254 genes, which account for 28.9%
of the E. chaffeensis str. Arkansas genome (Figure 2A). The gene
sparse region (GSR, genes with 5′ FIR ≥202 bp and 3′ FIR ≥203
bp) includes 255 genes, which account for 29% of the genome
(Figure 2A). y
Identified candidate T4Es were sorted according to their
S4TE score, which ranged from 72 (corresponding to the S4TE
algorithm threshold) to 229 (Table 2). Eight candidate T4Es
showed homology with known T4Es (17% of predicted T4Es) as
indicated by the number “1” in the Homology column in Table 2. Among these candidates, one is ECH_RS01385 previously called
ECH0825 (old NCBI locus_tag) (Liu et al., 2012; Table S1). This effector was predicted with the second highest S4TE score
of 164. The first predicted T4E, ECH_RS01940, matched the
homologous gene of A. phagocytophilum AnkA (IJdo et al.,
2007; Lin et al., 2007; Garcia-Garcia et al., 2009; Table S1). ECHLIB_RS02190,
ECHLIB_RS01065,
ECHLIB_RS01605,
and ECHLIB_RS01860 are four candidate T4Es presenting
homologies with known Coxiella burnetii effectors (Table
S1). ECHLIB_RS022545
shows
homology
with
a
known
Legionella pneumophila T4E (lpg2936, 16S ribosomal RNA
methyltransferase
RsmE)
while
ECHLIB_RS00490
shows
homology with a Brucella effector (Table S1). The other two quadrants define in-between regions (IBRs)
grouping genes with a 5′ FIR shorter than the median length and
a longer 5′ FIR, and inversely. In the E. chaffeensis str. Arkansas
genome, 370 genes, which account for 42% of the genome, fall
into IBRs (Figure 2A). This genome architecture of E. chaffeensis
str. Arkansas is representative of other strains of E. chaffeensis
(Figure 2B). Prediction of Type IV Effectors for
E. chaffeensis Identifies the Core Type IV
Effectome among Four Human Isolates We then performed a detailed analysis of the distribution of
predicted E. chaffeensis T4Es according to local gene density. We found that the predicted T4Es of all isolates of E. chaffeensis
frequently had both FIRs above the genome median value. Although 29% of E. chaffeensis genes belong to GSRs, 42.2%
to 46.8% of predicted type IV effector genes fall in GSRs
(Figures 2A,B). Thus, compared to the whole genome, the GSRs
showed a 1.5-fold enrichment in candidate type IV effector genes. Consequently, the proportion of candidate T4Es in the GDRs and
IBRs is lower than the proportion of genes of the whole genome
(Figure 2B). These results suggest that plastic regions with low
gene density harbor pathogenicity genes and could play a role in
host-bacteria interactions. Besides homology with known effectors, several other
candidate T4Es had interesting features (Table 2). Indeed, 59.6%
of predicted T4Es had a promoter motif such as PmrA upstream
of the effector genes of Coxiella spp. and Legionella spp. Furthermore, 8.5% of putative T4Es harbored eukaryotic-like
domains such as AnkA (Ankyrin repeat-containing domain)
and BRCT (phospho-protein binding domain) domains. Only
two putative T4Es contained domains of unknown function
(DUF). It is interesting to note that 38.3 and 61.7% of candidate
T4Es had a tyrosine phosphorylation domain (EPIYA) and a
nuclear localization signal (NLS), respectively. Moreover, nearly
38% of the proteins harboring an NLS also had an EPIYA
phosphorylation domain. None of the predicted T4Es had a
prenylation domain or a coiled-coil domain. Thirty-four percent
of the candidate T4Es harbored the canonical L. pneumophila
secretion domain (E-block). Putative Type IV Effectors of E. chaffeensis
are Overrepresented in Gene Sparse
Regions of the Genome However in our study, the S4TE algorithm correctly
predicted the two known type IV effectors in Anaplasmataceae
family with E. chaffeensis mitochondrial effector ECH0825
(ECHLIB_RS01385) and the homolog of A. phagocytophilum
nucleomodulin
AnkA
(ECHLIB_RS01940)
(Table
S1). In
addition, S4TE predicted effectors that are homologous to known
effectors in other bacteria, including C. burnetii, L. pneumophila,
and Brucella spp. S4TE also predicted some new candidate
T4Es that were not easy to identify ab initio, based solely on the
poor quality of automated genome annotations, especially for
bacteria harboring 30% or more unknown hypothetical proteins
like Anaplasmataceae. For example, S4TE identified some
bacterial enzymes as candidate effectors, including the annotated
acyltransferase ECHLIB_RS04455, which is in agreement with
current knowledge on bacterial effectors (Anderson et al., 2015). Among the other E. chaffeensis T4Es whose interaction with
human proteins was predicted, the transcriptional regulator
ECHLIB_RS01860 interacts with two putative targets. The first
is the nuclear factor NF-kappa-B, which plays a prominent
role in immune responses, responses to stress, and cell death. The second target is SDCB1, which is involved in cytoskeleton
organization, cell-cell signaling, locomotion, cell adhesion, and
growth (Table S2). g
(
)
Most of the predicted T4Es in E. chaffeensis belong to
the core type IV effectome, showing that effector repertoires
are highly conserved in this species. Thus, for bacteria with
compact genomes, the type IV effector repertoires may not
reflect the genetic diversity and the variations in pathogenesis
observed within a species. However, two candidate T4Es,
ECHLIB_RS02720 and ECHLIB_RS04640, are not present in
all E. chaffeensis strains and could explain some within-
strain variations in virulence. Indeed, in pathogens with
bigger genomes and more complex lifestyles, some authors
demonstrated that diversity in effector repertoires is linked to
host specificity (Cooke et al., 2012; Guyon et al., 2014; Schwartz
et al., 2015). The 45 T4Es predicted by S4TE in E. chaffeensis
account for about 5% of the genome. In comparison, in the
facultative intracellular L. pneumophila str. Philadelphia I, which
contains a well-characterized type IV effectome, 286 T4Es
account for about 9% of the genome (Lifshitz et al., 2013). Thus, in relation to the number of genes, the predicted type
IV effectome of E. chaffeensis is significantly smaller than that
of L. pneumophila. This could be explained by the reduced size
of the E. Putative Type IV Effectors of E. chaffeensis
are Overrepresented in Gene Sparse
Regions of the Genome In order to understand how genomic plasticity influences the
distribution of predicted T4Es, we first analyzed the genome
architecture of E. chaffeensis by looking at local gene density
(Figure 2). The median length of 3′ and 5′ flanking intergenic
regions (FIRs) delimits four coherent gene pools when combined
with the 2-variable binning representations (Figure 2A). January 2017 | Volume 6 | Article 204 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 10 Comparative genomics of Ehrlichia chaffeensis Noroy and Meyer The nuclear effectors ECHLIB_RS02315 and the DNA ligase
ECHLIB_RS03585 interact with several putative targets involved
in immune and stress responses, cell organization, and cell
death. Most of the proteins targeted by ECHLIB_RS00175 are
located in the nucleus and are involved in nuclear organization
(chromosomal protein HMG2) or biosynthetic process (proline-
rich and zinc finger proteins) (Table S2). The nuclear effector
ECHLIB_RS02435 interacts with kinases and with the nuclear
transcriptional repressor BCLF1 suggesting an important role in
signal transduction and stress response, particularly activation
of response to DNA damage. It is of note that this effector also
harbors a tyrosine phosphorylation domain that could play an
important role in the ATM/MAP kinases signaling pathway. differences in pathogenesis and symptoms between the Arkansas,
Liberty and Wakulla strains (Table 1) could be due to the absence
of certain genes in the core genome. The core type IV effectome of a bacterial species is defined
by the minimum set of type IV effectors conserved in all
strains within a species, which make it necessary for the
bacterium to develop inside the host cell. Using our comparative
genomics approach, we showed that the core type IV effectome
of E. chaffeensis contains 45 candidate T4Es. In addition, we
showed that the Liberty isolate of E. chaffeensis contains all
the 47 predicted T4Es. Although, S4TE software was designed
for optimal sensitivity (Meyer et al., 2013), the prediction of
false positives can occur and is inherent to any predictive
computational approach. A dihydropyrimidine dehydrogenase is the only target of
ECHLIB_RS00920 involved in catabolic and metabolic processes. The last putative nuclear effector with a target is a ligase
(ECHLIB_RS01395), which interacted with a protein associated
with cytochrome c oxidase and had one putative target on the
human genome. This protein plays a role in the organization
of mitochondria, the assembly of cell components and in the
generation of precursor metabolite and energy. Putative Type IV Effectors of E. chaffeensis
are Overrepresented in Gene Sparse
Regions of the Genome chaffeensis genome, linked to its obligate intracellular
lifestyle, thus leading to less functional redundancy in the type
IV effectome. Finally,
four
other
cytoplasmic
E. chaffeensis
T4Es
(ECHLIB_RS01605,
ECHLIB_RS02855,
ECHLIB_RS04290,
ECHLIB_RS01280) were predicted to interact with one or two
proteins involved in reticulum catabolic processes (HERP2),
protein folding (SACS), cytoskeleton organization (INF2) and
transcriptional repression (ARI4B), or in immune response
(antigen processing by HG2A), respectively. Frontiers in Cellular and Infection Microbiology | www.frontiersin.org DISCUSSION We demonstrated that predicted T4Es are preferentially
distributed in gene sparse regions of the genome. In addition,
some putative effectors harbor typical eukaryotic features such
as Ank or BRCT domains. These results suggest that some
effectors could be acquired via HGT from other bacterial species
(McAdam et al., 2014) or from the host cell (Lurie-Weinberger
et al., 2010). To guide the functional characterization of the candidate
T4Es of interest with respect to E. chaffeensis pathogenesis,
we tried to predict some putative host targets. Among the 47
candidate T4Es in E. chaffeensis str. Liberty, most of the proteins
with predicted NLSs were predicted to interact with human
proteins located in the nucleus. Moreover, several putative targets
of candidate T4Es affect human immunity-related proteins. Two predicted T4Es (ECHLIB_RS01550 and ECHLIB_RS01860)
could interact with the nuclear factor NF-kappa-B1. This is a
pleiotropic transcription factor induced by a vast array of stimuli
and which is linked to many biological processes, including
immunity, inflammation, and apoptosis. Another predicted
T4E (ECHLIB_RS01280) may play a role in controlling innate
immune responses by interacting with two human proteins in
particular, ARIA4B and HG2A. The first is a transcriptional
repressor, and the second plays a critical role in MHC class II
antigen processing by stabilizing peptide-free class II alpha/beta
heterodimers in a complex. Suppressing innate immunity of
the host cells is one of the necessary actions for the proper
development of this intracellular bacterium (Luo, 2012). The fact that our analysis of host-interacting proteins revealed
putative targets involved in cell signaling, transcriptional
regulation, and vesicle trafficking is of particular interest in
the context of Ehrlichia pathogenesis. Indeed, recent studies
on the cellular biology of E. chaffeensis infection demonstrated
that some E. chaffeensis type I effectors interact with similar
eukaryotic proteins (Wakeel et al., 2009; Luo et al., 2011). This
reinforces the interest of our approach to identify novel type IV
effectors and to facilitate their functional characterization, but
could also highlight a possible redundancy of action between type
I and type IV effectors of E. chaffeensis for better infection. Other putative T4Es could affect host cell transcription like
ECHLIB_RS01605, which targets two transcriptional repressors:
N42L2 and HERP2. On the other hand, some putative targets
involve the global organization of cell membranes. DISCUSSION At the whole genome
level, some horizontal gene transfer (HGT) of genes that control
advantageous phenotypic differences, might also have occurred
during evolution to explain the differing degrees of virulence
between Wakulla, Liberty and Arkansas isolates of E. chaffeensis
(Dorman et al., 2016). a role in vesicular trafficking (Zimmermann et al., 2001). This
modification of global membrane organization could be related
to the lysosome-like vacuole recruitment in intracellular bacteria,
as shown in C. burnetii (Moffatt et al., 2015). Our analysis of the protein-protein interaction network
also revealed that certain candidate T4Es could alter the
phosphorylation cascades by putatively interacting with protein
kinases (FYN, PTK7, TIE2, KAP2, ATM, MK14), enzymes
which catalyze phosphorylation reactions (Dhanasekaran and
Premkumar Reddy, 1998). Phosphorylation-dephosphorylation
mechanisms are extremely common in signaling pathways where
they regulate cell activity (Dhanasekaran and Premkumar Reddy,
1998). For example, PTK7 is a catalytically inactive receptor
tyrosine kinase which is upregulated in many common human
cancers. Knocking down this protein was shown to inhibit cell
proliferation and induce apoptosis (Meng et al., 2010). MK14
is a serine/threonine kinase, which is an essential component
of the MAP kinase signaling pathway. MK14 is one of the
four p38 MAPKs that play important roles in the cascade of
cell responses induced by extracellular stimuli, such as pro-
inflammatory cytokines or physical stress, leading to direct
activation of transcription factors (Lo et al., 2014). Blocking these
cascades could enable the bacterium to evade the innate immune
response of the host cell. ATM/MKA14 regulatory networks
have also been shown to regulate cytoplasmic targets, resulting
in extensive cytoskeletal rearrangements (Pines et al., 2011). Acting on these cascades could favor the maturation of Ehrlichia-
containing vacuoles, as shown for L. pneumophila which controls
vesicle trafficking to escape host defenses and counteract the
endocytic pathway (Michard et al., 2015). Finally, some candidate
T4Es could affect metabolic proteins, like SYVN1, which acts
as an E3 ubiquitin-protein ligase. Ubiquitination is a post-
traductional biochemical modification that mainly leads to
the degradation of ubiquitinated proteins by the proteasome. Moreover, it has been shown that ubiquitination of proteins in the
endoplasmic reticulum negatively regulates the stress-induced
apoptotic signaling pathway (Kaneko et al., 2002). Interestingly,
we found another candidate T4E predicted to interact with the
SACSIN molecular chaperone, which is highly expressed in the
central nervous system, which regulates HSP70 machinery and
interacts with the proteasome (Parfitt et al., 2009; Anderson et al.,
2011). DISCUSSION Motivated by the availability of eight genome sequences, we
explored the world of pathogenicity determinants in the species
E. chaffeensis. We hypothesized that variations in virulence
between some strains could be driven by genome plasticity and
the acquisition of different repertoires of type IV effectors (T4Es). Such mechanisms of evolution have already been observed in
plant pathogenic and non-pathogenic Xanthomonas (Cesbron
et al., 2015). The aim of our work was to show that computational
methods to identify and categorize putative T4Es, prior to
their functional characterization, could be a valuable approach
to better understand E. chaffeensis-host interactions. We also
aimed to identify novel candidate T4Es and their interactions
with host cell proteins to advance our current understanding of
E. chaffeensis pathogenesis. Interestingly, the E. chaffeensis Liberty strain contained one
specific candidate T4E, ECHLIB_RS02720, a hypothetical protein
exhibiting EPIYA and NLS domains as well as a classical type
IV secretion signal. These features strongly suggest this effector
could be phosphorylated in the cytoplasm, addressed to the We showed that E. chaffeensis genomes had low plasticity and
with few intra-species genomic rearrangements. We also showed
that the eight genomes of E. chaffeensis are highly conserved
with 96% genes present in the core genome. Hence, the observed January 2017 | Volume 6 | Article 204 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 11 Comparative genomics of Ehrlichia chaffeensis Noroy and Meyer nucleus, and play an important role inside the host cell, like the
AnkA effector of A. phagocytophilum (IJdo et al., 2007; Garcia-
Garcia et al., 2009). This effector could also be involved in the
differential virulence phenotypes described between the Arkansas
and Liberty strains in SCID mice (Miura and Rikihisa, 2007). Conversely, the identical putative type IV effectomes of the
Arkansas and Wakulla strains cannot explain their differential
pathogenesis in SCID mice. We cannot exclude the possibility
that the homologous T4Es repertoires of these two strains
contain point mutations in some effectors, which would alter
the pathogenesis of the strain, as shown in L. pneumophila
with the mutant protein kinase LegK2 (Hervet et al., 2011). Another explanation could be differences in the metabolisms or
the kinetics of infection of the Arkansas and Wakulla strains. Indeed, Marcelino et al. showed that virulent and attenuated
Gardel strains of E. ruminantium, which have the same gene
content, only differ in their proteome expression, yet have
different life cycles (Marcelino et al., 2015). Frontiers in Cellular and Infection Microbiology | www.frontiersin.org January 2017 | Volume 6 | Article 204 DISCUSSION Thus, COA6 is
involved in the maturation of the mitochondrial respiratory chain
complex IV; CO1A2 and CO4A1 are involved in the extracellular
membrane by forming fibrillar collagen, with SDCB1 playing In summary, our results are in accordance with the current
knowledge of Ehrlichia molecular pathogenesis (Moumène and
Meyer, 2016), and the T4Es we predicted using the S4TE
algorithm for E. chaffeensis are good candidates for further January 2017 | Volume 6 | Article 204 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 12 Comparative genomics of Ehrlichia chaffeensis Noroy and Meyer We identified 47 candidate T4Es in E. chaffeensis (45 belonging
to the core type IV effectome) with several of the above-
cited features. Some presented homologies with known type IV
effectors in other bacterial systems and others were annotated as
hypothetical proteins with no predicted function. We revealed
one strain to be a specific candidate effector in the Liberty
strain. The majority of predicted T4Es belonged to plastic
regions of the genome. Prediction of protein-protein interactions
between E. chaffeensis T4Es and human proteome revealed
host target proteins that could play a critical role in disease
development. Experimental characterization of E. chaffeensis
candidate T4Es and their targets is now required to confirm
these predictions. Yet, our study is the first to show the
power of comparative effectomics, even in the case of closely
related strains at the intra-species level, in deciphering new
cellular pathways potentially involved in host-Anaplasmataceae
interaction. biological analysis. In addition, the human interactome predicted
via HPIDB provides useful information on the possible mode
of action of these putative T4Es within the host cell. This
study is proof-of-concept that comparative effectomics allows
the identification of important host pathways targeted by the
bacterial pathogen. g
In addition to strain-level variations, allelic diversification
in type IV effectors should be further investigated along with
variations in regulation or protein expression of these genes. Because type IV effector repertoires are suggested to be major
determinants of virulence in Ehrlichia (Moumène and Meyer,
2016), it is also important to understand the diversity of type
IV effectors present in different species that infect common
hosts. Likewise, studying the evolution of type IV effector
repertoires among different bacterial species with different host
ranges or lifestyles could provide key information to identify the
determinants of host specificity. Based on our results, we hypothesize that the evolution
of E. CONCLUSION Using S4TE software, we predicted in silico the putative type IV
effectors from available complete genomes among E. chaffeensis
species. In particular, we searched for proteins with eukaryotic-
like domains, signals for addressing organelles, structural features
known to be involved in protein-protein interactions or type
IV secretion, and homolog to known T4Es in other bacteria. DISCUSSION chaffeensis intra-species pathogenicity occurs via the
acquisition of key regulatory genes. Ultimately, the successive
acquisition of type IV effectors could lead to the adaptation
of new environmental niches—hosts—resulting in a potential
host jump followed by the emergence of new strains in a
dynamic environment. However, functional evidence is still
lacking for many functions that are hypothetically involved in
host specificity. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found
online
at:
http://journal.frontiersin.org/article/10.3389/fcimb. 2016.00204/full#supplementary-material ACKNOWLEDGMENTS The authors acknowledge the financial support from European
project,
FP7-REGPOT-2012-2013-1,
grant
agreement
No. 31598, “EPIGENESIS,” One Health approach to integrate
Guadeloupe research on vector-borne and emerging diseases in
the ERA: From the characterization of emergence mechanisms
to innovative approaches for prediction and control (financial
support for CN). We are grateful to T. Lefrançois and N. Vachiéry
at CIRAD for their confidence and initial input into this project. We thank the reviewers whose insightful comments helped
improve our manuscript. This study, which focused on type IV effector repertoires
in several strains of E. chaffeensis, is a step forward in the
understanding of E. chaffeensis pathobiology. We propose an
original approach with rational targets to enable the design
of alternative therapies for ehrlichiae and other intracellular
pathogens. AUTHOR CONTRIBUTIONS CN and DFM conceived the paper, analyzed the results, and
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conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Copyright © 2017 Noroy and Meyer. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). Zhang, J. Z., Popov, V. L., Gao, S., Walker, D. H., and Yu, X. J. (2007). The
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chaffeensis. J. Clin. Microbiol. 37, 1137–1143. Frontiers in Cellular and Infection Microbiology | www.frontiersin.org January 2017 | Volume 6 | Article 204 Yu, X. J., McBride, J. W., and Walker, D. H. (1999). Genetic diversity of the
28-kilodalton outer membrane protein gene in human isolates of Ehrlichia
chaffeensis. J. Clin. Microbiol. 37, 1137–1143.
Zhang, J. Z., Popov, V. L., Gao, S., Walker, D. H., and Yu, X. J. (2007). The
developmental cycle of Ehrlichia chaffeensis in vertebrate cells. Cell. Microbiol.
9, 610–618. doi: 10.1111/j.1462-5822.2006.00812.x
Zimmermann, P., Tomatis, D., Rosas, M., Grootjans, J., Leenaerts, I., Degeest, G.,
et al. (2001). Characterization of syntenin, a syndecan-binding PDZ protein,
as a component of cell adhesion sites and microfilaments. Mol. Biol. Cell 12,
339–350. doi: 10.1091/mbc.12.2.339 REFERENCES The use,
distribution or reproduction in other forums is permitted, provided the original
author(s) or licensor are credited and that the original publication in this journal
is cited, in accordance with accepted academic practice. No use, distribution or
reproduction is permitted which does not comply with these terms. January 2017 | Volume 6 | Article 204 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 15
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Profile of Students’ Argumentation Ability Based On Adversity Quotient In Statistical Problem
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Journal of Medives : Journal of Mathematics Education IKIP Veteran Semarang/Journal of Medives : journal of mathematics education IKIP Veteran Semarang
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Journal of Medives : Journal of Mathematics Education IKIP Veteran Semarang
Volume 7, No. 1, 2023, pp. 106 - 116
https://doi.org/10.31331/medivesveteran.v7i1.2330
e-ISSN: 2549-5070
p-ISSN: 2549-8231 Journal of Medives : Journal of Mathematics Education IKIP Veteran Semarang
Volume 7, No. 1, 2023, pp. 106 - 116
https://doi.org/10.31331/medivesveteran.v7i1.2330
e-ISSN: 2549-5070
p-ISSN: 2549-8231 e-ISSN: 2549-5070
p-ISSN: 2549-8231 Journal of Medives : Journal of Mathematics Education IKIP Veteran Semarang
Volume 7, No. 1, 2023, pp. 106 - 116
https://doi org/10 31331/medivesveteran v7i1 2330 https://doi.org/10.31331/medivesveteran.v7i1.2330 How to Cite: Putra, Z., Rahardi, R., Sisworo, S., & Permadi, H. (2023). Profile of Students’
Argumentation Ability Based On Adversity Quotient In Statistical Problem. Journal Of Medives
: Journal Of Mathematics Education IKIP Veteran Semarang, 7(1), 106 - 116. *Zuhadur Ra’is Ariyono Putra1, Rustanto Rahardi2, Sisworo3, Hendro Permadi4
1, 2, 3, 4 Universitas Negeri Malang
*zuhad.rais@gmail.com Received: November 2022. Accepted: December 2022. Published: January 2023. INTRODUCTION interpreting; and (4) communication of
results (Chick & Pierce, 2012; Setiawan
& Sukoco, 2021). Statistics is also a
material taught from high school to
university. In the secondary school
curriculum in Indonesia, statistics are
integrated into mathematics, focusing on
the concepts of collecting, processing,
and interpreting observed data. At the
higher education level, statistics stands
alone, thus confirming that statistics has
an a vital role in life, which is not only
limited to theory but directly used in the
final project research. The development of education
always follows the changing times and
needs. The rapid growth and complexity
of today's problems require parallel
skills, known as 21st-century skills (Kim
et al., 2019). The development of the
21st century has given rise to the skills
needed
by
students,
namely
communication skills,
collaboration,
complex thinking, and creativity (Soulé
& Warrick, 2015). 21st-century skills in
schools can be developed through
appropriate learning, namely student
centered learning. Several learnings that
can develop these skills include game-
based, project-based, problem-based,
and argument-based learning (Redhana,
2019). Argumentative ability is crucial
learning mathematics. This is because
one of the indicators that students
understand a mathematical concept is
that students can develop and evaluate
mathematical arguments and evidence
(NCTM,
2000). Argumentation
demonstrates the ability to express
reasons
logically,
accompanied
by
adequate data and theoretical support
both in oral and written form (Soekisno,
2015). As
Toulmin
explains,
argumentation involves a combination of
claims,
data,
warrants,
rebuttals,
qualifications, and backing (Conner et
al., 2014). The
ability
to
overcome
difficulties is one factor that influences a
person's way of thinking when solving
problems. This statement is based on the
results
of
research
conducted
by
(Khumairoh et al., 2020; Sukmaningrum
& Kurniasari, 2022), which found that
the ability to overcome difficulties can
affect a person's reasoning process. Various factors
influence students'
argumentative abilities in dealing with
problems. However,
according
to
(Stoltz, 2000)there is one factor that has
a major influence on a person's success,
namely intelligence in overcoming
issues which are known as the Adversity
Quotient (AQ). According to him, a
person's success living life is determined
by the level of AQ he has. Furthermore
(Hastuti et al., 2018) explained that AQ
is a person's ability to survive in
encountering difficulties and his efforts
to solve problems. Therefore, AQ has the
potential to help students strengthen their
abilities and resilience in dealing with a
problem they have. ABSTRACT Argumentative ability can be seen from the argumentation pattern that appears. This
pattern needs to be evaluated to look over the quality of the argumentation to make the
right problem-solving. This evaluation can be done by recognizing the components that
make up the argument. This study goals to describe students' argumentation abilities in
solving statistical problem based on Adversity Quotients (AQ). This qualitative descriptive
research elaborated 39 students taking a statistical methods course. Subjects were grouped
into three types of Adversity Quotient based on the ARP (Adversity Response Profile)
questionnaire results. Data were obtained using statistical problem tests and interviews. The outcomes showed three levels of AQ found in students, namely Camper, Toward
Climber, and Climber. Camper-type students bring up the Claim-Data-Warrant pattern. Students with AQ levels towards climbers tend to have the same pattern as the Camper
type. In comparison, students with the AQ Climber type have a Claim-Data-Warrants-
Backing pattern. Based on the outcomes of the study, it can see that students'
argumentation skills are determined by the Adversity Quotient level they have when solving
statistical problems. The discussions about how to improve the quality of argumentation
can be the subject of further research in the future Keywords: argumentative ability, adversity quotient, statistical problem How to Cite: Putra, Z., Rahardi, R., Sisworo, S., & Permadi, H. (2023). Profile of Students’
Argumentation Ability Based On Adversity Quotient In Statistical Problem. Journal Of Medives
: Journal Of Mathematics Education IKIP Veteran Semarang, 7(1), 106 - 116. 107 Zuhadur Ra’is Ariyono Putra, Rustanto Rahardi, Sisworo, Hendro Permadi - Profile Of Students’ Argumentation Ability … METHOD climbers,
campers,
and
quitters
(Abdiyani et al., 2019). People with the
climber’s type tend to choose to keep
fighting and don’t give up easily in the
face of various kinds of problems, so the
results obtained are better than people of
the campers type (Septianingtyas &
Jusra, 2020). People with the campers
type tend to be easily satisfied with what
has been achieved, so the results are not
optimal. Meanwhile, people with the
quitter’s type tend not to be enthusiastic
about solving a problem and even give
up before trying, so the results obtained
are also unpredictable (Kartikaningtyas
et al., 2018). From the description above,
it can conclude that the Adversity
Quotient is an ability that a person has to
observe difficulties and processing them
with their intelligence so that they
become a challenge to be solved. This research is a qualitative
descriptive study with the aim of
obtaining an overview of students'
mathematical arguments based on their
adversity quotient in statistics material in
the form of a case study. The subjects of
this study were 39 students of the
Mathematics Education Study Program
at Malang state university who took
methods of statistics lesson. Selection of
subjects using sampling with maximum
variation, namely looking for samples of
cases or individuals with differences in
characteristics or traits (Moleong, 2009). The differences referred to in this
study are based on the diversity and
correctness of the methods used by
students based on the level of adversity
quotient they have, namely the types of
climbers, campers, and quitters. The
instruments in this study were 1) an
argumentation test on statistical material
in the amount of one question adopted
from (Riki Andriatna & Kurniawati,
2021). This question contains a critical
question, where according to (Gal,
2005)this aspect can emphasize a
person's ability to criticize statistical
information
related
to
data
and
arguments. 2) AQ questionnaire that
measures the level of AQ found in
students. The questionnaire was adopted
from
(D. F. Hidayat,
2020)The
instrument
adopted
from
previous
research has been tested for validity and
reliability, so repeated tests for this
component are no longer needed. Much research has been carried
out regarding exploring one's arguments
in the field of mathematics (Conner et
al., 2014; Indrawati & Febrilia, 2019;
Pramesti & Rosyidi, 2020). INTRODUCTION Statistics is one of the materials in
mathematics that requires the integration
of argumentation skills in learning. This
is because learning statistics has the
following
competencies:
(1)
understanding the importance of data
and the basic concepts of statistics; (2)
collecting and describing data; (3) The adversity quotient (AQ) level
is classified into three categories: 108 | 108 Zuhadur Ra’is Ariyono Putra, Rustanto Rahardi, Sisworo, Hendro Permadi - Profile Of Students’ Argumentation Ability … METHOD However,
previous research was minimal, which
raised statistical material in assessing
argumentation abilities and further
examined the significance of differences
in argumentation abilities based on
students' adversity quotients. Based on
the description above, this study goals to
describe the argumentation abilities of
prospective teacher students based on
their adversity quotient in statistical
problem. 109 109 | Zuhadur Ra’is Ariyono Putra, Rustanto Rahardi, Sisworo, Hendro Permadi - Profile Of Students’ Argumentation Ability … e. Interpret the possible elements of
an
argument
and
write
a
reconstructed argument like the
following image. Figure 2. Toulmin's Argumentation Model Figure 1. Statistical Problem Test Table 1. AQ Categorization Based on
(Z. R. A. Putra & Oktaviane, 2022) Table 1. AQ Categorization Based on
(Z. R. A. Putra & Oktaviane, 2022) Figure 2. Toulmin's Argumentation Model No
Adversity
Response Profile
Score
Adversity
Quotient
category
1
69-80
Climbers
2
44-55
Campers
3
20-31
Quitters Data analysis technique refers to
(Miles, M. B., Huberman, A. M., &
Saldaña, 2018), which contains data
reduction,
data
presentation,
and
conclusion and verification. At the data
reduction stage, the authors make
reductions to getting the right data or
information to conclude. At stage data
presentation, the author analyzes the data
or information obtained from the first
stage to present it in a table or other so
that the writer can describe the data
accurately brought. The last stage is the
conclusion to look for meaning and
explanation. The validity of the data is
done by triangulation theory. Then, for analyzing argumentation
abilities in this study, it refers to the
Toulmin argumentation model, as shown
in Figure 2 (Conner et al., 2014), which
is as follows. a. Identifying claims by recording
statements in the written test as
well
as
the
results
of
subject interviews. RESULT AND DISCUSSION b. Look for possible data to support
each claim. Adversity
Analysis c. Look for warrants that show how
the data relate to a particular
claim. If no warrant can be
identified, the claim is not part
of the research because it is not
considered an argument. The table below shows the selected
subjects based on the results of the
Adversity
Response
Profile
(ARP)
questionnaire: d. Looking for possible backing for
the warrants 110 | Zuhadur Ra’is Ariyono Putra, Rustanto Rahardi, Sisworo, Hendro Permadi - Profile Of Students’ Argumentation Ability … Figure 5 Warrants of PAP Subjects Table 2 Selected Subjects Based on ARP Results
No
Subject
Code
ARP
Score
AQ
Category
Code
1
PAP
53
Campers
S-Ca
2
AAJ
60
Toward
Climbers
S-
MC
3
LAS
73
Climbers
S-Cl
Analysis of the Argumentative Ability
of Prospective Teacher Students in the
AQ Campers (S-Ca) Category Figure 5 Warrants of PAP Subjects Figure 5 Warrants of PAP Subjects To clarify the written results of the
PAP subject, the following interviews
were conducted. Researcher: "Try to briefly explain your
steps in responding to the DPS
Director's statement!"
S-Ca: " So it's like this, first I first record
the
information
contained
in
the
problem. It is known that the problem
shows a bar chart of the percentage of
accidents in 5 years, as well as the DPS
director's statement that 15% of the
percentage of accidents that occur. After
that I calculated the average percentage
that occurred in 5 years, and got a result
of 12.8%. From the results of my
calculations and the director's statement
it's not the same. So I can conclude that
the DPS director's statement is invalid." Analysis of the Argumentative Ability
of Prospective Teacher Students in the
AQ Campers (S-Ca) Category Analysis of the Argumentative Ability
of Prospective Teacher Students in the
AQ Campers (S-Ca) Category Analysis of the Argumentative Ability
of Prospective Teacher Students in the
AQ Campers (S-Ca) Category The claim (in this case, the answer
to the problem) made by the subject of
the PAP, namely the statement of the
DPS director, is invalid. The claim is
shown in Figure 3 below. Figure 3. Claims of PAP Subjects Based on the results of the PAP
subject's written test, data was obtained
to support the claim: the percentage of
accidents each year and statements from
the DPS director. Figure 4 below is the
data used by the PAP. Based on the written results and
interviews, initially, the PAP subject
collected existing information on the
problems given. The details of the data
are rewritten in the arguments presented. After getting the current information,
PAP calculates the average of the
existing data. After obtaining the
average, the PAP subject compares the
computed results with the statements in
the problem. It was found that there was
a discrepancy between the calculation
results and the information given. Therefore the subject of the PAP
submitting a claim for the distinction
was that the DPS director's statement
was invalid. Figure 4 PAP Subject Data Figure 4 PAP Subject Data While PAP uses warrants as a
bridge between data and claims in the
form of an average data concept. Figure
5 shows the written result of the PAP
subject showing the warrant. 111 | Zuhadur Ra’is Ariyono Putra, Rustanto Rahardi, Sisworo, Hendro Permadi - Profile Of Students’ Argumentation Ability … Based on Toulmin's model, the
following summarizes the arguments for
the subject of PAP. Figure 7 AAJ Subject Claims Figure 6 Summary of DPS Subject Arguments Figure 7 AAJ Subject Claims There are two claims given,
because in the arguments given the AAJ
subject examined the DPS director gave
two statements. Figure 6 Summary of DPS Subject Arguments The chart above
shows the
argumentation patterns of students with
AQ-level campers. This is in line with
research (Aaidati et al., 2022)where
students in the camper category have
patterns of claims, data, and warrants
arguments. In solving problems students
do not fully pay attention to any
information contained in the problem,
and do not fully use the concepts and
knowledge they have before. This is in
accordance with what is stated (R. P. Analysis of the Argumentative Ability
of Prospective Teacher Students in the
AQ Campers (S-Ca) Category Putra et al., 2022; Stoltz, 2000),
explained that someone with AQ
campers category will try to face
challenges but only up to a certain level
then stop when they are satisfied enough
whit what they have got even though it is
not optimal. The data in the question support
AAJ's claim. Here the subject only
mentions the data without rewriting it. Figure 8 AAJ Subject Data Figure 8 AAJ Subject Data Meanwhile, the warrant used by
the AAJ subject was a comparison of the
compatibility between the statements
given by the director of DPS and the data
in the problems given. For the first
statement, the subject assumes that there
is a contradiction and for the second
statement there is compatibility. Figure 9 AAJ Subject Warrants Analysis of the Argumentative Ability of
Prospective Teacher Students in the AQ
Category Towards Climbers (S-MC) Analysis of the Argumentative Ability of
Prospective Teacher Students in the AQ
Category Towards Climbers (S-MC) Figure 9 AAJ Subject Warrants The following are the results of the
AAJ subject's mathematical argument
analysis. AAJ's subject claim (in this
case, the answer to the given problem) is
regarding the statement given by the
DPS director. The claim is shown in
Figure 7 below. The following interviews were
conducted to confirm the AAJ subject’s
written results. Researcher: "Do you give 2 conclusions
in this case? Try to explain the strategy
you used in responding to the DPS
Director's statement!” 112 | Zuhadur Ra’is Ariyono Putra, Rustanto Rahardi, Sisworo, Hendro Permadi - Profile Of Students’ Argumentation Ability … Based on Toulmin's model, the
following is a summary of AAJ's subject
arguments. Figure 10 Summary of AAJ Subject Arguments Based on Toulmin's model, the
following is a summary of AAJ's subject
arguments. S-MC : " Yes, the reason I answered
that way is because I referred to the data
in the question, namely the percentage of
accidents in 5 years and the DPS
director's statement. I separated the DPS
director's statement into two, namely: Figure 10 Summary of AAJ Subject Arguments 1. Commercial vehicles are responsible
for about 15% of accidental deaths in
Texas. - From that statement, if I refer to the
data, the statement will be false because
it does not match the data or there is no
correlation where the data shows the
percentage
of
accidents
involving
commercial
vehicles
in
Texas. Meanwhile, the statement from the DPS
director of commercial vehicles is
responsible for deaths due to accidents. 2. Those who use unsafe commercial
vehicles or drive commercial vehicles
unsafely pose a serious threat to traffic. - From this statement, I conclude that the
statement is true because the statement
can be the cause of accidents involving
commercial vehicles which logically fits
the data because the statements above
are synonymous with data on the
percentage
of
accidents
involving
commercial vehicles in Texas." Figure 10 Summary of AAJ Subject Arguments Even though in concluding the
problems given, the subject gave two
claims, the argument pattern of students
with AQ levels towards climbers is
Claims-Data-Warrants. Students have
been careful in reviewing the claims
presented by the DPS director, but they
need to be more careful in utilizing the
information provided in the questions. This is in line with (R. P. Analysis of the Argumentative Ability of
Prospective Teacher Students in the AQ
Category Towards Climbers (S-MC) Putra et
al., 2022; Stoltz, 2000)where students
who are included in the category of
climbers are people who may have
survived enough to penetrate challenges
and take advantage of most of their
potential that develops every day. So it
can solve the problem well, but there are
some solutions that need to be corrected
because students need to be more careful
in solving problems. Based on the written results and
interviews, the AAJ subject did not write
down the data he used in his argument. But what he uses as data in his argument
is the percentage of accidents in 5 years
that exist in the problems given. After
that, AAJ observed that the DPS director
made two statements. From these
statements, AAJ compares the keywords
between the statements given and the
existing data. Statement 1 is considered
wrong because there is no match
between the words death and accident. Then statement 2 is considered accurate
because of the causality of the statement
with the data. Analysis of the Argumentative Ability of
Prospective Teacher Students with the
AQ Climbers (S-Cl) Category Following are the results of the
LAS subject argument analysis. LAS
subject claim (in this case the answer to
the given problem) i.e., agree with the
statement given by the DPS director. The
claim is shown in the following figure. 113 | Zuhadur Ra’is Ariyono Putra, Rustanto Rahardi, Sisworo, Hendro Permadi - Profile Of Students’ Argumentation Ability … Figure 11 LAS Subject Claims the problem I was looking for averaged
12.8% then in the last year on the graph
(2010) there was a large increase,
therefore there is a high probability of an
increase in the following year. In
addition, there is a statement in the
question that prohibits 1 in 5 vehicles
from operating in 2012, which is several
years after the existing graphic data. This confirms that 15% is legitimate and
that the vehicle did have a role in the
accident.” Figure 11 LAS Subject Claims LAS claims are supported by data
showing the average percentage of
accidents in Texas in the last five years. The data used by the subject is shown in
the following figure. Figure 12 LAS Subject Data LAS claims are supported by data
showing the average percentage of
accidents in Texas in the last five years. The data used by the subject is shown in
the following figure. Figure 12 LAS Subject Data LAS claims are supported by data
showing the average percentage of
accidents in Texas in the last five years. The data used by the subject is shown in
the following figure. Figure 12 LAS Subject Data Based on the written results and
interviews above, a summary of the LAS
subject arguments can be summarized in
the Toulmin model as follows: LAS uses warrants in the form of
its observations of increasing data
patterns. The following image shows the
LAS
subject's
written
results
demonstrating the warrant. Figure 13 LAS Subject Warrants LAS uses warrants in the form of
its observations of increasing data
patterns. The following image shows the
LAS
subject's
written
results
demonstrating the warrant. Figure 13 LAS Subject Warrants Figure 14 Summary of LAS Subject Arguments Figure 13 LAS Subject Warrants Figure 13 LAS Subject Warrants After that, LAS added backing to
support the warrants granted. The
backing presented by the subject was
information contained in the question
regarding 1 out of 5 vehicles not
permitted in 2012. Analysis of the Argumentative Ability of
Prospective Teacher Students with the
AQ Climbers (S-Cl) Category This backing is
indeed not visible from the written
results of the LAS but can be
demonstrated by LAS's explanation in
the following interview. Figure 14 Summary of LAS Subject Arguments Based on the chart above, the
pattern of student argumentation at the
climbers level is Claim-Data-Warrant-
Backing. Compared to the two students
with the AQ level below, it can be seen
that there is a difference in the pattern of
argumentation of students with the AQ
Climber level. This is following research
by (W. Hidayat et al., 2018)that students
with AQ climber levels have more
developed argumentation patterns. In the
process, students use the concepts and
knowledge
they
have
previously
acquired to solve the problems given. Then from the answers and interviews,
the students looked very careful and To clarify the written results of the
LAS subject, the following interview
was conducted. Researcher: "Try to briefly explain your
steps in responding to the DPS
Director's statement!"
S-Cl : " I agree, with the statement given
by the DPS director that commercial
vehicles are responsible for 15% of
existing accidents. The data shown by Researcher: "Try to briefly explain your
steps in responding to the DPS
Director's statement!" S-Cl : " I agree, with the statement given
by the DPS director that commercial
vehicles are responsible for 15% of
existing accidents. The data shown by 114 Zuhadur Ra’is Ariyono Putra, Rustanto Rahardi, Sisworo, Hendro Permadi - Profile Of Students’ Argumentation Ability … Kemampuan Pemecahan Masalah
Matematika Siswa SMP Negeri 1
Jogoroto Berdasarkan Langkah-
langkah
Polya
Ditinjau
dari
Adversity Quotient. Al-Khwarizmi:
Jurnal
Pendidikan
Matematika
Dan Ilmu Pengetahuan Alam, 7(2),
123–134. https://doi.org/10.24256/jpmipa.v7
i2.774 maximal in
solving problems,
as
evidenced by the more complete results
of students' mathematical arguments. In
line with this, (Stoltz, 2000) suggests
that
people
with
the
climber’s
personality type are very persistent,
conscientious and never give up. She
determination is to strive to the pinnacle
of success. She will continue to try his
best to achieve the set targets. Chick, H. L., & Pierce, R. (2012). TEACHING FOR STATISTICAL
LITERACY:
UTILISING
AFFORDANCES
IN
REAL-
WORLD
DATA. International
Journal
of
Science
and
Mathematics
Education,
10(2),
339–362. https://doi.org/10.1007/s10763-
011-9303-2 CONCLUSION This study found that prospective
teacher students had AQ levels of
Camper, Towards a Climber, and
Climber. For student argumentation
patterns with camper AQ levels are
Claims-Data-Warrants. While
the
argumentation pattern of students with
AQ levels towards climbers is Claim-
Data-Warrants, this pattern tends to be
the same as campers. Then for the
argumentation pattern of the climber
type student is Claim-Data-Warrant-
Backing. The results showed contrast in
the
quality
of
the
argumentation
structure
based
on
the
Adversity
Quotient level when solving a problem. Differences in quality can be caused by
various factors and discussions about
how
to
improve
the
quality
of
argumentation can be the subject of
further research in the future. Conner, A., Singletary, L. M., Smith, R. C., Wagner, P. A., & Francisco, R. T. (2014). Teacher support for
collective
argumentation:
A
framework for examining how
teachers
support
students’
engagement
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mathematical
activities. Educational Studies in
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86(3),
401–429. https://doi.org/10.1007/s10649-
014-9532-8 Gal, I. (2005). Statistical literacy:
Meanings,
components,
responsibilities, the challenge of
developing
statistical
literacy. Reasoning and Thinking, 1, 47–78. Hastuti, T. D., S, D. R. S., & Riyadi. (2018). Student profile with high
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https://openalex.org/W2389712248
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https://europepmc.org/articles/pmc5400534?pdf=render
|
English
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Differential DNA methylation patterns of polycystic ovarian syndrome in whole blood of Chinese women
|
Oncotarget
| 2,016
|
cc-by
| 6,589
|
Differential DNA methylation patterns of polycystic ovarian
syndrome in whole blood of Chinese women Shuxia Li1,*, Dongyi Zhu2,3,*, Hongmei Duan4, Anran Ren2,3, Dorte Glintborg5, Marianne
Andersen5, Vibe Skov6, Mads Thomassen1, Torben Kruse1, Qihua Tan1,7
1Unit of Human Genetics, Department of Clinical Research, University of Southern Denmark, Odense, Denmark
2Center of Reproductive Medicine, Linyi People’s Hospital, Linyi, China
3Department of Obstetrics and Gynecology, Shandong Medical College, Linyi, China
4Department of Medicine, Kolding Hospital, Kolding, Denmark
5Department of Endocrinology, Odense University Hospital, Odense, Denmark
6Department of Hematology, Roskilde Hospital, Roskilde, Denmark
7Epidemiology, Biostatistics, and Biodemography, Department of Public Health, University of Southern Denmark, Odense,
Denmark 7Epidemiology, Biostatistics, and Biodemography, Department of Public Health, University of Southern Denmark, Odense,
Denmark 7Epidemiology, Biostatistics, and Biodemography, Department of Public Health, University of Southern Denmark, Odense,
Denmark *These authors contributed equally to this work *These authors contributed equally to this work Correspondence to: Qihua Tan, email: qtan@health.sdu.dk Correspondence to: Qihua Tan, email: qtan@health.sdu.dk
Keywords: polycystic ovarian syndrome, DNA methylation, genome-wide association study, clinical heterogeneity
Received: January 26, 2016 Accepted: April 26, 2016 Published: May 12, 2016 Keywords: polycystic ovarian syndrome, DNA methylation, genome-wide association study, clinical heterogeneity
Received: January 26, 2016 Accepted: April 26, 2016 Published: May 12, 2016 Keywords: polycystic ovarian syndrome, DNA methylation, genome-wide association study, clinica
Received: January 26, 2016 Accepted: April 26, 2016 Published: May 12, 2016 ABSTRACT As a universally common endocrinopathy in women of reproductive age, the
polycystic ovarian syndrome is characterized by composite clinical phenotypes
reflecting the contributions of reproductive impact of ovarian dysfunction and metabolic
abnormalities with widely varying symptoms resulting from interference of the genome
with the environment through integrative biological mechanisms including epigenetics. We have performed a genome-wide DNA methylation analysis on polycystic ovarian
syndrome and identified a substantial number of genomic sites differentially methylated
in the whole blood of PCOS patients and healthy controls (52 sites, false discovery
rate < 0.05 and corresponding p value < 5.68e–06 ), highly consistently replicating
biological pathways extensively implicated in immunity and immunity-related
inflammatory disorders (false discovery rate < 0.05) that were reportedly regulated
in the DNA methylome from ovarian tissue under PCOS condition. Most importantly,
our genome-wide profiling focusing on PCOS patients revealed a large number of DNA
methylation sites and their enriched functional pathways significantly associated with
diverse clinical features (levels of prolactin, estradiol, progesterone and menstrual
cycle) that could serve as novel molecular basis of the clinical heterogeneity observed
in PCOS women. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget/ www.impactjournals.com/oncotarget/ Epigenetic association with PCOS We first performed EWAS for single CpGs
(Manhattan plot shown in Supplementary Figure S1A). After correction for multiple testing, a total of 699 CpGs
(13 X-linked) were found with false discovery rate (FDR) <
0.20 with corresponding p value < 3.05e-04 (Supplementary
Table S1), among them 52 CpGs (1 X-linked) with FDR
< 0.05 with corresponding p value < 5.68e-06. As shown
in Supplementary Table S1, the mean methylation levels of
significant CpGs range from low to high but are dominated
by sites of high DNA methylation levels. Figure 1 is a
volcano plot displaying p value (in log scale) plotted against
corresponding difference in the mean methylation levels
between PCOS patients and controls. The coloured spots
represent 699 CpGs with FDR < 0.2, among them the red
spots stand for the 52 genome-wide significant CpGs with
FDR < 0.05 in Supplementary Table S1. The figure displays
the significance level for hyper- and hypo-methylated CpGs
without a predominant pattern of increased or decreased
methylation in patient or control group. The figure also
shows a symmetric pattern although the top significant
CpGs tend to be hypermethylated (i.e. increased in mean
methylation level) in the patient group. Both Supplementary
Table S1 and Figure 1 show that the significant CpGs are
those with only small differences in their DNA methylation
levels between the two groups. This paper reports our recent epigenome-wide
association study (EWAS) on a relatively large sample
size of 30 PCOS patients and 30 age-matched healthy
controls. We present results from analysis on single CpG
sites (5′—C—phosphate—G—3′, cytosine and guanine
separated by one phosphate) followed by findings on
enriched biological pathways significantly associated
with PCOS condition. Furthermore, we report our novel
analysis in associating genomic DNA methylation
with levels of reproductive hormones in PCOS patients
including estradiol (E2), luteinising hormone (LH), follicle
stimulating hormone (FSH), progesterone (P), thyroid
stimulating hormone (TSH), prolactin (PRL), testosterone
(TST), which could reveal the molecular basis of the
observed clinical heterogeneity in PCOS patients. g
p
Based on the EWAS results, we conducted a gene-
set enrichment analysis (GSEA, see Methods section)
on the 273 genes linked to the 699 significant CpGs in
Supplementary Table S1. A total of 22 functional pathways
were significantly enriched with FDR < 0.05 (Table 2). INTRODUCTION environmental contributions [3]. For example, a Dutch
twin study [4] estimated a high genetic component in
PCOS with a heritability estimate of over 60%. Moreover,
previous genetic studies identified multiple genomic
loci associated PCOS [5–6]. Meanwhile, animal studies
provided evidence that prenatal exposure to excessive
androgen induced similar phenotypes to PCOS [7–9]
and ovarian dysfunction suggesting the important role of
environment in PCOS pathogenesis. The multifactorial
nature of PCOS calls for biological functional studies at
molecular level to elucidate the integrative mechanisms in
the development of PCOS. Polycystic ovarian syndrome (PCOS) is a common
endocrinological disorder that affects about 10% women
of reproductive age [1–2]. The syndrome has a complex
mode of inheritance, in which genomic variants interfere
with important environmental factors, including diet,
life style, leading to heterogeneous expression of the
syndrome characterized by chronic anovulation or
infrequent ovulation, obesity, hirsutism, hyperandrogenism
and polycystic ovaries. As a complex disorder the
pathophysiology of PCOS involves both genetic and www.impactjournals.com/oncotarget Oncotarget 20656 Epigenetics focuses on molecular mechanisms in
the regulation of gene expression not caused by DNA
sequence variation. It represents a new frontier in functional
genomics of complex diseases and serves as a potential
molecular bridge linking the environment to the genetic
materials. Among the various mechanisms of epigenetic
regulation, genomic DNA methylation patterns have been
widely analysed to investigate the molecular basis of
complex disorders mediated by genetic and environmental
factors. In the literature, genome-wide association analyses
have been performed on PCOS patients and controls by
comparing DNA methylation levels between the two
groups measured using high-throughput techniques
[10–13]. However, results from the different genome-
wide analyses have been highly inconsistent. For example,
while Xu et al. [10] reported no significant difference
in the DNA methylome of peripheral blood cells of 20
PCOS patients and 20 controls, multiple differentially
methylated genes were identified by Shen et al. [11] in peripheral blood from even a smaller sample size. By
targeting the ovarian tissue, large numbers of differentially
methylated genes were found by both Wang et al. [12]
and Yu et al. [13] in their small studies. Nonetheless, the
detected genes from each study were associated with
different molecular functions even though both studies were
conducted on the ovarian tissues. The situation calls for well-
designed studies on relatively large sample sizes to validate
and update current findings to look for novel genomic sites
and biological pathways associated with PCOS. RESULTS Table 1 presents the basic statistics for both PCOS
and control samples on phenotypes of interest including
anthropometric measurements: weight, height, body mass
index (BMI), waist and hip circumference, waist-hip-
ratio (WHR); blood pressure: systolic and diastolic blood
pressure (SBP, DBP); menstrual cycle (MC); reproductive
hormones: E2, LH, FSH, P, TSH, PRL and TST; and
metabolic variables: fasting immunoreactive insulin (IRI),
immunoreactive insulin at 2 hours after ingestation of
75 gram dextrose (IRI2), fasting blood glucose (GLU), INTRODUCTION blood glucose at 2 hours after ingestation of 75 gram
dextrose (GLU2), homeostatic model assessment of insulin
resistance (HOMA-IR). Highly significant differences
were found for MC, LH and TST between the two groups;
statistical or borderline differences were also found for
WHR (p = 0.037), IRI2 (p = 0.026) and GLU2 (p = 0.031),
all with higher levels in PCOS patients than in controls. Epigenetic association with PCOS Among the 22 pathways, the top significant ones are
mainly those involved in immune and inflammatory
processes with the rest pertaining to biological processes
including metabolism of proteins and carbohydrates. There are in total 4 pathways with FDR < 0.01(the very
top of Table 2), all are involved in immunity (intestinal
immune network for IgA production; asthma; O-Glycan
biosynthesis) and inflammation (viral myocarditis). Epigenetic association with clinical features in
PCOS patients In addition to comparing DNA methylation between
PCOS patients and controls, we also conducted EWAS on the
30 PCOS patients for their clinical features including BMI, MC,
reproductive hormones (E2, LH, FSH, P, TSH, PRL, TST),
and metabolic variables (IRI, IRI2, GLU, GLU2, HOMA- www.impactjournals.com/oncotarget Oncotarget 20657 Table 1: Descriptive statistics of PCOS and control samples
PCOS, n = 30
Control, n = 30
Median
2.5%
97.5%
Median
2.5%
97.5%
P value
Age, year
25
23
30
27
24
31
Weight, kg
61
47.5
116.9
63
47.8
86
0.90
Height, cm
160
149.8
175.3
160
154.3
168.3
0.19
BMI, km/m2
23
19.4
38
23.4
18.1
33.6
0.67
Waist, cm
80
56.1
114.8
81
66.3
101.5
0.75
Hip, cm
97
84.8
123.3
98
88.8
115
0.68
WHR, %
83.7
75.7
102.8
81.1
72.7
94.5
0.04
SBP, mmHg
110
100
130
110
90
132.5
0.95
DBP, mmHg
70
60
83.8
70
66
90
0.07
MC, day
75
28
407
30
25
35
7.09e–10
E2, pg/ml
49.9
13.8
151.5
45.5
27.9
115.9
0.38
LH, mIU/ml
14.1
3
25
4.4
1.6
12
5.12e–08
FSH, mIU/ml
6.4
4.8
8.9
6.3
3.4
11.1
0.63
P, ng/ml
0.4
0.2
1.1
0.4
0.1
0.9
0.99
TSH, uIU/ml
1.8
0.5
5.4
1.7
0.6
4.7
0.79
PRL, ng/ml
10.4
4
55
11.3
5.8
27.2
0.97
TST, ng/dl
50.6
6.2
94.7
31.4
7.6
62.8
3.30e–04
IRI, uIU/ml
15.3
2.1
51.6
10.5
5.6
27.3
0.11
IRI2, uIU/ml
66.4
10.4
300
46.5
13.7
196.5
0.03
GLU, mmol/l
5.2
4.8
5.7
5.3
4.8
5.9
0.31
GLU2,
mmol/l
6.7
4.5
8.8
6.0
3.5
8.4
0.03
HOMA-IR
3.5
0.5
12.4
2.3
1.3
6.2
0.15
Abbreviations: WHR: waist to hip ratio; SBP: systolic blood pressure; DBP: diastolic blood pressure; MC: menstrual cycle; E2:
t di l LH l t i i i
h
FSH f lli l
ti
l ti
h
P
t
TSH th
id ti
l ti
h
PRL Table 1: Descriptive statistics of PCOS and control samples Abbreviations: WHR: waist to hip ratio; SBP: systolic blood pressure; DBP: diastolic blood pressure; MC: menstrual cycle; E2:
estradiol; LH: luteinising hormone; FSH: follicle stimulating hormone; P: progesterone; TSH: thyroid stimulating hormone; PRL:
Prolactin; TST: Testosterone; IRI: immunoreactive insulin; IRI2: IRI at 2 hours after ingestion of 75 g dextrose; GLU: glucose;
GLU2: GLU at 2 hours after ingestion of 75 g dextrose; HOMA-IR: homeostatic model assessment of insulin resistance. IR). Epigenetic association with clinical features in
PCOS patients Multiple CpGs reaching genome-wide significance
(FDR < 0.05) were found for E2 (87 CpG sites, corresponding
p value < 8.36e-06, Supplementary Table S2); for PRL
(199 CpG sites, corresponding p value < 2.02e-05,
Supplementary Table S3); and borderline significant for P
(3 CpG sites, FDR = 0.06, corresponding p value < 4.30e-
07, Supplementary Table S4). Only one CpG was found to
show genome-wide significance for menstrual cycle (1 CpG
site, cg08916385 on chromosome 4 near gene GNRHR,
p = 5.09e-10, FDR = 2.47e-04). Manhattan plots for E2, PRL
and P are shown in Supplementary Figure S1B, S1C and S1D
respectively. In Supplementary Figure S1C, CpGs in the HLA
(human leukocyte antigen) region of chromosome 6 are highly
associated with PRL in PCOS patients. This is more clearly
illustrated by the Manhattan plot for chromosome 6 with
the HLA region highlighted with red colour (Figure 2). No
genome-wide significant association was found for the other
clinical features. Genes linked to CpGs in Supplementary Tables S2–
S4 were submitted to GSEA to look for gene-sets
enriched. Significant pathways were found for E2 (2
pathways) and PRL (10 pathways) (Table 3) with no
significantly enriched pathways for progesterone. The
2 significant functional pathways for E2 include steroid
hormone biosynthesis and metabolism of xenobiotics
by cytochrome P450 while the 10 pathways enriched
for PRL are dominated by immunity and inflammation
processes which largely overlap with the pathways in
Table 2. DISCUSSION Although there have been sizable genome-wide
studies reporting significant associations between genetic
variations and PCOS, [5, 6, 14–17] the regulatory patterns
in the molecular pathogenesis of PCOS has been, to www.impactjournals.com/oncotarget Oncotarget 20658 date, rarely investigated with only a handful epigenomic
studies performed on small sample sizes [10–13]. We have
conducted a larger EWAS on DNA methylation in whole
blood of 30 PCOS patients and 30 controls. In contrast to
previous studies, we engaged a stringent adjustment for
multiple testing in our EWAS and were able to identify
multiple CpGs reaching genome level significance for
their association with PCOS or with clinical features in
PCOS patients. Meanwhile, results from our single site
analysis also implicate biological pathways that either
reconfirming previous studies or representing novel
findings. i in the top ten pathways from both studies although each
targeting at a very different tissue type (whole blood
versus ovarian tissue). While these results are strongly
confirmatory and supportive, the high conformity also
provides further evidence that the easy-to-access whole
blood could serve as a useful surrogate to hard-to-access
tissues like ovary to enable non-invasive large scale
epigenetic studies on human diseases [18].i Many of the significant pathways in Table 2 are
enriched by genes pertaining to immunity (e.g. adaptive
immune system, class I MHC mediated antigen processing
and presentation), or biological pathways directly related
to certain diseases including inflammatory diseases (viral
myocarditis, asthma, Leishmania infection), autoimmune
diseases (type 1 diabetes mellitus, systemic lupus
erythematosus, autoimmune thyroid disease) and immune
reaction (allograft rejection, graft versus host disease). The
predominant involvement of immunity-related biological
pathways emphasizes the crucial role of inflammation
and immune reaction in the pathogenesis of PCOS. By
comparing the nationwide Danish population of PCOS
with a large control group, Glintborg et al. [19] recently
reported a significantly increased prevalence for diseases Although our genome-wide epigenetic profiling
was targeted at whole blood DNA, results are however
surprisingly highly consistent with a previous study
using ovarian tissue [12]. Among the top ten significantly
enriched pathways in this study (Table 2), six overlap with
the pathways found by Wang et al. [12]. Likewise, in their
list of top ten most significant pathways, five can be found
in our 22 functional pathways listed in Table 2. It is even
more interesting to see that, there are four pathways, i.e. www.impactjournals.com/oncotarget DISCUSSION viral myocarditis, allograft rejection, graft versus host
disease and type I diabetes mellitus, appearing consistently Figure 1: A volcano plot for the negative log10-transformed p values plotted against the difference in the mean levels
of DNA methylation at each CpG site between PCOS and controls groups. CpGs reaching genome-wide significance with
FDR < 0.05 are coloured red and those with 0.05 < FDR < 0.2 coloured purple. Figure 1: A volcano plot for the negative log10-transformed p values plotted against the difference in the mean levels
of DNA methylation at each CpG site between PCOS and controls groups. CpGs reaching genome-wide significance with
FDR < 0.05 are coloured red and those with 0.05 < FDR < 0.2 coloured purple. Oncotarget DISCUSSION www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 20659 Oncotarget
20660
www.impactjournals.com/oncotarget
Table 2: The 22 functional pathways enriched (FDR < 0.05) by genes linked to CpG sites in
Supplementary Table S1
Gene Set Name [# Genes (K)]
Description
# Genes in Overlap (k)
p-value
FDR (q-value)
KEGG: VIRAL
MYOCARDITIS [73]
Viral myocarditis
6
5.07 e−6
6.59 e−3
KEGG: INTESTINAL
IMMUNE NETWORK FOR
IGA PRODUCTION [48]
Intestinal immune network
for IgA production
5
9.91 e−6
6.59 e−3
KEGG: ASTHMA [30]
Asthma
4
2.96 e−5
9.83 e−3
KEGG: O–GLYCAN
BIOSYNTHESIS [30]
O–Glycan biosynthesis
4
2.96 e−5
9.83 e−3
KEGG: LEISHMANIA
INFECTION [72]
Leishmania infection
5
7.21 e−5
1.46 e−2
REACTOME: METABOLISM
OF PROTEINS [518]
Genes involved in
Metabolism of proteins
12
7.33 e−5
1.46 e−2
KEGG: ALLOGRAFT
REJECTION [38]
Allograft rejection
4
7.67 e−5
1.46 e−2
REACTOME: ADAPTIVE
IMMUNE SYSTEM [539]
Genes involved in Adaptive
Immune System
12
1.06 e−4
1.69 e−2
KEGG: GRAFT VERSUS
HOST DISEASE [42]
Graft–versus–host disease
4
1.14 e−4
1.69 e−2
KEGG: TYPE I DIABETES
MELLITUS [44]
Type I diabetes mellitus
4
1.37 e−4
1.82 e−2
REACTOME: POST
CHAPERONIN TUBULIN
FOLDING PATHWAY [19]
Genes involved in Post–
chaperonin tubulin folding
pathway
3
1.87 e−4
2.22 e−2
KEGG: SYSTEMIC LUPUS
ERYTHEMATOSUS [140]
Systemic lupus
erythematosus
6
2.01 e−4
2.22 e−2
REACTOME: HEPARAN
SULFATE HEPARIN HS GAG
METABOLISM [52]
Genes involved in Heparan
sulfate/heparin (HS–GAG)
metabolism
4
2.64 e−4
2.54 e−2
KEGG: AUTOIMMUNE
THYROID DISEASE [53]
Autoimmune thyroid disease
4
2.84 e−4
2.54 e−2
REACTOME: ANTIGEN
PROCESSING
UBIQUITINATION
PROTEASOME
DEGRADATION [212]
Genes involved in Antigen
processing: Ubiquitination &
Proteasome degradation
7
2.97 e−4
2.54 e−2
KEGG: INOSITOL
PHOSPHATE METABOLISM
[54]
Inositol phosphate
metabolism
4
3.05 e−4
2.54 e−2
REACTOME: O–LINKED
GLYCOSYLATION OF
MUCINS [59]
Genes involved in O–linked
glycosylation of mucins
4
4.29 e−4
3.36 e−2
PID AR PATHWAY [61]
Coregulation of Androgen
receptor activity
4
4.87 e−4
3.6 e−2
REACTOME: IMMUNE
SYSTEM [933]
Genes involved in Immune
System
15
5.26 e−4
3.68 e−2 Oncotarget www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 20660 REACTOME: METABOLISM
OF CARBOHYDRATES [247]
Genes involved in
Metabolism of carbohydrates
7
7.37 e−4
4.9 e−2
REACTOME: CLASS
I MHC MEDIATED
ANTIGEN PROCESSING
PRESENTATION [251]
Genes involved in Class
I MHC mediated antigen
processing & presentation
7
8.1 e−4
4.98 e−2
REACTOME: HS GAG
BIOSYNTHESIS [31]
Genes involved in HS–GAG
biosynthesis
3
8.24 e−4
4.98 e−2 Table 3: The functional pathways enriched (FDR < 0.05) by genes linked to CpG sites in
Supplementary Tables S2–S4
Gene Set Name [# Genes (K)]
Description
# Genes in Overlap (k)
p-value
FDR (q-value)
Estradiol
KEGG:STEROID HORMONE
BIOSYNTHESIS [55]
Steroid hormone biosynthesis
3
3.64 e−5
4.84 e−2
KEGG:METABOLISM
OF XENOBIOTICS BY
CYTOCHROME P450 [70]
Metabolism of xenobiotics by
cytochrome P450
3
7.51 e−5
4.99 e−2
Prolactin
KEGG: ALLOGRAFT
REJECTION [38]
Allograft rejection
3
3.52 e−5
2.43 e−2
KEGG: GRAFT VERSUS
HOST DISEASE [42]
Graft-versus-host disease
3
4.76 e−5
2.43 e−2
KEGG: TYPE I DIABETES
MELLITUS [44]
Type I diabetes mellitus
3
5.48 e−5
2.43 e−2
KEGG: AUTOIMMUNE
THYROID DISEASE [53]
Autoimmune thyroid disease
3
9.59 e−5
2.57 e−2
REACTOME: ENDOSOMAL
VACUOLAR PATHWAY [9]
Genes involved in Endosomal/
Vacuolar pathway
2
9.64 e−5
2.57 e−2
REACTOME:
NEF MEDIATED
DOWNREGULATION
OF MHC CLASS I
COMPLEX CELL SURFACE
EXPRESSION [10]
Genes involved in Nef mediated
downregulation of MHC class I
complex cell surface expression
2
1.2 e−4
2.67 e−2
REACTOME: INTERFERON
SIGNALING [159]
Genes involved in Interferon
Signaling
4
1.46 e−4
2.67 e−2
REACTOME: INTERFERON
GAMMA SIGNALING [63]
Genes involved in Interferon
gamma signaling
3
1.61 e−4
2.67 e−2
KEGG: VIRAL
MYOCARDITIS [73]
Viral myocarditis
3
2.49 e−4
3.32 e−2
KEGG: ENDOCYTOSIS [183] Endocytosis
4
2.5 e−4
3.32 e−2 www.impactjournals.com/oncotarget Oncotarget 20661 manifestations of PCOS has been investigated using high-
throughput omics approaches and reported molecular
biomarkers for metabolic heterogeneity [27]. We point out
that their reported findings were based on statistical testing
without correction for multiple testing thus missing an
important procedure in analysing very high dimensional
omics data. Based on genomic DNA methylation
profiles measured in our PCOS patients, we were able
to conduct association analysis of DNA methylation
with multiple clinical features including metabolic
parameters and reported significant findings after strict
adjustment for multiple testing. www.impactjournals.com/oncotarget Although no genome-
wide significant results were found for correlating DNA
methylation with any of the metabolic features (BMI,
IRI, IRI2, GLU, GLU2, HOMA-IR), highly significant
epigenetic associations were observed in our PCOS
subjects on multiple reproductive hormones including E2
(Supplementary Table S2), PRL (Supplementary Table S3),
and progesterone (Supplementary Table S4). From Table 1,
we see that the three hormones have comparable mean
levels in PCOS patients and healthy controls. However,
for the two hormones with large number of significant
CpGs, i.e. E2 (87 CpGs, Supplementary Table S2) and
PRL (199 CpGs, Supplementary Table S3), their hormone
levels in the blood displayed larger dispersions in PCOS
samples as compared with the controls. In Table 3, the
methylation regulated pathways for E2 include steroid
hormone biosynthesis and metabolism of xenobiotics by
cytochrome P450. The two significant biological pathways such as diabetes, thyroid disease and asthma. In the
literature, high prevalence of autoimmune thyroiditis was
also observed in PCOS patients [20–24]. We think that
our EWAS results based on DNA methylation profiling
provide strong molecular evidence for the epidemiological
findings based on clinical data. Overall, both epigenetic
and epidemiological data suggest a possible autoimmune
basis in the pathogenesis of PCOS. With interest in the genome-wide distribution of
PCOS related CpGs, we calculated the proportion of CpGs
in Supplementary Table S1 for their genomic locations
(open sea, shelf, shore, island) among hyper- and hypo-
methylated CpGs (Supplementary Figures S2A, S2B) and
compared them with corresponding proportions in all
CpGs on the Illumina 450 K array (Supplementary Figure
S2C). Compared with the whole array, the distribution of
genomic location for the CpGs in Supplementary Table
S1 was significantly different characterized by high
proportions of hypermethylated CpGs on both north and
south shelves, a low proportion of hypermethylation on
the island, and a high proportion of hypomethylated CpGs
in the open sea representing isolated CpGs in the genome. The implication of differential genomic distribution of
hyper- and hypo-methylated CpGs in the transcriptional
regulation of PCOS requires further investigation. Women with PCOS demonstrate markedly clinical
heterogeneity with the commonly associated features
neither uniform nor universal [25–26]. Recently, the
molecular basis underlying the heterogeneous clinical Figure 2: A Manhattan plot for the negative log10-transformed p values for PRL in PCOS patients plotted against
chromosome location (Mb) for each CpG alone chromosome 6. The HLA region highlighted with red colour harbours CpGs
highly associated with PRL. www.impactjournals.com/oncotarget Figure 2: A Manhattan plot for the negative log10-transformed p values for PRL in PCOS patients plotted against
chromosome location (Mb) for each CpG alone chromosome 6. The HLA region highlighted with red colour harbours CpGs
highly associated with PRL. Figure 2: A Manhattan plot for the negative log10-transformed p values for PRL in PCOS patients plotted against
chromosome location (Mb) for each CpG alone chromosome 6. The HLA region highlighted with red colour harbours CpGs
highly associated with PRL. www.impactjournals.com/oncotarget Oncotarget 20662 for E2 reveal, for the first time, the differential regulation
in the synthesis of reproductive hormone and in drug
metabolism [28] by DNA methylation mechanism in
PCOS patients.i and prostate. After binding of gonadotropin-releasing
hormone, the receptor associates with G-proteins that
activate a phosphatidylinositol-calcium second messenger
system. Activation of the receptor ultimately causes the
release of gonadotropic luteinizing hormone (LH) and
follicle stimulating hormone (FSH). Our result, for the
first time, points to the important role of DNA methylation
mediated epigenetic regulation in controlling menstrual
cycle in PCOS patients which could impact individualized
treatment and management of the disease. i p
Perhaps the most important and novel finding in
this study is the genome-wide significant patterns of DNA
methylation in association with prolactin level in our
PCOS group (Supplementary Table S3). Nearly all of the
10 functional pathways significantly enriched by GSEA
(Table 3) are involved in immune function and immune-
mediated inflammatory conditions. The strong involvement
of immune system in the epigenetic regulation of PRL
under PCOS condition is further illustrated by Figure
2 where a remarkably distinct pattern of association is
shown in the region of major histocompatibility complex
(MHC) on band 6p21.3 of the short arm of chromosome 6,
a region harbouring the human leukocyte antigen (HLA)
genes (marked in red in Figure 2). Diaz et al. [29] recently
summarized the multiple actions of PRL unrelated to
reproduction including its role in the immune system in
promoting proliferation and in inhibiting apoptosis that
could help to maintain the appropriate number of immune
cells in physiological conditions and to maintain immune
tolerance. Meanwhile, clinical studies failed to associate
higher levels of PRL with PCOS [30] and suggested that
PCOS and hyperprolactinemia are two distinct entities
[31] although low prolactin can be a metabolic risk
marker in PCOS patients [32]. The study samples Sample collection was conducted at the Centre
of Reproductive Medicine, Linyi People’s Hospital,
Shandong, China. First, 30 patients aged from 22 to 33
years were recruited from patients diagnosed as PCOS
according to the 2003 revised diagnostic criteria of
Rotterdam consensus [34]. Based on the age and BMI of
the 30 PCOS patients, 30 controls aged from 23 to 32 years
were then recruited from healthy females of reproductive
age who volunteered to participate. All participants were
free from medication and hormone therapy. A written
informed consent was obtained from each participant. All
experiments were conducted according to the principles
of the Declaration of Helsinki. The research was approved
by the Reproductive Ethics Committee of Linyi People’s
Hospital. It has been suggested that epigenetics may be
involved in the regulation of endometrial gene expression
during the menstrual cycle in healthy individuals [33]. Our study provides new data on DNA methylation and
menstrual cycle in PCOS patients. Although only one CpG
(cg08916385) was found, its significance level remained
extremely high even at genome level after adjustment for
multiple testing. Most importantly, the CpG is located
within 1500 bps of the transcription start site (TSS) in
the promotor region of gonadotropin-releasing hormone
receptor (GNRHR) gene on chromosome 4. This gene
encodes the receptor for type 1 gonadotropin-releasing
hormone. The gene is expressed on the surface of pituitary
gonadotrope cells as well as lymphocytes, breast, ovary, www.impactjournals.com/oncotarget Based on these results, one
could assume that our observed significant association
between DNA methylation and PRL levels could be a
phenomenon independent of PCOS and thus should be
also observable in non-PCOS subjects. To validate the
assumption, we conducted a EWAS on DNA methylation
and PRL levels in the 30 control samples of this study. No CpG site was significantly associated with PRL levels
in the healthy controls suggesting that the significant
association between methylation and PRL is a conditional
result only observable in PCOS patients. Although our
conclusion requires further validation, it already provides
novel suggestive evidence in linking differential DNA
methylation and immune responses with PRL regulation
in PCOS samples. g
In conclusion, we have identified a substantial
number of CpGs differentially methylated in the whole
blood samples of PCOS patients and healthy controls,
highly consistently replicating biological pathways
extensively implicated in immunity and immunity-
related inflammatory conditions that were differentially
regulated in the DNA methylome of ovarian tissue from
PCOS women. Most importantly, our genome-wide DNA
methylation profiling focusing on PCOS patients revealed
a large number of CpG sites and their enriched functional
pathways significantly associated with diverse clinical
features (levels of prolactin, estradiol, progesterone and
menstrual cycle) that could serve as novel molecular basis
of clinical heterogeneity observed in PCOS women. Clinical data Comparison of clinical features between PCOS
patients and controls was done by the non-parametric
Wilcoxon rank sum test (equivalent to the Mann-Whitney
test) with consideration that some of the measurements
(e.g. menstrual cycle) may not follow the normal
distribution. Likewise, dispersion of clinical data was
described by calculating the 2.5% and 97.5% quantiles. Genome-wide DNA methylation profiling For each CpG site, statistical association of DNA
methylation with clinical features was tested using the
dmpFinder function provided by the free R package minfi
with type of analysis specified as “categorical” for PCOS
status (1 for cases and 0 for controls) and as “continuous”
for anthropometric and clinical measurements. Continuous
measurements were tested with linear regression, while
an F-test was used for categorical features (here PCOS
status, equivalent to a t-test), both performed on the logit
transformation of the methylation β values. Multiple
testing was adjusted by calculating the false discovery
rate (FDR) using the Benjamini–Hochberg method [36]. Genome-wide significance in differential methylation was
defined as FDR < 0.05. Genome-wide DNA methylation level was measured
using the Illumina’s Infinium HumanMethylation450
Beadchip assay (Illumina, San Diego, CA, USA) at
CapitalBio Corporation (http://www.capitalbio.com), a
certified Illumina service provider in Beijing, China. The
array interrogates over 480,000 CpG sites across and
beyond gene and CpG island regions in the human genome. All laboratory work for the assay was performed according
to the manufacturer’s instructions. Data normalization
was done using the free R package minfi which employs
the quantile normalization [35]. At each CpG site, DNA
methylation level was summarized by calculating a
methylation “beta” value defined by the Illumina’s formula
as β = M/(M + U + 100) where M and U are signal intensities
measured by the methylated and unmethylated probes at a
CpG site. Probe quality was controlled by the detection p
value calculated using minfi. A β value with its assigned
detection p value > 0.01 was treated as missing. CpG sites
with more than 5% missing data across the samples were
dropped from the subsequent analysis. Based on the DNA
methylation data measured in whole blood of each sample
and published cell-type-specific DNA methylation data,
the package minfi estimated blood cell composition in each
individual for 6 blood cell types: CD8T, CD4T, natural killer
cell, B cell, monocyte, and granulocyte. The estimated cell
type proportion was used by minfi to adjust the effects of
cell composition on DNA methylation levels in each sample
before downstream statistical analysis. Data analysis i.e. luteinising hormone (LH), follicle stimulating hormone
(FSH), estradiol (E2), total testosterone (TST), prolactin,
progesterone, and thyroid stimulating hormone (TSH) were
determined by direct chemiluminescence immunoassay
(Siemens Healthcare Diagnostics Inc; East Walpole,
Massachusetts, U.S.A) according to the manufacturer’s
instructions. Serum fasting blood-glucose (GLU) and
GLU at 2 hours after ingestion of 75 gram dextrose
(GLU2) were determined by the oxygen electrode method
(Beckman Coulter, Inc., Suzhou, China). Homeostatic
model assessment IR (HOMA-IR) was calculated by the
equation HOMA-IR = GLU*IRI/22.5. Clinical biochemistry and reproductive hormone From each participant, blood was taken from
antecubital venous for blood biochemical test following
routine protocol and meanwhile the blood for DNA
methylation analysis was immediately stored under −80°C
at the central laboratory of Linyi People’s Hospital. Fasting
immunoreactive insulin (IRI) and immunoreactive insulin
at 2 hours after ingestion of 75 gram dextrose (IRI2) were
assayed by radioimmunoassay (Beckman Coulter, Inc.,
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Papanastasiou L, Bergiele A, Panidis D. Polycystic ovary
syndrome: the influence of environmental and genetic
factors. Hormones (Athens). 2006; 5:17–34. 3. Diamanti-Kandarakis E, Piperi C, Spina J, Argyrakopoulou G,
Papanastasiou L, Bergiele A, Panidis D. Polycystic ovary
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Azziz R, Goodarzi MO. Further investigation in europeans
of susceptibility variants for polycystic ovary syndrome
discovered in genome-wide association studies of Chinese
individuals. J Clin Endocrinol Metab. 2015; 100:E182–186. 19. ACKNOWLEDGMENT AND FUNDING 12. Wang XX, Sun BF, Jiao J, Chong ZC, Chen YS, Wang XL,
Zhao Y, Zhou YM, Li D. Genome-wide DNA methylation and
gene expression patterns provide insight into polycystic ovary
syndrome development. Oncotarget. 2014; 5:6603–6610. doi:
10.18632/oncotarget.2224. This work was supported by the Region of
Southern Denmark 2012 research grant project no. 12/6629 and partially supported by the Novo Nordisk
Foundation Medical and Natural Sciences Research Grant
NNF13OC0007493. 13. Yu YY, Sun CX, Liu YK, Li Y, Wang L, Zhang W. Genome-
wide screen of ovary-specific DNA methylation in polycystic
ovary syndrome. Fertil Steril. 2015; 104:145–153. Software packages 9. Wu XY, Li ZL, Wu CY, Liu YM, Lin H, Wang SH, Xiao WF. Endocrine traits of polycystic ovary syndrome in prenatally
androgenized female Sprague-Dawley rats. Endocr J. 2010;
57:201–209. All statistical analyses were conducted under the
R software environment for statistical computing and
graphics (https://www.r-project.org/). DNA methylation
data were analysed using the R package minfi (http://
bioconductor.org/packages/release/bioc/html/minfi.html)
[35] for raw data preprocessing (quality control and
normalization) and for statistical analysis. 10. Xu N, Azziz R, Goodarzi MO. Epigenetics in polycystic
ovary syndrome: a pilot study of global DNA methylation. Fertil Steril. 2010; 94:781–783. 11. Shen HR, Qiu LH, Zhang ZQ, Qin YY, Cao C, Di W. Genome-wide methylated DNA immunoprecipitation
analysis of patients with polycystic ovary syndrome. PLoS
One. 2013; 8:e64801. The authors report no conflicts of interest. The authors report no conflicts of interest. Biological pathway analysis To identify biological pathways differentially
regulated by DNA methylation, we used the Gene-Set
Enrichment Analysis (GSEA) which is a bioinformatics
tool for determining whether an a priori defined set
of genes shows statistically significant, concordant
differences between two biological states (http://
software.broadinstitute.org/gsea/index.jsp) [37]. Based
on a collection of the Molecular Signatures Database
(MSigDB) of GSEA, we computed overlaps between our
genes linked to significant CpGs identified in our analysis
and gene sets in MSigDB. Statistical significance of the
overlap with each gene set in MSigDB is obtained from
the hypergeometric distribution of number of overlapping
genes (k), number of genes in the query set (n), total
number of genes in a MSigDB set (K) and number of all
known genes (N). For each individual, DNA methylation levels were
measured on a total of 485512 CpG sites across the
genome. We first filtered out 728 CpGs with detection
p value > 0.01 in more than 5% of the overall samples
(i.e. 3 samples). Different from other genome-wide
analysis that removed all CpGs on sex chromosomes,
we only dropped Y-linked CpGs (147 sites) but kept
all X-linked CpGs (11229 sites) considering that all
our samples are females. A total of 484637 CpG sites
were available for subsequent analysis. The raw and
normalized DNA methylation data are deposited in the
Gene Expression Omnibus database (http://www.ncbi. nlm.nih.gov/geo/; accession no. GSE80468).
−
−
−
=
∑
−
=
n
N
i
n
K
N
i
K
p
k
i
1
0
1 Correction for multiple testing was done by
calculating the false discovery rate according to Benjamini
and Hochberg [36]. www.impactjournals.com/oncotarget Oncotarget 20664 CONFLICTS OF INTEREST 14. Shi Y, Zhao H, Shi Y, Cao Y, Yang D, Li Z, Zhang B, Liang X,
Li T, Chen J, Shen J, Zhao J, You L, et al. Genome-wide
association study identifies eight new risk loci for polycystic
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Andersen M. Prolactin is associated with metabolic risk and
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multiple testing under dependency. Ann. Stat. 2001;
4:1165–1188. 37. Subramanian A, Tamayo P, Mootha VK, Mukherjee S,
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and metabolism of xenobiotics. Cell Mol Life Sci. 2001;
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Typing Analysis and Normalization Construction of Administrative Rating
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Frontiers in business, economics and management
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Frontiers in Business, Economics and Management
ISSN: 2766-824X | Vol. 6, No. 3, 2022 Frontiers in Business, Economics and Management
ISSN: 2766-824X | Vol. 6, No. 3, 2022 1. Meaning of Administrative Rating In recent years, administrative rating has been widely used
in administrative management, and administrative rating has
begun to enter the vision of administrative law scholars. The
so-called administrative rating refers to administrative
activities in which the administrative subject evaluates the
previous performance of the counterpart in the relevant field
according to certain standards and procedures, and marks and
publishes them with concise symbols. [1] Administrative
rating is widely used because it simplifies complex
administrative
supervision,
improves
administrative
efficiency, increases administrative transparency, and realizes
law
enforcement
optimization. On
the
one
hand,
administrative rating, as a means of classified supervision,
corresponds and simplifies the administrative scheme to
symbols, and plays an irreplaceable role in the field of
administrative supervision; On the other hand, the widespread
use and abuse of rating activities may also trigger problems
such as unwarranted rating and abuse of administrative
discretion, which raises questions about the legality and
rationality of administrative rating. Whether the act of an
administrative organ can bring administrative relief depends
on whether it has the attribute of administrative decision. [2]
Whether an act is "subject to legality review" mainly depends
on the judgement of the nature of the act. Therefore, it is
crucial to clarify the nature of administrative rating in
administrative law. At the same time, to solve the legal
problems arising in administrative rating, we must first return
to the origin of the problem to explore the own legal attributes
of administrative rating. Only by distinguishing different
types of administrative rating according to legal attribute
differences can we explore the applicable boundaries of
administrative rating, implement procedures and remedies for
rights in rating behaviors. From the point of view of rating
process, for administrative rating, through the formulated or
recognized standards set by administrative organs, the
administrative counterpart is evaluated and assigned a certain
value, so there is no doubt that rating activities have
administrative attributes. From the applicable areas of rating
and the rating results, administrative rating can be legally Mi Liu1, a 1Central University of Finance and Economics, Beijing 100081, China
aliumiray@126.com 1Central University of Finance and Economics, Beijing 100081, China
aliumiray@126.com Abstract: Administrative rating is widely used in administrative management activities as a means of classifying supervision,
which not only investigates the previous performance or status of rating objects in established fields, but also provides a reference
basis for subsequent regulatory behaviors, resulting in the risk of abuse. Administrative rating should have its legality and
legitimacy if it is to operate under the framework of administrative rule of law. Therefore, it is necessary to introduce principle
of legality, principle of proportionality and principle of remedies to construct the scope of application areas of administrative
rating, information collection and processing system in rating and right relief based on the lack of administrative rating norms
in application, procedure and relief. Keywords: Administrative rating, Administrative management, Administrative verification, Administrative fact action. attributed to administrative verification behavior or
administrative fact action. Typing Analysis and Normalization Construction of
Administrative Rating Mi Liu1, a 3. Fact Rating First, the scope of application is not clear. As one of the
governance tools, with the wide application and continuous
development of digital technology, administrative rating is
often implemented by making certain evaluation and
assignment of governance objects after integrating and
processing data according to certain rules or criteria, and
further perform differential governance based on it. Specific
to the application of administrative rating, on the one hand, it
is confirmed that verification rating shall take relevant laws
and regulations or regulatory documents as the legitimacy
basis for rating acts; on the other hand, although the framed
order set by the Constitution justifies the transfer of individual
rights for public interests under fact rating, it does not mean
that excessive restrictions or even infringement of citizens'
relevant rights are imposed. Accordingly, not all fact ratings
are justifiable. Administrative rating is the boundary of the
scope of application and is actually the boundary of social
governance. Admittedly administrative rating plays an
irreplaceable role in the process of differential governance,
but if administrative rating is applied beyond the boundaries
of social governance, it will lead to illegality and irrationality
of rating behavior. The applicable expansion of administrative
rating is essentially the expansion of public power, which can
bring direct and indirect, objective and subjective benefits of
control and support, with natural expansion. The expansion of
public power also means the transfer of private rights, and the
excessive expansion of public power will certainly lead to the
restriction or even violation of private rights. For "code
governance", as a large-scale exercise of big data governance
by administrative organs, it evaluates and assigns the
governance objects through different colors or different forms
of "code", which is not only a typical form of data governance,
but also a typical form of administrative rating and belongs to
the fact rating in administrative rating. Typical code
governance is to prevent and control the emergence of the
COVID-19 epidemic "accompany code." That is, after the
"code", some regions have launched similar "civilization
code" and "color change code" that eager to play the value of
data and cross the boundary of social governance - a
governance form that unilateral pursuit governance efficiency
and excessively restrict private rights. As a form of fact rating,
"civilization code" and "discoloration code" map out a series
of problems applicable to rating. 3. Fact Rating It also shows that not all
fields are suitable for "evaluation", and the scope of
administrative rating should be standardized. The decision-making basis of traditional social risk
response is mostly established facts or realistic state. At this
time, although the behavior basis is sufficient, it is easy to
appear ineffective governance or regulation failure caused by
delayed decision-making. As public decision makers,
administrative subjects tend to rely on big data to make real-
time judgments or make certain predictions in the face of
emergencies, thereby accelerating the speed of response and
controlling possible losses to a minimum. In this context, one
of the tasks of administrative subjects lies in how to collect
all kinds of information related to citizens in time or in real
time through various forms to provide intuitive and objective
basis for scientific and reasonable decision-making. In face of
emergencies or other special circumstances, in order to make
scientific
and
reasonable
decisions
and
responses,
administrative authorities have set or recognized certain
rating criteria, and obtain rating results through certain data
tools based on the collection and handling of relevant
information
of
administrative
counterparts,
and
administrative rating of administrative counterparts to
distinguish and control administrative counterparts based on
this result, which conforms to the characteristics of
administrative fact action and should be attributed to
administrative fact action. The so-called administrative fact
action refers to administrative subjects acts implemented by
authority that cannot directly produce, change or eliminate
administrative legal relationships. administrative fact action
is only a content uncertainty about the rights and obligations
of the parties, and is not a consequence that does not affect
the rights and obligations of the parties. At present
administrative fact action not means that it dose not produce
legal effects, however, it does not directly produce legal
effects. That is, administrative fact action may affect the
rights and obligations of the parties, but this is not the
intention of the administrative authorities. Direct fact effects
are characteristic of administrative fact action. For administrative rating (hereinafter referred to as fact
rating) attributed to administrative fact action, taking
accompany code as an example. 2. Verification Rating Through
relevant
evaluation
criteria
set
by
the
administrative organ and in combination with the information
of the counterpart or previous behavior, qualitative evaluation
is made in the established fields, on this basis, administrative
rating behavior of corresponding classified supervision
carried out confirms the extent to which the counterparts
complies with the legal norms for a certain period of time, and
the behavior shall be attributed to administrative verification
behavior. The so-called administrative verification refers to
the administrative act that the administrative subject
examines and confirms the legal status, specific legal
relationship or relevant legal fact of the administrative
counterpart in accordance with the law, and declares them in
a legal manner. [3] Although administrative verification does
not directly create or change the status of rights and
obligations of administrative counterparts as administrative
acts such as administrative penalty, licensing, and compulsory
expropriation, it only confirms the existing legal relationship,
legal status, and legal facts. Administrative verification
confirms social relationships and facts of legal significance,
and it is an administrative act made by administrative subjects
enjoying administrative verification power in accordance
with legal authority, procedures, standards, forms, etc.. It has
public law effectiveness, and is the basis for administrative
counterparts to claim rights against third parties. Taking the credit rating of tax payment as an example,
administrative
rating
attributed
to
administrative
verification acts (hereinafter referred to as verification rating)
is to improve the administrative efficiency of tax
administration, improve taxpayers' awareness of tax payment
in good faith according to law and compliance with tax law,
and tax authorities evaluate the credit status of enterprise
taxpayers according to four dimensions (subjective attitude,
compliance ability, actual results and dishonesty) and nearly
100 evaluation indicators. According to the evaluation results,
it is divided into five levels: A, B, M, C and D. The result of 249 4. Normalization Deficiency of
Administrative Rating this verification rating is a recognition evaluation of the law-
abiding status of the tax payer over a period of time. 3. Fact Rating As a verification rating, the rating
as administrative verification act is an independent
administrative act and shall belong to the category of
"believing that an administrative organ infringes upon other
lawful rights and interests such as personal rights and
property rights" listed in Item 12 of Article 12 of the
Administrative Procedure Law, and the administrative
counterpart has the right to seek relief through administrative
litigation. In judicial practice, administrative rating has ruled
that administrative rating has not increased the rights or
obligations of its counterparts, thus dismissing the plaintiff 's
prosecution. In fact rating, the application of fact rating is
usually government-led automation rating under special
circumstances. In the face of emergencies, the government
must respond promptly and assume responsibility. Because
the government department's basic technical ability and data
operation ability are limited, the government is relatively
inferior in technology. It is difficult to realize from scratch to
develop intelligent and digital rating tools in a short time
because it requires manpower, material resources and time
costs. Therefore, governments at all levels have to rely on the
resources and platforms of large enterprises as well as
technical capacity and data operation capacity to achieve the
implementation of fact rating. However, because of profit-
seeking quality, enterprises may rely on their technical
advantages and capital advantages, causing infringement on
the basic rights of citizens. In the case of impaired rights, there
is no clear right relief mechanism to remedy citizens' rights. q
Second, principle of proportionality should be used to
standardize information collection and processing in
administrative rating. Principle of proportionality is one of the
basic principles of restricting public power in the field of
administrative law, which is initially manifested as the
necessary restriction of Germany on the exercise of police
power. Principle of proportionality is set based on the
restriction of state power, following the modern public law
concept of human rights protection, justice and rationality,
and has become the basic principle of rationality review of
public power exercise. Restrictions on private rights for the
purpose of protecting the public interest are legally justified
only if they are in line with principle of proportionality. 3. Fact Rating Although the neutrality of the
technology itself can ensure the objectivity and impartiality
of rating results to a certain extent, due to opaque or difficult
transparent decision-making algorithms with trade secret
attributes, there may exist problems such as the design of the
algorithm does not fully reflect the spirit of legal fairness and
justice or the purpose of the rating, the lack of supervision of
the algorithm in terms of scientific rationality. grade the counterpart within the scope of legal authority, in
accordance with legal standards and legal procedures and in
legal form. The primary concern about the legality of an act
is whether there is a basis for relevant laws and regulations as
a support. "It is prohibited without authorization by the law."
When administrative rating is implemented, administrative
authorities shall clarify the applicable fields of administrative
rating in strict accordance with the provisions of relevant laws
and regulations. Where administrative rating is required under
special circumstances without relevant legal basis, the scope
of administrative rating shall be set in strict accordance with
the legal procedures for administrative decision-making. In
terms of specific procedures, the decision-making process of
whether administrative rating is applicable in a certain field
shall include decision-making initiation, social investigation,
protocol drafting, public participation, expert demonstration,
risk assessment, legality review, collective discussion, public
release and other links. For administrative rating, digital
technology governance should not only control social
problems, but also govern administrative power itself to
prevent the expansion of administrative power. The
application of new technologies merely changes specific
governance modalities and governance tools and does not
compromise the rights that citizens should enjoy. Therefore,
we should adhere to and implement the clear requirements of
existing laws and regulations. Administrative rating process
only uses technology to achieve certain administrative
purposes, and does not newly set restrictions on the rights of
administrative counterparts, nor exempts the obligation
requirements for administrative authorities. g
y
Third, rights relief is not perfect. Administrative
counterparts refusing to accept administrative rating results
shall enjoy the right to relief. Given the different legal
attributes of verification ratings and fact ratings in
administrative rating, there should be different remedies for
different types of rating counterparts. Because the mechanism
of right relief is not perfect and the subject of responsibility is
not clear, the right relief of administrative counterpart is often
difficult to be guaranteed. 3. Fact Rating [5]
Therefore, the justification of information collection and
processing in administrative rating also requires that the
exercise of public power behind it should be highly restrained,
and restrictions on the relevant rights and interests of
administrative counterparts must be in line with principle of
proportionality. In administrative rating's information
collection and processing process, one side is the public value
of public power and the other side is the relevant right of
private rights. For the way and content of information
collection, the external system and internal algorithm of
information processing, it is necessary to introduce principle
of proportionality to select and choose. There is little doubt
that, information collection and processing systems should be
open and transparent. In particular, the public disclosure of
information in the verification rating, or the hidden protection
of information in the fact rating, should achieve public value,
but also as far as possible to minimize the losses caused by
personal rights. 3. Fact Rating accompany code is a
response plan made by administrative authorities in a timely
manner in face of the realistic dilemma of accurately
preventing and controlling the COVID-19 epidemic and
timely promoting the resumption of work and production
nationwide and restoring the socio-economic order,
combining personal health information of citizens, personal
identification information of citizens, personal interpersonal
relationship information of citizens, and personal trajectory
information of citizens to make a personal health risk rating
judgment on citizens. Unlike verification rating, the rating of
the accompany code is not associated with whether the
administrative counterpart adheres to legal norms, but rather
is a physical health dimension of legal development. Moreover, the color conversion of accompany QR code is a
real-time and dynamic process, no matter whether the result
of this fact rating is positive or negative, the generation of
accompany code itself has not directly caused the generation,
change or elimination of administrative legal relations. g
Second, information collection and processing are opaque. No matter what legal nature administrative rating, it is
inseparable from the collection and processing of information. Collection and processing of information is the basis for
administrative rating. In order to avoid the risks existing in
the collection and processing of information in the rating, it is
necessary to unify the formal standards and content standards
for the collected information with the help of the effectiveness
of relevant normative documents, construct a safe information
data transmission and sharing path, and reasonably handle the
information after using the information. In the information
collection stage of rating, administrative counterparts
certainly enjoy the right to know and inquire based on
administrative rating, which is an inevitable requirement of
proper administration. However, in actual operation, the
information collection in rating has lower transparency. [4] 250 For administrative counterparts, on the one hand, the types
and scope of information collected have not been fully
informed; on the other hand, if information leakage occurs
during the process of information collection, there is also a
lack of relevant mechanisms to clearly inform administrative
counterparts of how to produce evidence and how to relieve
them. In addition, in the information processing stage of
rating, the processing of information includes identification,
convergence, coding, encryption, storage, transmission,
mining, application, management and control, deletion of
information,
so
the
processing
of
information
in
administrative rating is usually realized in the form of
algorithm administration. 5. Normalization Construction of
Administrative Rating For verification rating, it shall be
clearly confirmed that the it is an independent administrative
act, which is neither an internal administrative act nor a
procedural administrative act, and does not belong to the acts
listed in Article 1 of the Interpretation of the Supreme
People's Court on the Application of the “Administrative
Procedure Law of the People's Republic of China” that do not
have external legal effect, procedural acts and acts that do not
have actual impact on the rights and obligations of the
counterparts, so they are litigable that explicating the review
and litigation system for verification rating. For fact rating,
the complaint system and state compensation system of fact
rating should be established. Although the complaint system
of fact rating is also partially provided for in some regions of China, in general, the complaints of fact rating in most regions
have not been put in place. Where any damage of fact rating
act is caused by breach of law, the specific compensation
standards for damages may be compensated by referring to
the provisions on the compensation methods and calculation
standards in Chapter IV of the State Compensation Law. China, in general, the complaints of fact rating in most regions
have not been put in place. Where any damage of fact rating
act is caused by breach of law, the specific compensation
standards for damages may be compensated by referring to
the provisions on the compensation methods and calculation
standards in Chapter IV of the State Compensation Law. 5. Normalization Construction of
Administrative Rating First, the applicable boundaries of administrative rating
should be determined by principle of legality. Administrative
rating shall be conducted within the framework of the
administrative rule of law. The administrative subject shall Third, we should rely on principle of remedies to improve
the rights relief mechanism in administrative rating. "No
remedy means no right". From the point of protecting the 251 lawful rights and interests of the people, citizens or
organizations must be granted the right to enjoy adequate
relief in the event that their lawful rights and interests are
violated by administrative violations or undue violations. First, we should establish the error correction mechanism of
administrative rating. During the implementation of
administrative rating, administrative organs should not only
clearly inform the rating criteria, contents and procedures, but
also inform administrative counterparts of the ways and
means of rights relief. For verification rating, it shall be
clearly confirmed that the it is an independent administrative
act, which is neither an internal administrative act nor a
procedural administrative act, and does not belong to the acts
listed in Article 1 of the Interpretation of the Supreme
People's Court on the Application of the “Administrative
Procedure Law of the People's Republic of China” that do not
have external legal effect, procedural acts and acts that do not
have actual impact on the rights and obligations of the
counterparts, so they are litigable that explicating the review
and litigation system for verification rating. For fact rating,
the complaint system and state compensation system of fact
rating should be established. Although the complaint system
of fact rating is also partially provided for in some regions of lawful rights and interests of the people, citizens or
organizations must be granted the right to enjoy adequate
relief in the event that their lawful rights and interests are
violated by administrative violations or undue violations. First, we should establish the error correction mechanism of
administrative rating. During the implementation of
administrative rating, administrative organs should not only
clearly inform the rating criteria, contents and procedures, but
also inform administrative counterparts of the ways and
means of rights relief. References [1] Wang Ruixue: On Administrative Rating and its legal control,
Legal Business Research, Issue 3, Year 2018, Page 28. [2] Chen Xinmin: Principles of Administrative Law in China,
China University of Political Science and Law Press, 2002
Edition, Page 133. [3] Ying Sonian: Administrative Law and Administrative
Procedure Law, Higher Education Press, 2018 Edition, Page
194. [4] Zhu Chunhua: Information Regulation Tools for Government
ruled by Law, China Social Science Press, 2022 Edition, Page
155. [5] Liu Quan: "Principle of Proportionality", Tsinghua University
Press, 2022 Edition, Page 33. 252
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The Role Of Actual Units Of Division In The Compositional-Syntactic Structure Of Poetic Speech
|
The American journal of social science and education innovations
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cc-by
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The American Journal of Social Science and Education Innovations
(ISSN – 2689-100x) (
)
Published: June 30, 2021 | Pages: 171-179
Doi : https://doi.org/10.37547/tajssei/Volume03Issue06-29 Journal
Website:
http://theamericanjour
nals.com/index.php/taj
ssei
Copyright:
Original
content from this work
may be used under the
terms of the creative
commons
attributes
4.0 licence. The Role Of Actual Units Of Division In The Compositional-
Syntactic Structure Of Poetic Speech
Mukhammadtokhir Tojimamatovich Abdupattoev
Candidate Of Philology, Associate Professor, Fergana State University, Fergana, Uzbekistan The Role Of Actual Units Of Division In The Compositional-
Syntactic Structure Of Poetic Speech ABSTRACT The article presents theoretical views on the compositional-syntactic structure of poetic speech,
which is one of the problematic issues in the field of poetic syntax and the study of its actual division. Within the framework of the Uzbek poetic syntax, the importance of studying the issue of actual
division as a separate category in the study of Uzbek poetic speech based on the experience of world
linguistics. KEYWORDS Linguopoetic, syntax, poetic syntax, poetic speech, actualization, subject-material relations,
expressiveness, communication, predicate, subject. The American Journal of Social Science and Education Innovations
(ISSN – 2689-100x)
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(ISSN – 2689-100x)
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(ISSN – 2689-100x)
Published: June 30, 2021 | Pages: 171-179
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The Role Of Actual Units Of Division In The Compositional-
Syntactic Structure Of Poetic Speech
Mukhammadtokhir Tojimamatovich Abdupattoev
Candidate Of Philology, Associate Professor, Fergana State University, Fergana, Uzbekistan The American Journal of Social Science and Education Innovations
(ISSN – 2689-100x) INTRODUCTION should be noted that poetic speech has many
ways and means of expression, because it is a should be noted that poetic speech has many
ways and means of expression, because it is a Any poetic speech means a certain dialogue
between the listener and its author, in which
the listener acts only as the addressee. With a
communicative approach to speech analysis,
the position of the author of the speech as an
addressee is of great importance in the
compositional-syntactic structure of speech. It complex and multifaceted syntactic structure. Accordingly, in the compositional-syntactic
formation of poetic speech content, various
methods and means are used. The question of
the role of actual units of division in the The USA Journals Volume 03 Issue 06-2021 MAIN PART Another view of the semantic-syntactic
relations between the parts of poetic speech
is manifested in the form of actual division and
plays an important role in the compositional-
syntactic structure of speech, since in the
syntactic whole, the relation of the subject
and the rheme constitutes the problem of the
actual division of speech. The actual division
was originally applied to speech in linguistics. Czech scientist V. Matezius, who founded the
theory of the actual division of speech as a
special linguistic direction [1]. Another view of the semantic-syntactic
relations between the parts of poetic speech
is manifested in the form of actual division and
plays an important role in the compositional-
syntactic structure of speech, since in the
syntactic whole, the relation of the subject
and the rheme constitutes the problem of the
actual division of speech. The actual division
was originally applied to speech in linguistics. Czech scientist V. Matezius, who founded the
theory of the actual division of speech as a
special linguistic direction [1]. A person often interacts not with words taken
separately, but with the help of statements in
which
the
content
is
interconnected,
extracted from each other and communicates
with
each
other
in
a
certain
way. Consequently, speech comes first in terms of
performing
the
communicative
function. Consequently,
the
compositional-syntactic
structure of speech is associated with the
problem of actual division. There is also a
thematic division in a speech: the theme
(theme), the topic of the speech (what is at
stake), and the message (rheme) is the
coverage of the topic, what it consists of. According to him, the sentence consists of
two parts: the starting point of thought - the
part (theme) known from the situation, easily
understood
from
the
previous
speech
situation, and the communicative center - the
part (rheme), which is the main information in
the sentence. I.P. Raspopov is a scientist
dealing with the problem of actual division in
Russian linguistics [2]. The problem of the actual division into
syntactic units has attracted the attention of
Turkologists since the 70s of the last centure. It should be noted the articles and main
studies
of
R.S.Amirova,
F.M.Agaeva,
K.N.Valiev[3]. In large poetic works, the theme is revealed
throughout the entire work. In small volumes
(poems, quartets), a new message on the
topic (rheme) is announced in the same small
volume. The American Journal of Social Science and Education Innovations
(ISSN – 2689-100x) The American Journal of Social Science and Education Innovations
(ISSN – 2689-100x) (
)
Published: June 30, 2021 | Pages: 171-179
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)
Published: June 30, 2021 | Pages: 171-179 issues
using
the
example
of
complex
sentences and complex speech units remains
relevant. compositional-syntactic structure of poetic
speech is of great importance. It is well known, that in syntactic construction
as units of actual division, most scientists
determine the basis of the expression of a
sentence - the theme and the core of the
expression - rheme. The author of the speech
and the listener or reader are part of the
communication mechanism. A subject is a new
message, information that is transmitted to
the rest of the addressee, often if the subject
is known to both of them in advance. Apparently, it is also studied in the semantic
aspect as a syntactic category and serves as
the basis for the compositional-syntactic
formation of poetic speech. The USA Journals Volume 03 Issue 06-2021 171 The American Journal of Social Science and Education Innovations
(ISSN – 2689-100x)
Published: June 30, 2021 | Pages: 171-179
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(ISSN – 2689-100x)
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OCLC - 1121105668 MAIN PART Coverage of the problem of the actual division
of speech in Uzbek linguistics dates back to
the 70s of the last century, and as a result, A. Hayitmetov's monographic research appeared
[4]. In Uzbek linguistics, the units of division
proper - the relationship between the theme
and the rheme - study mainly in the context of
simple sentences, and the study of these The world is like a market, The world is like a market, The world is like a market. The world is like a market. I have not seen any of these two, I have not seen any of these two, The USA Journals Volume 03 Issue 06-2021 The American Journal of Social Science and Education Innovations
(ISSN – 2689-100x) The American Journal of Social Science and Education Innovations
(ISSN – 2689-100x) IMPACT FACTOR
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)
Published: June 30, 2021 | Pages: 171-179
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Doi : https://doi.org/10.37547/tajssei/Volume03Issue06-29 expressed through an addition. The topic of a
sentence corresponding to the topic of poetic
speech is interpreted and expanded by a
series of sentences equivalent to rheme. In
this case, no matter what concept the subject
of
the
subject
expresses,
the
words
expressing rhyme continue, that is, expand
the ideas related to this subject. Based on the
nature
of
the
collected
and
analyzed
examples, we considered it necessary to
highlight the following types of subject
meanings. Who badly called theirs things. (A.Oripov) In the compositional and syntactic structure of
this quartet, the theme is clarified first of all -
the world, and the rest of the lines appear in
the rheme function, that is, new author's
messages about the world - experiences,
feelings - are expressed. As you can see, in the
compositional-syntactic structure of poetic
speech, the theme-rheme relation, showing
the semantic connection between parts
(lines), played an important role in ensuring
the integrity of poetic speech, the consistent
development and conclusion of thought. ... In
addition, to ensure a strong semantic and
syntactic
connection
between
sentences
(lines) and to enhance the emotional impact, a
syntactic figure was used - repetition (multiple
use of a combination similar to the market
one), synonyms (market, product). ... 1. Comment related to the topic of the
action: Occasionally riding on a horse of anger, Occasionally riding on a horse of anger, Terrible crying madman Kokand’s wind Terrible crying madman Kokand’s wind
Your eyes are narrowed, your ears are
pierced, Your eyes are narrowed, your ears are
pierced, It is well known that every sentence is based
on judgment. In other words, judgment is
expressed through judgment. As the linguist
A. Nurmonov noted, judgment and speech
form a dialectical unity. Typically, each
sentence contains three main elements:
subject, predicate and glue [5,72]. Therefore, a
sentence semantically consists of a subject, a
predicate, and a linking relation. Since this
attitude exists in any form of speech, it also
forms the basis of sentences equivalent to the
topic of speech. You cannot separate the sky from the ground.
(Iqbol Mirzo) From the quoted passage of poetic speech, it
is clear that the subject of speech (subject of
action), which is equivalent to the subject, is
explained by the "wind", and its action is
rheme. The influence of an object on
existence is widely and comprehensively
revealed in rhyme. This gives the reader the
opportunity to complete a specific idea. Known and new - in the above-mentioned
manifestation
of
the
theme-rheme
relationship, mainly the object and its action
play an important role RESULTS AND DISCUSSIONS 2. Comment on the topic of the case: The USA Journals Volume 03 Issue 06-2021 172 The American Journal of Social Science and Education Innovations
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(ISSN – 2689-100x) When we semantically
examine sentences that are equivalent to the
theme of poetic speech, we see that the
existing subject is important in relation to the
subject-rem of speech. You cannot separate the sky from the ground. (Iqbol Mirzo) The American Journal of Social Science and Education Innovations
(ISSN – 2689-100x) The American Journal of Social Science and Education Innovations
(ISSN – 2689-100x) IMPACT FACTOR
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Published: June 30, 2021 | Pages: 171-179
Doi : https://doi.org/10.37547/tajssei/Volume03Issue06-29 Bruises on her face every day. (X. Davron) Even in the cited poetic discourse, as noted
above, rheme serves as an explanation,
enumerating the distinguishing features of the
qualitative subject in the subject. At the same
time, words characteristic of the adjective
phrase, as well as adjectives and adverbial
forms of the verb, are more often used (since
the main emphasis is on the interpretation of
the subject sign). Our example above uses a
feminine theme and its characteristic features
are interpreted rhymingly. The white flowers are already in full bloom 2. Comment on the topic of the case: It is well known that under the term subject
different concepts are combined, that is, the
predicate is interpreted as the owner of
different symbols. Linguists note that the
subject of speech grammatically masters or is Gust slowly touches to my face, Sight buries in heart of the day The USA Journals Volume 03 Issue 06-2021 173 The American Journal of Social Science and Education Innovations
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Doi : https://doi.org/10.37547/tajssei/Volume03Issue06-29 (Usmon Nosir) The theme of this poetic discourse is the
poet's momentary experiences, which, as you
can see, constitute the subject of the situation
(static subject) in accordance with the subject
of the subject. Rheme begins with the first
sentence in the speech itself. The main
emphasis is on the state of the subject in
predicative units, which constitute the rhyme
used to provide, complete and interpret a
complete
description
of
the
subject's
situation. 4. Annotation related to a quantitative
object. In poetic speech, in which theme is
a quantitative defining subject in the
subject and which is interpreted in theme,
the subject indicates the quantitative
attribute of the subject. However, in
rheme the same amount is explained and
justified. To complete it, evidence is
provided: The state of the object is explained by such
verb forms as “buried”, “touches”, “picking”,
“full”, “rise”, “full”. It is known that the verb
forms denoting the passive action of the
subject are characteristic of the figurative-
speech type, in which the situation prevails
over the action. A similar situation can be seen
in the passage above. There are many thousand year old ruins in the
world, There are many thousand year old ruins in the
world, Memory of the wars, memory of the floods Memory of the wars, memory of the floods The graves are stacked high 3. A comment related to the object on which
the sign stands. In cases where the subject
in the sentence expressing the topic is of a
qualitative nature, the main emphasis in
the topic is placed on the nature of the
subject. The same law is preserved in the
sentences that make up rheme. That is the
subject
sign
is
expanded
by
the
interpretation: In fact, they are also large ruins. (A.Oripov) As you can see from this example, the number
of subjects is justified and explained (plural)
rheme. In rhyme, the use of quantitative (top-
down) tokens also provides a certain semantic
link, as well as a special integrity of the topic-
rheme relationship. 5. Comment related to the subject of
ownership. In poetic discourse, in which
the subject in the subject has a positive
character, it is implied that the subject has Woman's hands are rough, Woman's dress is rough. Woman's dress is rough. (Usmon Nosir) The USA Journals Volume 03 Issue 06-2021 174 The American Journal of Social Science and Education Innovations
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9
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Doi : https://doi.org/10.37547/tajssei/Volume03Issue06-29 Uzbeks have as long as been hospitable something. In
the
rheme
however,
explains this with additional information. Uzbeks have as long as been hospitable They say the guest is important than your
father. Putting all the things what have to a daytime
guest And after eaten dried apricots for a long time. (Sh. Rahmon) In the given example, the subject expresses
the existence of the Uzbek people and
through rheme this existence reflects the
interpretation of the characteristics of the
subject, additional information about his
specific impact on the object - the outside
world. In poetic speech, this type of meaning of the
object is manifested through From the above poetic speech, it is clear that
the subject of the theme - "my" - "my sky, my
hot heart, my sun, my faith, my joy, my
consolation, my aspiration, my regret, my
suffering ..." is explained in rheme. The
reasons for possessing these characteristics
are specified. The continuation of the use of
possessive suffixes in rheme, denoting and
indicating
property,
serves
to
explain,
complement, while providing a connection
between the theme-rheme, as well as the
general characteristics of the subject of
ownership in the subject. From the above poetic speech, it is clear that
the subject of the theme - "my" - "my sky, my
hot heart, my sun, my faith, my joy, my
consolation, my aspiration, my regret, my
suffering ..." is explained in rheme. The
reasons for possessing these characteristics
are specified. The continuation of the use of
possessive suffixes in rheme, denoting and
indicating
property,
serves
to
explain,
complement, while providing a connection
between the theme-rheme, as well as the
general characteristics of the subject of
ownership in the subject. 7. Commentary related to the subject speech
situation. The context, situation (situation). This topic
constituting
the
theme,
requires
an
explanation of the situation. The situational
topic is given in the thematic part of the
poetic speech and its essence is revealed in
the theme. (Usmon Nosir) The blue sky was covered with clouds, The old man's face was covered with anger,
A cloud came and threw three or four drops
To the old man's head
The old man was full of anger and moan,
His pale eyes were burning,
He was on fire, in a nervous tone,
His words were on his lips ... ( Uygun) The old man's face was covered with anger, 6. Commentary related to the topic of
accessibility: In the theme of such speech, the existence of
an object from the point of view of the
present is expressed by a statement and it is
asserted that the subject of poetic speech is
about this subject. In the rheme the
characteristics of the subject of existence are
referred to through interpretation. The USA Journals Volume 03 Issue 06-2021 175 The American Journal of Social Science and Education Innovations
(ISSN – 2689-100x)
Published: June 30, 2021 | Pages: 171-179
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IMPACT FACTOR
2021: 5. 857
OCLC - 1121105668 The American Journal of Social Science and Education Innovations
(ISSN – 2689-100x) IMPACT FACTOR
2021: 5. 857
OCLC - 1121105668 IMPACT FACTOR
2021: 5. 857 (
)
Published: June 30, 2021 | Pages: 171-179 (
)
Published: June 30, 2021 | Pages: 171-179
Doi : https://doi.org/10.37547/tajssei/Volume03Issue06-29 Published: June 30, 2021 | Pages: 171-179
D i htt
//d i
/
/t j
i/V l Published: June 30, 2021 | Pages: 171-179
Doi : https://doi.org/10.37547/tajssei/Volume03Issue06-29 In the rheme of this poetic passage, a
situation is given where the rhetoric is
explained in rhyme: "The blue sky is clouded,
the face of the old man is slowly overflowing
with rage." Thus, the content of the topic
arises
in
connection
with
the
general
contextual situation (situation). From this
point
of
view
on
the
subject-object
relationship in poetic speech, this type of
meaning of the subject in the subject plays an
important function. Because figurative and
affective lexemes related to the subject
require a general thematic (contextual)
situation in this context. This function is
performed by the rheme type of comment. comparable subject. In such poetic speeches,
since the signs of a person or an object are
compared (a sign in the broadest sense),
words belonging to the category of adjectives
are more often used in sentences that make
up speech, and thus a goal is set. (Usmon Nosir) In the
example above, the antonyms of good, evil,
helplessness, useness, day and night, life and
death were used to reinforce the comparison. 9. Commentary related to the theme of the
gang. The subject of a gang in the SSB topic refers to
his belonging to a certain group, gang or class,
profession. In this case, an important role is
played by the predicate related to the subject
in the theme. As A. Nurmanov points out, in
this case the predicate is represented by a
noun denoting the type (class) of the
subject[5,74]. 8. Note referring to the compared item. When this view of the subject is included in
the theme of speech, it is compared with
something - another subject, person, etc.,
Comparison expressed in the theme is further
expanded and explained in rheme. In the rheme of poetic speech, specific
common features of the type or class to which
the subject belongs are expressed in the form
of comments: The USA Journals Volume 03 Issue 06-2021 11. Commentary related to the perceived
subject. When this type of subject matter of poetic
speech is interpreted in rheme, the theme in
the subject basically refers to the perceived
person or certain creatures. Perception,
cognition, perception are shown as the main
subjective attribute of an object. The rheme of
poetic speech explains the result of the same
perception, knowledge, perception (of what
they know): A real actor is an artist whose heart is
pounding. Almost sees the day. There are people who cry with one eye
One eye smiles. I know them, they will be pure, One eye smiles. We live without realizing You're good I am bad, Do not be surprised by the poets
The poet's people are diverse. Sometimes proud, sometimes diltang,
Sometimes sad, sometimes gloomy. (E. Vohidov) Do not be surprised by the poets If you leave, I am safe. The poet's people are diverse. The poet's people are diverse. The killer life that made me Sometimes proud, sometimes diltang, Sometimes proud, sometimes diltang, Useless, helpless who created you. (E. Vohidov) Sometimes sad, sometimes gloomy. (E. Vohidov) Apparently, as a comparable topic in the
above poetic speech, “you” are involved in
this theme. The analogy expressed in the
theme continues in the rest of the speech, i.e. the author relies on the characters of two
faces (you and the lyrical protagonist) by the
method of opposition. It is characterized by
the presence of lexical elements such as
words and relative, while the subject is
comparable in sentences expressing both the
theme
and
rheme
in
speech
with
a From the given example, we can conclude
that the characteristic features of the type to
which the qualifying subject belongs in the
subject of poetic speech have been expanded
and explained. The first sentence of the above
passage implies that the subject is poets, and
the revised one gives general characteristics
of the owners of this profession (poetry) and
also explains that there are other similar signs. The USA Journals Volume 03 Issue 06-2021 176 The American Journal of Social Science and Education Innovations
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IMPAC
202 The American Journal of Social Science and Education Innovations
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(ISSN – 2689-100x) 10. A comment related to a functioning
object. The American Journal of Social Science and Education Innovations
(ISSN – 2689-100x) (
)
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Doi : https://doi.org/10.37547/tajssei/Volume03Issue06-29 certain emotional coloring, a significant effect
is provided in poetic speech. certain emotional coloring, a significant effect
is provided in poetic speech. In this case, the main function (task) of the
subject is reflected in the theme of poetic
speech. For example: harvester, harvester,
thresher. The teacher teaches students,
shares knowledge. How a hairdresser gets his
hair. However, in the rheme explains this
feature; 11. Commentary related to the perceived
subject. Next to us Evaluation on the theme of
poetic speech or attitude and evaluation of
the subject in an emotional relationship is
explained on the basis of rhyme. In this example, the field, or rather the field,
which was built as an ownerless courtyard,
acted as a locative subject, and poetic speech
played an important role in the compositional
and syntactic construction of the theme. In
the rheme describes the image of the field and
the
author's
feelings
and
experiences
associated with the field. Consequently, the
indicated system of meanings of an object is
observed in the subject of poetic speech. Rheme on the other hand, is realized mainly
through the interpretation of the meanings of
the object in the object. From the above, we
can conclude that in poetic speech, as in the
case of speech, there is a theme-rheme
relationship - an actual division. 13. Commentary related to the subject of
kinship or social relations. The theme in
the theme of poetic speech is used in this
case together with the predicate horses,
which express the meanings of kinship,
social connection. The rheme reveals the
symbols associated with the person or
object represented by the predicate, and
provides additional information about
him: 13. Commentary related to the subject of
kinship or social relations. The theme in
the theme of poetic speech is used in this
case together with the predicate horses,
which express the meanings of kinship,
social connection. The rheme reveals the
symbols associated with the person or
object represented by the predicate, and
provides additional information about
him: The synonym of the word "heart" is "sister". My heart warms when I tell you. Next to us But then, at that second These tall people will be humiliated. (Sh. Rahmon) Standing leg on the breast of the mother. (A.Oripov) In this example, it is also observed that the
subject of the theme - knowledge and
understanding of the lyrical protagonist - is
explained in rheme with the help of emotional
means and provides compositionally and
syntactically correct construction of poetic
speech. The above points are also confirmed by the
quoted passage from the poetic speech. The
functional subject in the subject of speech is
assigned the main function (function) of the
characters. In the rheme some aspects of this
function are expanded, and the characters of
the "characters" in life are shown figuratively. 12. Evaluation or comment related to the
subject in emotional relationships: in such
cases, the subject of poetic speech
expresses the value, attitude, influence
and inner feelings of the subject to
another object, subject or person. M: I
love my brother. As if he loves everyone. However, at rheme, this relationship is
gradually evolving, and their reasons are
explained and explained. The above points are also confirmed by the
quoted passage from the poetic speech. The
functional subject in the subject of speech is
assigned the main function (function) of the
characters. In the rheme some aspects of this
function are expanded, and the characters of
the "characters" in life are shown figuratively. Thus, we can observe that the presence of a The USA Journals Volume 03 Issue 06-2021 177 The American Journal of Social Science and Education Innovations
(ISSN – 2689-100x)
Published: June 30, 2021 | Pages: 171-179
Doi : https://doi.org/10.37547/tajssei/Volume03Issue06-29
IMPACT FACTOR
2021: 5. 857
OCLC - 1121105668 The American Journal of Social Science and Education Innovations
(ISSN – 2689-100x)
Published: June 30, 2021 | Pages: 171-179
Doi : https://doi.org/10.37547/tajssei/Volume03Issue06-29
IMPACT FACTOR
2021: 5. 857
OCLC - 1121105668 The American Journal of Social Science and Education Innovations
(ISSN – 2689-100x) (
)
Published: June 30, 2021 | Pages: 171-179
Doi : https://doi.org/10.37547/tajssei/Volume03Issue06-29 sister, and details and events related to the
sister in the rheme are described. 14. Commentary related to the subject of the
seat. The plot of a place is also common in poetic
speech, because logically an event or an image
is associated with a certain place: I am sorry when I see happy people. (Iqbol
Mirzo) The above points can be continued in the
quoted passage. CONCLUSION Rhema, on the other hand, consists of
sentences (lines) that form the following parts
of poetic speech. The relationship between
them serves as an important tool in the
formation of the compositional and syntactic
structure of poetic speech. This, in turn,
ensures the logical coherence of poetic
speech and the formation of poetic speech as Lovely, sister! (Iqbol Mirzo) In the above example, the subject of kinship in
the theme (the theme of poetic speech) is the The USA Journals Volume 03 Issue 06-2021 The USA Journals Volume 03 Issue 06-2021 178 The American Journal of Social Science and Education Innovations
(ISSN – 2689-100x)
Published: June 30, 2021 | Pages: 171-179
Doi : https://doi.org/10.37547/tajssei/Volume03Issue06-29
IMPACT FACTOR
2021: 5. 857
OCLC - 1121105668 The American Journal of Social Science and Education Innovations
(ISSN – 2689-100x)
IMPACT FACT
2021: 5 8 The American Journal of Social Science and Education Innovations
(ISSN – 2689-100x) (
9
)
Published: June 30, 2021 | Pages: 171-179
Doi : https://doi.org/10.37547/tajssei/Volume03Issue06-29
2021: 5. 857
OCLC - 1121105668 (
)
Published: June 30, 2021 | Pages: 171-179
Doi : https://doi.org/10.37547/tajssei/Volume03Issue06-29 REFERENCES 1. Mattius V. About the so-called actual
division of the proposal. "Praisky linguistic
circle" M., 1967. 2. Raspov
I.P. Actual
members
and
communicative
syntactic
types
of
declarative sentences in Russian. ADD. M.,
1964. 3. Amirov R.S. Stosob of an actual member in
the Kazakh language. "Soviet Turkology". 1970. No. 6, Agaeva F.M. The syntax of
Azerbaijani colloquial speech. ADD. M.,
1979. Valiev
K.N. Semantic
(actual)
purpose in the Turkic languages. Sh All-
Union Turk. Conf (abstracts and reports)
"Linguistics", Tashkent, 1980. 3. Amirov R.S. Stosob of an actual member in
the Kazakh language. "Soviet Turkology". 1970. No. 6, Agaeva F.M. The syntax of
Azerbaijani colloquial speech. ADD. M.,
1979. Valiev
K.N. Semantic
(actual)
purpose in the Turkic languages. Sh All-
Union Turk. Conf (abstracts and reports)
"Linguistics", Tashkent, 1980. 4. Khaitmetov A. Actual separation of speech
and positional issues in the Uzbek
language. Tashkent. Fan, 1984. 5. Nurmonov A.N. Syntactic theories of
speech (special course for students). Tashkent. 1988 p. 72. 6. Abdupattoev, M.(2021). COMPOSITIONAL
STRUCTURE
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MICROMATES. Конференции,
1(2). https://doi.org/10.47100/.v1i2.153. 7. Abdupattoev M. T. Unusual Connections
As Forming Literary Text //The American
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https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0191739&type=printable
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English
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Comparison of the efficacy of a commercial inactivated influenza A/H1N1/pdm09 virus (pH1N1) vaccine and two experimental M2e-based vaccines against pH1N1 challenge in the growing pig model
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PloS one
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RESEARCH ARTICLE OPEN ACCESS ¤a Current address: Animal Science, School of Environmental and Rural Science, University of New
England, Armidale, New South Wales, Australia
¤b Current address: Complexo Educacional Faculdades Metropolitana Unidas, Veterina´ria, Real Parque,
Morumbi, São Paulo, São Paulo, Brazil
* Tanja.Opriessnig@roslin.ed.ac.uk Citation: Opriessnig T, Gauger PC, Gerber PF,
Castro AMMG, Shen H, Murphy L, et al. (2018)
Comparison of the efficacy of a commercial
inactivated influenza A/H1N1/pdm09 virus (pH1N1)
vaccine and two experimental M2e-based vaccines
against pH1N1 challenge in the growing pig model. PLoS ONE 13(1): e0191739. https://doi.org/
10.1371/journal.pone.0191739 Comparison of the efficacy of a commercial
inactivated influenza A/H1N1/pdm09 virus
(pH1N1) vaccine and two experimental M2e-
based vaccines against pH1N1 challenge in
the growing pig model Tanja Opriessnig1,2*, Phillip C. Gauger2, Priscilla F. Gerber1¤a, Alessandra M. M. G. Castro2¤b, Huigang Shen2, Lita Murphy1, Paul Digard1, Patrick G. Halbur2, Ming Xia3,
Xi Jiang3,4, Ming Tan3,4 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 1 The Roslin Institute and The Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian,
United Kingdom, 2 Department of Veterinary Diagnostic and Production Animal Medicine, College of
Veterinary Medicine, Iowa State University, Ames, Iowa, United States of America, 3 Division of Infectious
Diseases, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States of America,
4 Department of Pediatrics, University of Cincinnati, College of Medicine, Cincinnati, Ohio, United States of
America ¤a Current address: Animal Science, School of Environmental and Rural Science, University of New
England, Armidale, New South Wales, Australia
¤b Current address: Complexo Educacional Faculdades Metropolitana Unidas, Veterina´ria, Real Parque,
Morumbi, São Paulo, São Paulo, Brazil
* Tanja.Opriessnig@roslin.ed.ac.uk Abstract Swine influenza A viruses (IAV-S) found in North American pigs are diverse and the lack of
cross-protection among heterologous strains is a concern. The objective of this study was to
compare a commercial inactivated A/H1N1/pdm09 (pH1N1) vaccine and two novel subunit
vaccines, using IAV M2 ectodomain (M2e) epitopes as antigens, in a growing pig model. Thirty-nine 2-week-old IAV negative pigs were randomly assigned to five groups and rooms. At 3 weeks of age and again at 5 weeks of age, pigs were vaccinated intranasally with an
experimental subunit particle vaccine (NvParticle/M2e) or a subunit complex-based vaccine
(NvComplex/M2e) or intramuscularly with a commercial inactivated vaccine (Inact/pH1N1). At 7 weeks of age, the pigs were challenged with pH1N1 virus or sham-inoculated. Nec-
ropsy was conducted 5 days post pH1N1 challenge (dpc). At the time of challenge one of
the Inact/pH1N1 pigs had seroconverted based on IAV nucleoprotein-based ELISA, Inact/
pH1N1 pigs had significantly higher pdm09H1N1 hemagglutination inhibition (HI) titers com-
pared to all other groups, and M2e-specific IgG responses were detected in the NvParticle/
M2e and the NvComplex/M2e pigs with significantly higher group means in the NvComplex/
M2e group compared to SHAMVAC-NEG pigs. After challenge, nasal IAV RNA shedding
was significantly reduced in Inact/pH1N1 pigs compared to all other pH1N1 infected groups
and this group also had reduced IAV RNA in oral fluids. The macroscopic lung lesions were
characterized by mild-to-severe, multifocal-to-diffuse, cranioventral dark purple consoli-
dated areas typical of IAV infection and were similar for NvParticle/M2e, NvComplex/M2e Introduction Influenza A virus (IAV), a genus within the family Orthomyxoviridae, is a negative sense, sin-
gle-stranded RNA virus. The IAV genome consists of eight segments that encode a minimum
of 10 viral proteins. The glycoproteins hemagglutinin and neuraminidase are located on the
surface of the virus and often used for characterization of IAV strains [1]. These glycoproteins
are responsible for viral entry and subsequent viral release from the infected cells, are highly
variable and are the major targets of the host humoral immune response [1]. Swine IAV (IAV-S) is considered one of the most important causes of acute respiratory dis-
eases in U.S. swine herds. Infection of pigs with IAV-S typically manifests as an acute, highly
contagious disease that often affects an entire herd in a short period of time [2]. The main clin-
ical signs of IAV-S infection are depression, fever, anorexia, coughing and dyspnea [2]. Mor-
tality can vary from 1–4%. In addition, IAV-S is considered a zoonotic pathogen and
transmission between pigs and humans occurs on a regular basis [3,4] making IAV-S an
important public health consideration. The populations of IAV found in North American pigs
are very diverse and include H3N2, H1N1 and H1N2 viruses with a more limited distribution
of other subtypes. During 2009, a worldwide influenza pandemic with a novel swine-derived
A/H1N1/pdm09 (pH1N1) occurred in humans [5] and the virus also re-entered the pig popu-
lation where it continues to spread. Commercial inactivated vaccines against pH1N1 are avail-
able to date for pigs and it has also been shown that vaccination of swine workers had a
beneficial effect on minimizing IAV-S transmission between farms [6]. Vaccination, while often used in U.S. pigs, is not always effective due to the heterogeneity of
circulating IAV-S strains. All of the licensed, commercial IAV-S vaccines currently available in
the U.S. contain inactivated virus or subunit virus particles. Influenza virus is cultivated in
eggs or cell culture and inactivated with chemical agents, such as formaldehyde or binary ethy-
lenimine for use in vaccines [7]. There is a need for novel and universal vaccine strategies capa-
ble of cross-protection against genetically different IAV-S strains. Recently, two polyvalent vaccine platforms based on the norovirus (Nv) capsid protein have
been developed [8,9] and utilized for the development of a universal IAV-S vaccine [10,11]. Influenza A virus vaccination in the pig model and SHAMVAC-IAV pigs. Lesions were significantly less severe in the SHAMVAC-NEG and
the Inact/pH1N1pigs. Under the conditions of this study, a commercial Inact/pH1N1 specific
vaccine effectively protected pigs against homologous challenge as evidenced by reduced
clinical signs, virus shedding in nasal secretions and oral fluids and reduced macroscopic
and microscopic lesions whereas intranasal vaccination with experimental M2e epitope-
based subunit vaccines did not. The results further highlight the importance using IAV-S
type specific vaccines in pigs. 1; BBS/E/D/20241864) and support from a US-UK
BBSRC-NIFA Collaboration grant (BB/M027163/1). 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. Competing interests: The authors have declared
that no competing interests exist. Editor: Victor C Huber, University of South Dakota,
UNITED STATES Editor: Victor C Huber, University of South Dakota,
UNITED STATES Received: October 27, 2017
Accepted: January 10, 2018
Published: January 30, 2018 Copyright: © 2018 Opriessnig 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 and its Supporting Information
files. Funding: The study was funded by the Iowa
Livestock Health Advisory Council (ILHAC). Additional funding was received by the
Biotechnology and Biological Sciences Research
Council (BBSRC) Institute Strategic Programme
Grant awarded to the Roslin Institute (BB/J004324/ 1 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0191739
January 30, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0191739
January 30, 2018 Ethical statement The Iowa State University Institutional Animal Care and Use Committee approved this study
(Approval number 3-16-8220-S). The efforts to alleviate suffering in this study included
defined endpoints for each pig and independent veterinary supervision for the duration of the
project. Pigs and experimental design Thirty-nine 3-week-old pigs were obtained from an IAV-S-free herd and were randomly
assigned to five rooms with 7–8 pigs in each group (Fig 1). Water and feed were provided ad
libidum. Vaccination or sham-vaccination was done at 3 and 5 weeks of age followed by chal-
lenge or sham-challenge at 7 weeks of age. All pigs were euthanized 5 days post challenge
(dpc). Blood was collected in serum separator tubes (Fisher Scientific, Pittsburgh, Pennsylva-
nia, USA) weekly until challenge and at dpc 5. Blood tubes were centrifuged at 3000×g for 10
min at 4˚C and serum was stored at -80˚C until testing. Nasal swabs were collected individu-
ally from each pig on dpc 0, 1, 2, 3, 4 and 5 using polyester swabs and were stored in 5 ml plas-
tic tubes containing 1 ml of sterile saline solution at -80˚C until testing. Clinical assessment All pigs were weighed at vaccination 1 (dpc -28), at challenge (dpc 0) and at necropsy (dpc 5)
and the average daily weight gain was calculated. Rectal temperature, nasal discharge, cough
and respiratory scores were assessed every day starting on dpc 0 (Fig 1). Nasal discharge was
recorded as 0 = none, 1 = mild and 2 = severe and if present the location (left nostril, right nos-
tril, both), color (clear, yellow, white) and consistency (watery, mucoid) were also noted. The
cough score ranged from 0 = absent, 1 = single cough, and 2 = persisting cough, and respira-
tory scores ranged from 0 = normal to 6 = severe respiratory distress when at rest [15]. Influenza A virus vaccination in the pig model with large, higher order complexes assembling spontaneously through dimerization and/or
oligomerization of the homologous antigen [11,14]. Both P particle- (NvParticle/M2e) and
large complex-based (NvComplex/M2e) vaccines containing the M2e epitope have been pro-
duced in E. coli with high yield and high stability. Mouse experiments revealed that both vac-
cines were highly immunogenic and were able to fully protect vaccinated mice against IAV
lethal challenge using a mouse-adapted human IAV (H1N1, A/PR/8/34 [PR8] strain) [11,14]. The objective of this study was to determine the efficacy of a commercial inactivated
pH1N1 vaccine administered intramuscularly and two experimental M2e subunit vaccines
administered intranasally in protecting pigs from the effects of pH1N1 challenge. with large, higher order complexes assembling spontaneously through dimerization and/or
oligomerization of the homologous antigen [11,14]. Both P particle- (NvParticle/M2e) and
large complex-based (NvComplex/M2e) vaccines containing the M2e epitope have been pro-
duced in E. coli with high yield and high stability. Mouse experiments revealed that both vac-
cines were highly immunogenic and were able to fully protect vaccinated mice against IAV
lethal challenge using a mouse-adapted human IAV (H1N1, A/PR/8/34 [PR8] strain) [11,14]. The objective of this study was to determine the efficacy of a commercial inactivated
pH1N1 vaccine administered intramuscularly and two experimental M2e subunit vaccines
administered intranasally in protecting pigs from the effects of pH1N1 challenge. Introduction The M2 protein is integrated into the viral envelope of IAV and its ion channel activity is
required for efficient viral uncoating during virus invasion of cells. The M2e epitope, which is
23 amino acid residues in length, is the extracellular moiety of the M2 protein. This epitope is
abundantly expressed on the surface of IAV-infected cells and M2e domains are highly con-
served among IAV strains, making it an attractive target for a universal vaccine [12,13]. The
protruding domain (P) particle platform is based on P particles which each contain 24 copies
of Nv P domains with three exposed loops on the outermost surface in which the M2e epitope
has been inserted [8]. The large complex-based platform is based on fusion of 2–3 dimeric/
oligomeric antigens that are fused into one molecule through recombinant DNA technology 2 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0191739
January 30, 2018 Vaccines and vaccination Vaccine details are summarized in Table 1. The vaccine used for the NvParticle/M2e group
was a P particle-based vaccine [8,10] containing a pig IAV M2e epitope (SLLTEVETPIRNG
WECKCNDSSD) [16]. The vaccine used for the NvComplex/M2e group was an agglomerate
polymer-based (GST-P+) vaccine [11] containing a human IAV consensus M2e epitope
(SLLTEVETPIRNEWGCRCNDSSD) [8,17]. Both vaccines were expressed in E. coli. The two
M2e epitope sequences share 82% identity or 91% similarity. The vaccine used for the Inact/
pH1N1 group was a commercially available inactivated IAV-S vaccine purchased from MWI
Veterinary Supply (Boise, ID, USA). 3 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0191739
January 30, 2018 Influenza A virus vaccination in the pig model Fig 1. Experimental design. Vaccinations and sham-vaccinations were done when the pigs were 3 weeks old and repeated at 5 weeks
of age. All pigs except the SHAMVAC-NEG group were challenged with IAV at 7 weeks of age. Necropsy was done at 5 days post
challenge. Fig 1. Experimental design. Vaccinations and sham-vaccinations were done when the pigs were 3 weeks old and repeated at 5 weeks
of age. All pigs except the SHAMVAC-NEG group were challenged with IAV at 7 weeks of age. Necropsy was done at 5 days post
challenge. https://doi.org/10.1371/journal.pone.0191739.g001 https://doi.org/10.1371/journal.pone.0191739.g001 All pigs in the NvParticle/M2e group, NvComplex/M2e group and Inact/pH1N1group
were vaccinated at dpc -28 and again at dpc -14. Pigs in the NvParticle/M2e and NvComplex/
M2e groups were vaccinated intranasally with 2 ml of the P-particle vaccine or the complex
vaccine, each containing 50 μg of protein vaccine by using a mucosal atomization device
(MAD300, American Medical; Green Bay, WI, USA). While in previous mouse studies no
adjuvants were used, in this study the vaccines were prepared by mixing 500 μl of the respec-
tive protein stock at a concentration of 1 mg/ml and 1.5 ml Polyinosinic-polycytidylic acid Table 1. Details on the vaccines or sham vaccines used in the different treatment groups. Group
Type
Antigen
Platform
Vaccination route
NvParticle/M2e
Subunit
M2e epitope
Nv P particle3
Intranasally
NvComplex/M2e
Subunit
M2e epitope
Nv Complex4
Intranasally
Inact/pH1N1
Commercial inactivated1
Whole pH1N1 virus2
NA5
Intramuscularly
SHAMVAC-IAV
Sham
Saline
NA
Intramuscularly
SHAMVAC-NEG
Sham
Saline
NA
Intramuscularly
1 FluSure1 Pandemic (Zoetis) Table 1. Details on the vaccines or sham vaccines used in the different treatment groups. A/California/04/2009 (pH1N1). 3 Nv = norovirus, P = protruding domain. Influenza A virus vaccination in the pig model potassium salt adjuvant (PolyI:C; Sigma-Aldrich) at a concentration of 156 μg/ml shortly
before administration to the pigs. Previously, PolyI-C adjuvant has been shown to enhance
immune responses against IAV after intranasal administration [18]. Pigs in the Inact/
pH1N1group were vaccinated with 2 ml of FluSure1 Pandemic (Zoetis; serial numbers
149390 and 120823) intramuscularly into the neck area after rehydration of the freeze-dried
vaccine as recommended by the manufacturer. Serology To assess the antibody response against IAV-S in serum samples, a commercial IAV-S block-
ing ELISA (IDEXX Swine Influenza Virus Ab Test; IDEXX Inc., Westbrook, MA, USA) was
used according to the manufacturer’s instructions. This ELISA detects antibodies directed
against the IAV nucleoprotein (NP). A sample-to-negative control (S/N) ratio less than 0.6
was considered positive. In addition to serum, this assay was also used on oral fluids and
bronchoalveolar lavage (BAL) fluid with the following modifications: each plate was loaded
with 200 μl undiluted OF or BAL fluid and incubated for 16 h at 21˚C [20]. Serum, OF and
BAL fluid reactions were measured as optical density (OD) at a wavelength of 650 nm using an
ELISA plate reader. Sample-to-negative (S/N) ratios were calculated as described by the manu-
facturer, with S/N ratios of 0.60 considered antibody positive. For the OF test interpretation,
in addition to the S/N ratio cut-off of 0.60 as suggested for serum samples by the manufac-
turer, results were also obtained and evaluated using a S/N ratio cut-off of 0.65 as suggested
by a previous study [20]. In addition, serum samples collected at challenge (dpc 0) and nec-
ropsy (dpc 5) were also tested by a hemagglutinin inhibition (HI) assay. In brief, sera were heat
inactivated at 56˚C for 30 min to remove nonspecific hemagglutinin inhibitors and natural
serum agglutinins, according to standard techniques used at the Iowa State University Veteri-
nary Diagnostic Laboratory. The HI assay was performed using pH1N1antigen and turkey red
blood cells. pH1N1 challenge The pH1N1 isolate A/California/04/2009 was selected for challenge at dpc 0. The pigs were
anesthetized with an intramuscular administered cocktail of ketamine (8 mg/kg of body
weight), xylazine (4 mg/kg), and Telazol (6 mg/kg) as described [19] and challenged intratra-
cheally (2 ml) and intranasally (1 ml) with the pdm09H1N1 strain at a dose of 2 × 105 TCID50. Pigs in the SHAMVAC-NEG group were challenged by the same routes using similar amounts
of saline. Vaccines and vaccination Each P particle contains 24 copies of NvV P domains with three exposed loops on the outermost surface in which the M2e
epitope has been inserted. /
/
/
(p
)
3 Nv = norovirus, P = protruding domain. Each P particle contains 24 copies of NvV P domains with three exposed loops on the outermost surface in which the M2e
epitope has been inserted. p
p
4 Two to three dimeric/oligomeric antigens are fused into one molecule through recombinant DNA technology. The large, higher-order complexes of the fused antigens
assemble spontaneously through dimerization and/or oligomerization of the homologous antigen. 5 Not applicable 4 Two to three dimeric/oligomeric antigens are fused into one molecule through recombinant DNA technology. The large, higher-order complexes of the fused antigens
assemble spontaneously through dimerization and/or oligomerization of the homologous antigen. 5 Not applicable. 4 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0191739
January 30, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0191739
January 30, 2018 Detection and quantification of IAV-S specific nucleic acids and virus
titration Total nucleic acids were extracted from nasal swabs, oral fluids and bronchoalveolar lavage
(BAL) samples using a MagMAX-96 viral isolation kit (Applied Biosystems, Life Technologies,
Carlsbad, CA, USA) on a KingFisher™Flex platform (ThermoFisher Scientific, Pittsburgh, PA,
USA). Appropriate negative and positive controls were included in each extraction run. A
quantitative reverse transcriptase (RT) real-time PCR based on the N1 gene specific for pH1
NA was performed on the RNA extracts. In addition, selected samples were also tested by a
real-time PCR based on the M gene [22]. Virus titration was done on nasal swabs and BAL flu-
ids as described [23] and titers were presented as TCID50 per ml. Necropsy and gross lung lesions On dpc 5, all pigs were euthanized by intravenous pentobarbital overdose (FATAL-PLUS1,
Vortech Pharmaceuticals LTD, Dearborn, MI, USA) and necropsied. The percentage of lung
surface affected by visible lesions was assessed by a pathologist blinded to the pig treatment sta-
tus using an established protocol [15]. BAL fluids were collected from each pig using minimal
essential medium (MEM, Fisher Scientific). In addition, lung sections from the right cranial,
right middle and accessory lung lobes and a section of distal trachea were collected. The sections
were immersed in 10% neutral-buffered formalin and routinely processed for histopathology. Influenza A virus vaccination in the pig model (horse reddish peroxidase)-conjugated goat anti-Pig IgG (for serum IgG) and HRP-conjugated
goat anti-Pig IgA (for IgA in the lung wash) as secondary antibodies (Abcam, Cambridge, MA,
USA). Antigen-specific antibody titers were defined as the endpoint dilution with a cut off sig-
nal intensity of 0.2. Serum samples collected at the day of challenge were also characterized by western blot uti-
lizing lysates from MDCK cells that had been infected with either PR8 virus or a PR8 7:1 reas-
sortant with segment 7 from the pH1N1 strain A/California/ 07/2009 at an MOI of 5 as targets
[21]. Cell lysates were harvested 24 hours post infection and run on SDS-PAGE gradient gels
(4–20%) for separation of proteins before transferring to nitrocellulose membranes. Mem-
branes were probed with a rabbit polyclonal anti-NP serum [21] to confirm infection and
mouse monoclonal antibody 14C2 (Abcam) for detection of the PR8 M2 protein followed by
rabbit and mouse secondary antibodies (1:20,000) as appropriate. Serum samples were tested
using the Mini Protean II multiscreen (Biorad) with secondary swine antibodies (1:10,000). Membranes were developed by chemiluminescence using ECL Plus (Amersham). Histopathology and immunohistochemistry Tissue sections were fixed and microscopic lung lesions were assessed by a veterinary patholo-
gist blinded to treatment status and scored for presence and degree of tracheitis, tracheal epi-
thelial flattening or attenuation, bronchi and bronchiolar epithelial changes and
peribronchiolar lymphocytic cuffing ranging from 0 = none to 4 = severe as described [23]. Presence and amount of IAV-S specific antigen in tissue sections were assessed by immunohis-
tochemistry (IHC) as described [24,25]. The IAV antigen scores ranged from 0 = absent to
4 = abundant diffuse signals. Scoring was done blinded to the treatment status. Demonstration of M2e specific antibodies An in-house M2e ELISA to detect pig specific antibodies was performed as described elsewhere
[10] and with modifications tested on lavage samples collected at necropsy for presence of
anti-M2e IgA levels and on serum samples collected at challenge for anti-M2e IgG levels. Briefly, two synthetic M2e peptides, SLLTEVETPIRNGWECKCNDSSD for the NvParticle/M2e
vaccine and SLLTEVETPIRNEWGCRCNDSSDfor the NvComplex/M2e vaccine (3 μg/ml,
Ohio peptide, USA), were used as detection antigens for M2e specific antibodies. Sera and
lung washes were initially diluted to 1:25 (for lung washes) or 1:100 (for sera) and then 2-fold
serial dilutions were made in 5% non-fat dry milk in PBS (pH 7.4) for the end-point titer deter-
mination. The M2e peptides were coated on 96 well microtiter plates (Nunc MaxiSorp1,
USA) (200 ng/well) at 4˚C for overnight. Before detection, the coated plates were blocked with
200 μl of 5% non-fat dry milk in PBS for 1 h at 37˚C. After washing, diluted sera or lung washes
(100 μl) were added to the plates for 1 h at 37˚C. The bound antibody was detected by the HRP USA) (200 ng/well) at 4˚C for overnight. Before detection, the coated plates were blocked with
200 μl of 5% non-fat dry milk in PBS for 1 h at 37˚C. After washing, diluted sera or lung washes
(100 μl) were added to the plates for 1 h at 37˚C. The bound antibody was detected by the HRP 5 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0191739
January 30, 2018 Influenza A virus vaccination in the pig model treatment groups and pair-wise comparison were performed by least significant difference. The rejection level for the null hypothesis was P < 0.05. Pairwise testing was performed using
Tukey-Kramer adjustment to identify different groups. Non-repeated measures were assessed
using nonparametric Kruskal-Wallis ANOVA. When group variances were different, pair-
wise comparisons were performed using the Wilcoxon rank sum test. Differences in incidence
were evaluated by using Fisher’s exact test. Clinical disease No clinical signs of respiratory disease were observed in any of the pigs prior to challenge or in
the SHAMVAC-NEG pigs for the duration of the experiment. The average daily gain in
grams ± SEM from the time of challenge until necropsy was 279.7±59.2 for the SHAMVAC--
NEG pigs, 224.0±36.8 for the NvComplex/M2e pigs, 234.4±87.7 for the NvParticle/M2e pigs,
172.0±23.3 for the Inact/pH1N1 pigs and 207.9±54.6 for the SHAMVAC-IAV pigs which was
not significantly different (P = 0.715). In IAV infected pigs, coughing was first observed in
SHAMVAC-IAV and NvComplex/M2e pigs by dpc 2 (Fig 2A) in NvParticle/M2e pigs by dpc
3 and in Inact/pH1N1by dpc 5. Overall, 5/7 NvParticle/M2e pigs, 4/8 NvComplex/M2e pigs,
2/8 Inact/pH1N1pigs and 5/8 SHAMVAC-IAV pigs were observed coughing for 1–3 consecu-
tive days and the average number of days with coughing was significantly higher for SHAM-
VAC-IAV pigs (1.5±0.4) compared to SHAMVAC-NEG pigs (0). Nasal discharge was
observed in 4/7 NvParticle/M2e pigs, 5/8 NvComplex/M2e pigs, 2/8 Inact/pH1N1pigs and 8/8
SHAMVAC-IAV pigs for 1–3 days. The average number of days a pig was scored with a respi-
ratory score of 1 or greater was 2.0±0.7 for NvParticle/M2e pigs, 1.8±0.5 for NvComplex/M2e
pigs, 1.4±0.3 for Inact/pH1N1pigs, 3.3±0.4 for SHAMVAC-IAV pigs and 0.3±0.2 for SHAM-
VAC-NEG pigs. The group mean respiratory scores over time are summarized in Fig 2B and
the mean group rectal temperatures are presented in Fig 2C. For the purpose of this study,
fever was defined as a rectal temperature greater than 40.5˚C. Two of 7 NvParticle/M2e pigs,
2/8 NvComplex/M2e pigs and 3/8 SHAMVAC-IAV pigs had fevers by dpc 4 and dpc 5 lasting
1–2 days without group mean differences among all groups. PLOS ONE | https://doi.org/10.1371/journal.pone.0191739
January 30, 2018 Statistics RNA genomic copies in nasal swabs and HI titers were log transformed prior to analysis. A
one-way analysis of variance (ANOVA) was utilized to detect significant differences among 6 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0191739
January 30, 2018 ntly (P < 0.05) different group mean S/N values or HI titers at a dpc. of pigs per group [26] (mean log2 transformed pH1N1 HI titers ± SEM). p
2 Pigs with an HI titer > 40/total number of pigs per group [26] (mean log2 transformed pH1N1 HI titers ± SEM). Serology SHAMVAC-NEG pigs remained seronegative for IAV NP throughout the duration of the
study (Table 2). At the dpc 0, 1/7 NvParticle/M2e and 1/8 Inact/pH1N1pigs had S/N ratios less
than 0.6 and were considered positive. By dpc 5, the group mean Inact/pH1N1 ELISA value
was significantly lower compared to all other groups which is indicative of seroconversion and
7/8 Inact/pH1N1 pigs were considered NP-seropositive based on the assay cutoff. In BAL
fluid, 1/8 SHAMVAC-IAV pigs were ELISA positive (Table 2). Using a pH1N1 specific HI
assay, SHAMVAC-NEG pigs remained seronegative for pH1N1 surface antigens throughout
the duration of the trial (Table 2). Inact/pH1N1 pigs had significantly higher pH1N1 HI titers
compared to all other groups by dpc 0 and 5. An in house assay was used to test for specific
M2e IgA (BAL fluid) and IgG (serum samples) antibodies and a western blot assay was used to
test for antibodies against the M2e PR8/Cal07 epitopes in serum samples. All samples were
negative by the M2e IgA assay but IgG M2e specific antibody responses were detected in the
NvParticle/M2e and the NvComplex/M2e pigs. Specifically, titers as high as 1:12800 were
observed in1/7 NvParticle/M2e pigs and in 2/8 NvComplex/M2e pigs at the time of challenge. Moreover, the log transformed group mean in the NvComplex/M2e group was significantly
higher compared to that of the SHAMVAC-NEG pigs (3.8±0.2 versus 3.5±0.1). 7 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0191739
January 30, 2018 p
2 Pigs with an HI titer > 40/total number of pigs per group [26] (mean log2 transformed pH1N1 HI titers ± SEM). 3 Different superscripts indicate significantly (P < 0.05) different group mean S/N values or HI titers at a dpc. p
2 Pigs with an HI titer > 40/total number of pigs per group [26] (mean log2 transformed pH1N1 HI titers ± SEM).
3 Different superscripts indicate significantly (P < 0.05) different group mean S/N values or HI titers at a dpc. roup (mean group sample-to-negative (S/N) nucleoprotein blocking ELISA ratios ± SEM). An S/N ratio less than 0.6 was pts indicate significantly (P < 0.05) different group mean S/N values or HI titers at a dpc. Virus levels in nasal secretions and in lungs In nasal swabs collected daily after challenge, virus was isolated from NvParticle/M2e pigs (5/7
on dpc 1, 0/7 on dpc 2, 3/7 on dpc 3, 2/7 on dpc 4 and on dpc 5), NvComplex/M2e pigs (5/8
on dpc 1, 5/8 on dpc 2, 2/8 on dpc 3, 2/8 on dpc 4 and 1/8 on dpc 4) and the SHAMVAC-IAV
pigs (7/8 on dpc 1, 7/8 on dpc 2, 2/8 on dpc 3, 5/8 on dpc 4 and 4/8 on dpc 5) at certain days. At necropsy, 85.7% (6/7) of the NvParticle/M2e pigs, 50% (4/8) of the NvComplex/M2e pigs
and 62.5% (5/8) of the SHAMVAC-IAV pigs had viable IAV virus in BAL fluids. All Inact/
pH1N1pigs were negative for infectious IAV in nasal swabs and BAL fluids. Influenza A virus vaccination in the pig model Fig 3. Mean group log10 IAV RNA genomic copies ±SEM. A. Nasal swabs were collected at day post challenge (dpc) 1–5 and bronchoalveolar lavage (BAL) fluid was
collected on dpc 5. Different superscripts (A,B) on certain dpc indicate significantly (P < 0.05) different group means. B. Pen/group based oral fluid samples collected at
dpc 1–5. Fig 3. Mean group log10 IAV RNA genomic copies ±SEM. A. Nasal swabs were collected at day post challenge (dpc) 1–5 and bronchoalveolar lavage (BAL) fluid was
collected on dpc 5. Different superscripts (A,B) on certain dpc indicate significantly (P < 0.05) different group means. B. Pen/group based oral fluid samples collected at
dpc 1–5. https://doi.org/10.1371/journal.pone.0191739.g003 Presence of IAV RNA The amounts of IAV RNA in individual pigs are summarized for each group in S1 Fig. The
group mean log10 IAV RNA levels in nasal swabs and BAL fluid are summarized in Fig 3A. IAV RNA was not detected in any of the SHAMVAC-NEG group samples and by dpc 2 mean
group IAV RNA levels in nasal swabs and also in BAL fluids were significantly lower in Inact/
pH1N1pigs compared to all other IAV infected groups. The group mean log10 IAV RNA levels
in oral fluids are summarized in Fig 3B. There was a similar trend as seen with nasal swabs
with Inact/pH1N1pigs having lower levels of IAV RNA in oral fluids compared to all other
IAV infected pigs. Influenza A virus vaccination in the pig model Fig 2. Mean group clinical assessment on the day of IAV challenge and on day post challenge (dpc) 1–5. A. Presence of cough ranging from 0 = absent to
2 = persisting cough. B. Respiratory score ranging from 0 = normal to 6 = severe respiratory distress when at rest. C. Rectal temperature. Different superscripts (A,B) on
certain dpc indicate significantly (P < 0.05) different group means. https://doi.org/10.1371/journal.pone.0191739.g002 Fig 2. Mean group clinical assessment on the day of IAV challenge and on day post challenge (dpc) 1–5. A. Presence of cough ranging from 0 = absent to
2 = persisting cough. B. Respiratory score ranging from 0 = normal to 6 = severe respiratory distress when at rest. C. Rectal temperature. Different superscripts (A,B) on
certain dpc indicate significantly (P < 0.05) different group means. htt
//d i
/10 1371/j
l
0191739 002 https://doi.org/10.1371/journal.pone.0191739.g002 by a commercial blocking ELISA or a pdm09H1N1 specific HI assay on serum samples or bronchoalveolar lavage (BAL)
8, 0, and 5 days after pdm09H1N1 challenge. Table 2. Antibody responses as determined by a commercial blocking ELISA or a pdm09H1N1 specific HI assay on s
fluid in the different treatment groups at -28, 0, and 5 days after pdm09H1N1 challenge. Table 2. Antibody responses as determined by a commercial blocking ELISA or a pdm09H1N1 specific HI assay on serum samples or bronchoalveolar lavage (BAL)
fluid in the different treatment groups at -28, 0, and 5 days after pdm09H1N1 challenge. Group
-28 (Serum)
0 (Serum)
5 (Serum)
5 (BAL fluid)
NP ELISA1
NP ELISA1
pH1N1 HI2
NP ELISA1
pH1N1 HI2
NP ELISA1
NvParticle/M2e
0/7 (0.96±0.02)A,3
1/7 (0.94±0.06)AB
0/7 (3.1±1.9)A
0/7 (0.80±0.02)A
0/7 (3.8±1.5)A
0/7 (1.20±0.13)A
NvComplex/M2e
0/8 (1.00±0.01)A
0/8 (0.93±0.02)AB
0/8 (1.8±2.0)A
0/8 (0.83±0.03)A
0/8 (3.4±1.9)A
0/8 (1.29±0.12)A
Inact/pH1N1
0/8 (0.95±0.02)A
1/8 (0.74±0.08)A
8/8 (8.3±1.1)B
7/8 (0.42±0.05)B
8/8 (8.5±1.0)B
0/8 (0.88±0.05)A
SHAMVAC-IAV
0/8 (0.96±0.02)A
0/8 (0.94±0.04)AB
0/8 (1.5±1.8)A
0/8 (0.84±0.03)A
0/8 (4.2±1.5)A
1/8 (1.10±0.17)A
SHAMVAC-NEG
0/8 (0.94±0.01)A
0/8 (0.95±0.02)B
0/8 (2.2±2.0)A
0/8 (0.94±0.02)A
0/8 (2.1±1.8)A
0/8 (0.94±0.05)A 1 Positive pigs/total number of pigs per group (mean group sample-to-negative (S/N) nucleoprotein blocking ELISA ratios ± SEM). An S/N ratio less than 0.6 was
considered positive. PLOS ONE | https://doi.org/10.1371/journal.pone.0191739
January 30, 2018 8 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0191739
January 30, 2018 Lesions and IAV antigen in tissue sections Macroscopic lung lesions associated with IAV infection were characterized by mild-to-severe,
multifocal-to-diffuse, cranioventral, dark-purple consolidated areas (Fig 4); however, lesions
were essentially absent in Inact/pH1N1pigs. Microscopically there was necrosis and flattening
of the epithelium lining airways associated with accumulation of inflammatory cells in the
lung and distal trachea. These lesions were almost always associated with large amounts of
IAV antigen as determined by IHC stains (Table 3). 9 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0191739
January 30, 2018 Influenza A virus vaccination in the pig model Fig 4. Macroscopic lung lesions at 5 days post IAV challenge. A. SHAMVAC-NEG pig 229: There are no visible lung lesions present (Score: 0). B. SHAMVAC-IAV
pig 244: Dorsoventral there is diffuse severe dark purple consolidation present (Score: 30.1). C. NvParticle/M2e pig 239: Dorsoventral there is diffuse severe dark
purple consolidation present (Score: 33.5). D. NvComplex/M2e pig 232: Dorsoventral there is diffuse severe dark purple consolidation present (Score: 31.1). E. Inact/
pH1N1pig 234: There are no visible lung lesions present (Score: 0). Fig 4. Macroscopic lung lesions at 5 days post IAV challenge. A. SHAMVAC-NEG pig 229: There are no visible lung lesions present (Score: 0). B. SHAMVAC-IAV
pig 244: Dorsoventral there is diffuse severe dark purple consolidation present (Score: 30 1) C NvParticle/M2e pig 239: Dorsoventral there is diffuse severe dark Fig 4. Macroscopic lung lesions at 5 days post IAV challenge. A. SHAMVAC-NEG pig 229: There are no visible lung lesions present (Score: 0). B. SHAMVAC-IAV
pig 244: Dorsoventral there is diffuse severe dark purple consolidation present (Score: 30.1). C. NvParticle/M2e pig 239: Dorsoventral there is diffuse severe dark
purple consolidation present (Score: 33.5). D. NvComplex/M2e pig 232: Dorsoventral there is diffuse severe dark purple consolidation present (Score: 31.1). E. Inact/
pH1N1pig 234: There are no visible lung lesions present (Score: 0). Fig 4. Macroscopic lung lesions at 5 days post IAV challenge. A. SHAMVAC-NEG pig 229: There are no visible lung lesions present (Score: 0). B. SHAMVAC-IAV
pig 244: Dorsoventral there is diffuse severe dark purple consolidation present (Score: 30.1). C. NvParticle/M2e pig 239: Dorsoventral there is diffuse severe dark
purple consolidation present (Score: 33.5). D. NvComplex/M2e pig 232: Dorsoventral there is diffuse severe dark purple consolidation present (Score: 31.1). E. Inact/
pH1N1pig 234: There are no visible lung lesions present (Score: 0). https://doi.org/10.1371/journal.pone.0191739.g004 https://doi.org/10.1371/journal.pone.0191739.g004 https://doi.org/10.1371/journal.pone.0191739.g004 https://doi.org/10.1371/journal.pone.0191739.g004 PLOS ONE | https://doi.org/10.1371/journal.pone.0191739
January 30, 2018 Influenza A virus vaccination in the pig model centage of the lung surface affected by lesions) and prevalence and group mean microscopic lung lesions and
al stains 5 days after pdm09H1N1 challenge. Table 3. Mean group macroscopic lung lesions (percentage of the lung surface affected by lesions) and prevalence and group mean microscopic lung lesions and
IAV antigen as determined by immunohistochemical stains 5 days after pdm09H1N1 challenge. Group
Macroscopic lung
lesions
Microscopic lung lesions1 and IAV antigen
Microscopic tracheal lesions1 and IAV antigen
Peribronchiolar cuffing Epithelial
necrosis
Lung IHC2
Tracheal epithelial
flattening
Tracheitis
Trachea IHC2
NvParticle/M2e
15.4±3.7A,3
6/7 (1.6±0.3)A
7/7 (2.1±0.2)A
7/7 (2.0
±0.3)A
3/7 (0.7±0.4)A
5/7 (1.0
±0.3)A
3/7 (0.6
±0.3)A
NvComplex/M2e
14.3±3.7A
8/8 (2.6±0.4)B
8/8 (2.9±0.3)A
8/8 (2.8
±0.3)A
5/8 (1.3±0.4)A
7/8 (1.3
±0.4)A
8/8 (1.6
±0.3)A
Inact/pH1N1
0.1±0.1B
1/8 (0.1±0.1)C
0/8 (0)B
0/8 (0)B
0/8 (0)B
0/8 (0)B
0/8 (0)B
SHAMVAC-IAV
11.8±3.2A
8/8 (1.8±0.3)AB
8/8 (2.3±0.3)A
8/8 (2.8
±0.3)A
3/8 (1.0±0.3)A
5/8 (1.0
±0.3)A
5/8 (0.6
±0.2)A
SHAMVAC-NEG
0.1±0.0B
0/8 (0.1±0.1)C
0/8 (0)B
0/8 (0)B
0/8 (0)B
0/8 (0)B
0/8 (0)B
1 Score range from 0 = normal to 4 = severe. 2 Score range from 0 = not present to 4 = abundant. 3 Different superscripts indicate significantly (P < 0.05) different group mean values. Having access to a broadly reactive “universal” IAV vaccine would be a great asset to pig
producers. Universal influenza vaccines have been reported previously, including self-assem-
bling protein nanoparticles displaying M2e and Helix C with incorporation of TLR5 agonist
flagellin, which has been shown to protect mice from lethal IAV challenge and also induced
high levels of antibodies in chickens [28]. A novel M2e-tetra-branched multiple antigenic pep-
tide based vaccine was evaluated in the mouse model and shown to be effective in protecting
mice against heterologous strains [29]. Baculovirus constructs expressing HA fused to swine
IgG2a Fc, displayed in a FeLV gag VLP, or displayed in the baculoviral envelope were gener-
ated and used to vaccinate pigs which elicited robust HI titers in serum and reduced lung
lesions and prevalence of virus after challenge [30]. Previously, the same subunit vaccines
tested in pigs in this study were used in the mouse model by vaccinating mice three times
intranasally at 2-week intervals and using the mouse adapted H1N1 PR8 strain. The results
demonstrated that the polyvalent GST-Nv P−-M2e vaccine fully protected mice against lethal
challenge [14]. Discussion In this study, two novel experimental and broadly-reactive subunit IAV vaccines previously
tested in mice [8–11] were compared to a commercially available conventional inactivated
type-specific IAV vaccine in the pig model. In the past, experimental work carried out in mice
has not translated well to humans limiting the usefulness of this model. The use of pigs models
that are more similar to humans may be more appropriate [27]. Pigs vaccinated with the commercial inactivated type-specific vaccine were protected
against pH1N1 challenge based on reduced shedding of IAV RNA in nasal secretions and oral
fluids and reduced macroscopic and microscopic lesions; however, pigs vaccinated with the
experimental broadly-reactive subunit vaccines were not, and developed clinical disease and
severe macroscopic and microscopic lung lesions similar to the non-vaccinated SHAMVA-
C-IAV pigs. 10 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0191739
January 30, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0191739
January 30, 2018 Influenza A virus vaccination in the pig model immunization induced significantly higher Ab levels in the serum compared to intranasal
[36,37] but we speculate that local mucosal protection against IAV is perhaps most important
compared to systemic protection. Future studies need to directly compare both administration
routes using the same products. In addition, several factors regarding the design and application of the two subunit vaccines
may also have contributed to the failure of protection observed in pigs in this study: (1) Unlike
the inactivated whole pH1N1 virus-based Inact/pH1N1vaccine that should contain all original
IAV epitopes, the 23 amino acid-M2e epitope of the two subunit vaccines represent only a
small fraction of the IAV proteome. Hence, Inact/pH1N1 can elicit complex immune
responses to inhibit various steps of IAV infection, while the subunit vaccines in this study did
not elicit any detectable M2e-specific immune response. Previously it was found that M2e-
based vaccines confer relatively weak protection mediated via non-neutralizing immune
mechanisms compared to an inactivated IAV vaccine in mice [38]. Therefore, it may be under-
standable that the Inact/pH1N1vaccine exhibited much higher protection than the two subunit
vaccines; however, studies comparing the Inact/pH1N1 vaccine to the experimental M2e vac-
cines in the mouse model are lacking. (2) Due to its small size, the M2e epitope constitutes
only a small portion of the subunit vaccines, accounting for 7% and 4% of the NvParticle/M2e
and the NvComplex/M2e total protein, respectively. As a result, higher vaccine dose and dos-
age than the currently studied ones may be required to induce a higher M2e-specific immune
response. In previous trials, 15–30 μg of the subunit vaccines were used to immunize mice
three times to show protection [10,11,14] and 50 μg of the same subunit vaccines for two
immunizations may not have been sufficient to elicit protective immunity in the current pig
study. Thus, future studies with higher doses of the subunit vaccine are necessary. (3) BLAST
searches indicated that both M2e vaccine sequences used in this study match numerous M2e
sequences in pig IAV strains recovered from the U.S. and other countries. For the particular
pH1N1 challenge virus used in this study, both vaccines have a change on position 10
(Thr!Ile), and on positions 12 and 19 (Ser!Asn). PLOS ONE | https://doi.org/10.1371/journal.pone.0191739
January 30, 2018 Similar results were also obtained with a GST-Nv P+-M2e polymer vaccine
[11]. Despite being efficacious in mice using certain models, the vaccines failed to protect pigs
in this study. This reinforces that findings in the murine model may not be predictive of
responses in other animal species [31,32]. In addition, comparative mapping of anti-M2e anti-
bodies obtained from different species revealed that one epitope was exclusively recognized by
antibodies generated in rabbits while another epitope was only recognized by mice and chick-
ens indicating differences in species recognition [33]. The previously observed protection of the two subunit vaccines in mouse trials and the lack
of protection in this pig study potentially indicate differences in immune responses between
species. The structural diversity and the abundance and distribution of nasal cavity-associated
lymphoid tissues in both species may explain some of these differences [27,34,35]. Differences
in immune responses were observed when comparing inbred (BALB/c, C57BL/6, C3H and
BALB/cxC57BL/6) and outbred (CD1/ICR and Swiss Webster) mouse strains, since the second
group exhibited poor anti-M2e Ab levels [36]. Another factor that may have influenced the
immune response is the immunization route used herein. Most commercial pig vaccines avail-
able today are applied via the intramuscular route but it is suspected that, especially for respira-
tory infection, the intranasal vaccination would be beneficial. Therefore, the experimental M2e
vaccines used in this study were given intranasally. Previous studies comparing intranasal ver-
sus subcutaneous administration routes in chickens and mice showed that subcutaneous PLOS ONE | https://doi.org/10.1371/journal.pone.0191739
January 30, 2018 11 / 15 Author Contributions Conceptualization: Tanja Opriessnig, Ming Tan. Conceptualization: Tanja Opriessnig, Ming Tan. Data curation: Tanja Opriessnig, Priscilla F. Gerber, Paul Digard, Ming Xia, Xi Jiang. Formal analysis: Tanja Opriessnig, Priscilla F. Gerber, Ming Xia. Funding acquisition: Phillip C. Gauger, Patrick G. Halbur, Ming Tan. Investigation: Tanja Opriessnig, Phillip C. Gauger, Alessandra M. M. G. Castro, Huigang
Shen, Lita Murphy, Patrick G. Halbur. Methodology: Tanja Opriessnig, Phillip C. Gauger, Alessandra M. M. G. Castro, Huigang
Shen. Project administration: Tanja Opriessnig, Phillip C. Gauger. Supervision: Phillip C. Gauger. Writing – original draft: Tanja Opriessnig. Writing – review & editing: Tanja Opriessnig, Phillip C. Gauger, Priscilla F. Gerber, Alessan-
dra M. M. G. Castro, Lita Murphy, Paul Digard, Patrick G. Halbur, Ming Tan. Acknowledgments The authors thank Kelsey Oakley, Eve Fontanella, Gustavo de-Sousa-e-Silva, Marcelo Nunes
de Almeida and Will Alberto Lopez for their assistance with the animal work. The authors thank Kelsey Oakley, Eve Fontanella, Gustavo de-Sousa-e-Silva, Marcelo Nunes
de Almeida and Will Alberto Lopez for their assistance with the animal work. Supporting information S1 Fig. Individual pig log10 IAV RNA genomic copies in each group. Nasal swabs were col-
lected at day post challenge (dpc) 1–5 and bronchoalveolar lavage (BAL) fluid was collected on
dpc 5. (TIF) S1 Fig. Individual pig log10 IAV RNA genomic copies in each group. Nasal swabs were col-
lected at day post challenge (dpc) 1–5 and bronchoalveolar lavage (BAL) fluid was collected on
dpc 5. (TIF)
S2 Fig. Minimal data set. (XLSX) In addition, the NvParticle vaccines has a
change on position 13 (Glu!Gly) and on position 17 (Ala!Lys), while the NvComplex vac-
cine has a change on position 15 (Glu!Gly). While the M2e epitope has been considered to
be very conserved posing an ideal universal IAV vaccine target [12,13], recognition of epitopes
could have been affected by changes in amino-acid side chain polarity, as occurred with
Thr!Ile on position 10 (both vaccines), and Glu!Gly on positions 13 (NvParticle vaccine)
and 15 (NvComplex vaccine), due to conformation of the resulting protein. However, this
needs to be further explored. When tested with a commercial IAV NP-protein based blocking ELISA, most pigs in this
study remained negative for IAV specific antibodies. Pigs vaccinated with experimental
M2-based subunit vaccines were not expected to react with this assay since antibody directed
against the NP protein do not cross react with those against the M2 protein. However, even in
the Inact/pH1N1group only 1/8 pigs had a detectable response by dpc 0. Previously it has been
determined that, while vaccination affected the S/N antibody response, data did not support
the use of the NP ELISA for monitoring IAV vaccination compliance or for differentiating
between IAV-infected animals and IAV-infected and vaccinated animals [26]. Because of this,
an HI assay specific for subtype A/H1N1/pdm09 was also used. At challenge, all Inact/
pH1N1pigs had titers of 80 or greater whereas in all other groups titers were between 0 and 20. This further confirms that all Inact/pH1N1pigs mounted a detectable immune response
against pH1N1 by the time of challenge. Mucosal immunity specific for M2e was measured by
IgA ELISA on lung lavage fluid collected at necropsy. However, none of the pigs had a detect-
able response. Nevertheless, results obtained with an IgG M2e ELISA on serum collected at
challenge indicated a weak but detectable M2e-specific IgG antibody response in the PLOS ONE | https://doi.org/10.1371/journal.pone.0191739
January 30, 2018 12 / 15 Influenza A virus vaccination in the pig model NvParticle/M2e and the NvComplex/M2e pigs with significantly higher group means in the
NvComplex/M2e pigs compared to the SHAMVAC-NEG pigs. NvParticle/M2e and the NvComplex/M2e pigs with significantly higher group means in the
NvComplex/M2e pigs compared to the SHAMVAC-NEG pigs. Conclusions Under the study conditions, pigs vaccinated with the commercial inactivated vaccine were
protected against pH1N1 challenge based on reduced shedding of IAV RNA in nasal secre-
tions and oral fluids and reduced macroscopic and microscopic lesions. Pigs vaccinated with
the experimental subunit vaccines were not protected. Vaccine genetic makeup, dose, protein
concentration and administration route need to be further investigated and better adjusted
from usage in mice to usage in pigs. References 1. Gaymard A, Le BN, Frobert E, Lina B, Escuret V. Functional balance between neuraminidase and hae-
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GAS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT STRESS AND POTASSIUM FERTILIZATION
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Revista Caatinga
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cc-by
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ADAAN SUDARIO DIAS2, GEOVANI SOARES DE LIMA3*, FRANCISCO WESLEY ALVES PINHEIRO2, HANS
RAJ GHEYI4, LAURIANE ALMEIDA DOS ANJOS SOARES3 ABSTRACT - Water resources in the semi-arid region of Northeast Brazil commonly contain high salt
concentrations, compromising the quality of water for agriculture. Thus, adopting techniques that make the use
of these resources feasible in agriculture is fundamental. The present study aimed to evaluate the gas
exchanges, quantum yield and photosynthetic pigments of grafted West Indian cherry subjected to salt stress
and potassium fertilization under greenhouse conditions in the municipality of Campina Grande, PB, Brazil. Treatments were distributed in randomized blocks, composed of two levels of electrical conductivity – ECw
(0.8 and 3.8 dS m-1) of water and four doses of potassium (50, 75, 100 and 125% of the dose recommended for
the crop), with three replicates. The dose relative to 100% corresponded to 19.8 g of K2O per plant. Gas
exchanges, chlorophyll a fluorescence and photosynthetic pigments of West Indian cherry are negatively
affected by irrigation using water with electrical conductivity of 3.8 dS m-1, which compromises the
photosynthetic apparatus of the plant, a situation evidenced by the reduction in photosystem II quantum
efficiency. Increasing potassium doses led to increments in transpiration, chlorophyll a maximum fluorescence
and chlorophyll b content in West Indian cherry grown under salt stress, but do not attenuate the negative
effects of irrigation with 3.8 dS m-1 water on its potential photochemical efficiency. Keywords: Malpighia emarginata. Physiology. Potassium. Salinity. ISSN 0100-316X (impresso)
ISSN 1983-2125 (online)
90/1983 21252019v32n216rc ISSN 0100-316X (impresso)
ISSN 1983-2125 (online)
90/1983 21252019v32n216rc ISSN 0100-316X (impresso)
ISSN 1983-2125 (online)
http://dx.doi.org/10.1590/1983-21252019v32n216rc Universidade Federal Rural do Semi-Árido
Pró-Reitoria de Pesquisa e Pós-Graduação
https://periodicos.ufersa.edu.br/index.php/caatinga Universidade Federal Rural do Semi-Árido
Pró-Reitoria de Pesquisa e Pós-Graduação _______________________________
*Corresponding author
1Received for publication in 02/13/2018; accepted in 02/04/2019.
Paper extracted from the postdoctoral research of the second author.
2Center of Technology and Natural Resources, Universidade Federal de Campina Grande, Campina Grande, PB, Brazil;
adaansudariodias@gmail.com – ORCID: 0000-0002-2247-1511, wesley.ce@hotmail.com – ORCID: 0000-0002-5589-6882.
3Center Academic Unit of Agricultural Sciences, Center of Agrifood Science and Technology, Universidade Federal de Campina Grande,
Pombal, PB, Brazil; geovani.soares@pq.cnpq.br – ORCID: 0000-0001-9960-1858, lauriane.soares@pq.cnpq.br – ORCID: 0000-0002-7689
-9628.
4Nucleus of Soil and Water Engineering, Universidade Federal do Recôncavo da Bahia, Cruz das Almas, BA, Brazil; hans@pq.cnpq.br –
ORCID: 0000-0002-1066-0315. Palavras-chave: Malphigia emarginata. Fisiologia. Potássio. Salinidade. GAS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF
WEST INDIAN CHERRY UNDER SALT STRESS AND POTASSIUM
FERTILIZATION1 ADAAN SUDARIO DIAS2, GEOVANI SOARES DE LIMA3*, FRANCISCO WESLEY ALVES PINHEIRO2, HANS
RAJ GHEYI4, LAURIANE ALMEIDA DOS ANJOS SOARES3 4Nucleus of Soil and Water Engineering, Universidade Federal do Recôncavo da Bahia, Cruz das Almas, BA, Brazil; hans@pq.cnpq.br –
ORCID: 0000-0002-1066-0315. Keywords: Malpighia emarginata. Physiology. Potassium. Salinity. p
of Technology and Natural Resources, Universidade Federal de Campina Grande, Campina Grande, PB, Brazi
udariodias@gmail.com – ORCID: 0000-0002-2247-1511, wesley.ce@hotmail.com – ORCID: 0000-0002-5589-6882. 1Received for publication in 02/13/2018; accepted in 02/04/2019 MATERIAL AND METHODS The experiment was conducted in 250-L
plastic pots adapted as drainage lysimeters under
greenhouse conditions, at the Center of Technology
and Natural Resources of the Federal University of
Campina Grande (CTRN/UFCG), located in the
municipality of Campina Grande, PB, Brazil,
situated at the local geographic coordinates of 7° 15’
18” S, 35° 52’ 28” W and an altitude of 550 m. Despite the good prospects for West Indian
cherry cultivation, the Brazilian semi-arid region
poses risks to its cultivation, due to the scarcity of
water resources both quantitatively and qualitatively. In addition, the water sources of this region
commonly contain high concentrations of salts,
especially sodium (PÁDUA et al., 2017). The experimental design was randomized
blocks, in 2 x 4 factorial arrangement, corresponding
to
two
levels
of
irrigation
water
electrical
conductivity – ECw (0.8 and 3.8 dS m-1) and four
doses of potassium – KD (50, 75, 100 and 125% of
K2O, based on the recommendation of Musser
(1995)), with three replicates. The dose relative to
100% corresponded to the application of 19.8 g
of K2O per plant. Using saline water in irrigation causes
deleterious effects on crops, due to the reduction in
water availability to plants resulting from the
decrease in the osmotic potential of the soil solution,
leading to stomatal closure and compromising
transpiration
and
photosynthesis
(SILVA et al., 2015). Additionally, it causes
alterations in the functional state of chloroplast
thylakoid membranes and modifications in the
characteristics of fluorescence signals in the leaves
(FREIRE et al., 2014) and inhibits the synthesis of 5-
aminolevulinate acid, which is the precursor
molecule of chlorophyll, causing changes in nitrogen
assimilation
and
protein
metabolism
(CAVALCANTE et al., 2011). Water
with
electrical
conductivity
of
3.8 dS m-1 was prepared by dissolving the salts NaCl,
CaCl2.2H2O
and
MgCl2.6H2O,
at
equivalent
proportions of 7:2:1, respectively. This is the average
composition of the contents of these cations in the
water commonly used for irrigation in the Brazilian
Northeast semi-arid region (MEDEIROS, 1992). The
ECw level of 0.8 dS m-1 was obtained by mixing
rainwater (ECw = 0.02 dS m-1) and water from the
municipal supply system (ECw = 1.40 dS m-1). A drain was connected to the base of each
lysimeter, using a 4-mm-diameter plastic tube to
drain the leachate into a container, determine the
water consumption by the plants and evaluate the
electrical conductivity of the drained water. A. S. DIAS et al. A. S. DIAS et al. agriculture (GURGEL; GHEYI; OLIVEIRA, 2010;
PRAZERES et al., 2015). This element favors the
formation and translocation of carbohydrates and
efficient water use by plants, equilibrates nitrogen
application (ARAÚJO et al., 2012), acts as an
enzymatic activator and osmoregulation agent,
controlling stomatal opening and closure, and its
management may result in greater competition of
this macroelement with other cations, especially Na+
(HEIDARI; JAMSHID, 2010). INTRODUCTION West Indian cherry (Malpighia emarginata)
fruits are known for their pleasant taste, high
contents of vitamin C, vitamin A, B-complex
vitamins,
iron,
calcium
(SÁ
et
al.,
2017),
anthocyanins and carotenoids, compounds which
have beneficial effects on human health for their
known antioxidant action (MACIEL et al., 2010). The crop is adapted to the most diverse climates and
can be found in several regions of the planet, but its
commercial cultivation is concentrated in tropical
and subtropical regions (ADRIANO et al., 2011),
such as those found in the Brazilian territory. In this context, the present study aimed to
evaluate the gas exchanges, quantum yield and
photosynthetic pigments of grafted West Indian
cherry subjected to salt stress and potassium
fertilization. y
Brazil is the largest producer, consumer and
exporter of this fruit in the world. In the country,
West Indian cherry is cultivated in the Northeast,
North,
South
and
Southeast
regions
(ADRIANO et al., 2011), and the Northeast region
stands out due to its soil and climatic conditions, to
which the crop is better adapted. In this region, its
production reaches 22,964 tons per year in an area of
7,237 ha, which represents approximately 70% of the
national production, making the crop a promising
option for the Northeast region, due to the prospects
for the fruit market, constituting a source of income
for rural producers (CAETANO, 2010). GAS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT
STRESS AND POTASSIUM FERTILIZATION GAS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT
STRESS AND POTASSIUM FERTILIZATION TROCAS GASOSAS, RENDIMENTO QUÂNTICO E PIGMENTOS FOTOSSINTÉTICOS DA
ACEROLEIRA SOB ESTRESSE SALINO E ADUBAÇÃO POTÁSSICA RESUMO - Os recursos hídricos da região semiárida do Nordeste Brasileiro comumente possuem elevadas
concentrações de sais, comprometendo a qualidade da água para agricultura. Desta forma, a adoção de práticas
de manejo que viabilizem o uso de tais recursos na agricultura é fundamental. Neste trabalho, objetivou-se
avaliar as trocas gasosas, o rendimento quântico e os pigmentos fotossintéticos da acerola enxertada submetida
ao estresse salino e adubação potássica em condição de casa de vegetação no município de Campina Grande-
PB. Os tratamentos foram distribuídos em blocos casualizados, sendo constituídos de dois níveis de
condutividade elétrica da água - CEa (0,8 e 3,8 dS m-1) e quatro doses de potássio (50, 75; 100 e 125% da dose
recomendada para a cultura), com três repetições. A dose de 100% correspondeu a 19,8 g de K2O por planta. As trocas gasosas a fluorescência da clorofila a e os pigmentos fotossintéticos da aceroleira são afetados
negativamente pela irrigação com água de condutividade elétrica de 3,8 dS m-1 que compromete o aparato
fotossintético da planta, situação observada através da redução da eficiência quântica do fotossistema II. As
doses de potássio crescentes promovem incremento na taxa de transpiração, fluorescência máxima da clorofila
a e no teor de clorofila b da aceroleira cultivada sob estresse salino, porém não atenuam os efeitos negativos da
irrigação com água de 3,8 dS m-1 sobre a eficiência quântica potencial da acerola. Rev. Caatinga, Mossoró, v. 32, n. 2, p. 429 – 439, abr. – jun., 2019 429 Rev. Caatinga, Mossoró, v. 32, n. 2, p. 429 – 439, abr. – jun., 2019
429 GAS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT
STRESS AND POTASSIUM FERTILIZATION A. S. DIAS et al. A. S. DIAS et al. Donagema et al. (2011): Ca2+ = 9.07 cmolc kg-1;
Mg2+ = 2.78 cmolc kg-1; Na+ = 1.64 cmolc kg-1; K+ =
0.23 cmolc kg-1; H+ + Al3+ = 8.61 cmolc kg-1; Al3+ = 0
cmolc kg-1; CEC = 22.33 cmolc kg-1; organic matter =
2.93 dag kg-1; P = 39.8 mg kg-1; pH in water (1:2.5)
= 5.58; electrical conductivity of the saturation
extract = 2.15 dS m-1; SAR = 0.67 (mmol L-1)0.5;
exchangeable sodium percentage = 7.34%; sand =
659.9 g kg-1; silt = 161.2 g kg-1; clay = 178.9 g kg-1;
moisture at 33.42 kPa = 25.91 dag kg-1; moisture at
1519.5 kPa = 12.96 dag kg-1. At 400 days after transplanting, the period of
transition between flowering and fruiting, the
stomatal conductance - gs (mol H2O m-2 s-1), CO2
assimilation rate - A (μmol m-2 s-1), transpiration - E
(mmol
of
H2O
m-2
s-1)
and
internal
CO2
concentration – Ci (μmol mol 1) were determined
using a portable infrared gas analyzer (IRGA), model
LCPro+ Portable Photosynthesis System®. After
collection, these data were used to quantify the
instantaneous water use efficiency - WUEi (A/E)
[(µmol m-2 s-1) (mmol H2O m-2 s-1)
-1] and
instantaneous carboxylation efficiency - CEi (A/Ci)
[(µmol m-2 s-1) (μmol mol 1) -1]. In the same period,
the initial fluorescence (Fo), maximum fluorescence
(Fm), variable fluorescence (Fv) and potential
quantum efficiency (Fv/Fm) were measured in leaves
pre-adapted to the dark using clips for 30 minutes,
between 7:00 and 8:00 A.M., using a modulated
fluorometer Plant Efficiency Analyser – PEA II®. g g
In the experiment, the rootstocks consisted of
heirloom seedlings of West Indian cherry produced
at EMBRAPA Tropical Agroindustry, in Pacajus-
CE. At transplanting, the seedlings were 240 days
old. During the acclimation period in the greenhouse,
the seedlings were irrigated with low-salinity water
(0.8 dS m-1). The cultivar BRS 366 Jaburu was used
as the scion variety. This cultivar is known for its
high yield, 57 t ha-1, and vitamin C content of 2,648
mg 100g-1. The plants are approximately 1.87 m tall,
with a crown diameter of 2.18 m on average. The
fruits are shiny when ripe and show a mean weight
of 4 to 5 g when unripe, appropriate to obtain
vitamin
C,
and
6
to
7
g
after
ripening
(EMBRAPA, 2012). A. S. DIAS et al. The contents of chlorophyll a, chlorophyll b
and carotenoids were determined according to the
methodology of Arnon (1949), using 5 discs from
the lamina of the third mature leaf from the apex,
which were collected, immersed in 80% acetone and
stored in the dark for 48 hours. The obtained extracts
were subjected to readings in a spectrophotometer at
the wavelengths of 470, 646 and 663 nm. The values
observed in the readings were subjected to the
following equations: Chlorophyll a (Chl a) = 12.21
ABS663 – 2.81 ABS646; Chlorophyll b (Chl b) =
20.13 A646 – 5.03 ABS663; total carotenoids (Car)
= (1000 ABS470 – 1.82 Chl a – 85.02 Chl b) /198, to
determine the contents of chlorophyll a, chlorophyll
b and carotenoids, expressed in mg g-1 of fresh
matter (FM). Before transplanting the seedlings, the soil
was brought to field capacity using the respective
waters of each treatment. After transplanting,
irrigation was performed daily by applying in each
lysimeter a water volume sufficient to maintain the
soil close to field capacity, and the applied volume
was determined according to the plants’ water needs,
estimated by the water balance: volume applied
minus volume drained in the previous irrigation, plus
a leaching fraction of 0.10. The data were subjected to analysis of
variance by the F test and, when significant, a means
comparison test (Tukey test at 0.05 probability level)
was carried out for the water salinity levels (SL), and
regression analysis was conducted for the potassium
doses (KD). When there was significant interaction
between factors, the SL factor was further analyzed
considering each KD using the statistical program
SISVAR-ESAL (FERREIRA, 2014). Fertilization with phosphorus and nitrogen
was applied as recommended by Musser (1955),
using single superphosphate and urea, respectively. The phosphorus was entirely applied before planting,
whereas nitrogen and potassium were split into 12
equal portions, applied monthly. To meet probable
deficiencies of micronutrients, the plants were
weekly sprayed on the adaxial and abaxial faces of
the
leaves
with
Ubyfol
solution
containing
1.5 g L-1 [(N (15%); P2O5 (15%); K2O (15%); Ca
(1%); Mg (1.4%); S (2.7%); Zn (0.5%); B (0.05%);
Fe (0.5%); Mn (0.05%); Cu (0.5%); Mo (0.02%)]. On average, 5 L were used in each application for
the entire experiment. MATERIAL AND METHODS The tip
of the drain inside the pot was wrapped in nonwoven
geotextile (Bidim OP 30) to avoid clogging by soil. Thus, considering that most cultivated species
are sensitive to the presence of salts in water and/or
in soil, it is necessary to conduct research aiming to
obtain techniques capable of minimizing the
deleterious effects of salt stress on plants, since the
use of saline waters in agriculture is almost
mandatory in the Brazilian Northeast semi-arid
region (FREIRE et al., 2014). The lysimeters were filled using a 1-kg layer
of crushed stone (size 0), followed by 250 kg of
Regolithic Neosol with sandy clay texture, properly
pounded to break up clods, from the rural area of the
municipality of Esperança, PB. Its chemical and
physical–hydraulic characteristics were obtained
according to the methodologies proposed by Potassium has been studied as an attenuator
of the deleterious effects of water salinity on crops,
aiming to allow these resources to be used in Rev. Caatinga, Mossoró, v. 32, n. 2, p. 429 – 439, abr. – jun., 2019 430 GAS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT
STRESS AND POTASSIUM FERTILIZATION GAS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT
STRESS AND POTASSIUM FERTILIZATION A. S. DIAS et al. Table 1. Summary of F test for stomatal conductance (gs), transpiration (E), CO2 assimilation rate (A), internal CO2
concentration (Ci), instantaneous carboxylation efficiency (CEi) and instantaneous water use efficiency (WUEi) of grafted
West Indian cherry plants grown under water salinity and potassium doses, at 400 days after transplanting. Source of variation
Test F
E
gs
A
Ci
CEi
WUEi
Saline levels (SL)
**
**
**
*
**
ns
K dose (KD)
*
ns
ns
ns
ns
ns
Interaction (SL x KD)
ns
ns
ns
ns
ns
ns
Blocks
ns
ns
ns
ns
ns
ns
CV (%)
17.28
13.59
19.01
23.59
15.37
13.29
ns,**, *, respectively not significant, significant at p < 0.01 and p < 0.05 probability levels. grown with waters of different salinity levels
(0.6, 1.2, 1.8, 2.4 and 3.0 dS m-1). According to these
authors, there is a relationship between transpiration
and stomatal conductance, so that a reduction in gs
leads to a reduction in E. Gonçalves et al. (2010)
also observed the existence of this relationship and
concluded that the water vapor flow to the
atmosphere
decreases
as
the
stomata
close. Therefore, a reduction in stomatal conductance
causes a decrease in leaf transpiration. The leaf transpiration rate of the West Indian
cherry plants was significantly reduced by the
increase in irrigation water salinity. According to the
means comparison test (Figure 1A), E was 46.95%
lower in plants irrigated using water of the highest
salinity (3.8 dS m-1) compared to those subjected to
the lowest ECw (0.8 dS m-1). Hussain et al. (2012)
subjected citrus to salt stress and reported that a
reduction in gs leads to a reduction in CO2 diffusion. The same trend observed in the present study was
also found by Sousa et al. (2016), in citrus plants
1 1
2 Means followed by the same letter do not differ by Tukey test (p<0.05). Figure 1. Transpiration – E of grafted West Indian cherry as a function of irrigation water salinity – ECw (A) and
potassium doses (B), at 400 days after transplantation. Means followed by the same letter do not differ by Tukey test (p<0.05). Figure 1. RESULTS AND DISCUSSION According to Table 1, the water salinity
significantly influenced the stomatal conductance
(gs), transpiration (E), CO2 assimilation rate (A),
internal CO2 concentration (Ci) and instantaneous
carboxylation efficiency (CEi). The potassium doses
had a significant effect only on leaf transpiration in
the West Indian cherry. The interaction between the
water salinity levels and K doses had no significant
influence on any of the variables evaluated at 400
days after transplanting. The cultural practices consisted of manual
weeding every week, superficial soil scarification
before each irrigation event and plant tutoring to
avoid lodging. In addition, insecticides of the
Neonicotinoid chemical group, fungicide of the
Triazole chemical group and acaricide from the
Abamectin chemical group were preventively
applied at doses of 4.0, 5.0 and 3.5 g L-1,
respectively. Rev. Caatinga, Mossoró, v. 32, n. 2, p. 429 – 439, abr. – jun., 2019 431 A. S. DIAS et al. A. S. DIAS et al. possible to note that gs in plants subjected to 3.8 dS
m-1 was estimated to be 90.98% lower than that
observed in plants irrigated with 0.8 dS m-1 water. Reduction of gs in the West Indian cherry is one of
the first responses to salt stress. This occurs as a
defense mechanism to reduce water loss through the
leaves to the atmosphere because water absorption
by roots becomes more difficult in saline soil
(SOUSA et al., 2016). possible to note that gs in plants subjected to 3.8 dS
m-1 was estimated to be 90.98% lower than that
observed in plants irrigated with 0.8 dS m-1 water. Reduction of gs in the West Indian cherry is one of
the first responses to salt stress. This occurs as a
defense mechanism to reduce water loss through the
leaves to the atmosphere because water absorption
by roots becomes more difficult in saline soil
(SOUSA et al., 2016). 2.70 to 2.82 in plants under K supply. These authors
also claimed that K application can minimize the
negative effects of water deficit. 2.70 to 2.82 in plants under K supply. These authors
also claimed that K application can minimize the
negative effects of water deficit. The means comparison test (Figure 2A)
shows that the West Indian cherry plants irrigated
using high-salinity water (3.8 dS m-1) had a drastic
reduction in stomatal conductance compared to those
irrigated using water of the lowest salinity level
(0.8 dS m-1). By comparing the results, it was Means followed by the same letter do not differ by Tukey test (p<0.05). Figure 2. Stomatal conductance - gs (A), CO2 assimilation rate - A (B), internal CO2 concentration – Ci (C) and
instantaneous carboxylation efficiency - CEi (D) of grafted West Indian cherry, as a function of irrigation water salinity –
ECw, at 400 days after transplanting. Means followed by the same letter do not differ by Tukey test (p<0.05). Figure 2. Stomatal conductance - gs (A), CO2 assimilation rate - A (B), internal CO2 concentration – Ci (C) and
instantaneous carboxylation efficiency - CEi (D) of grafted West Indian cherry, as a function of irrigation water salinity –
EC
400 d
f
l
i Means followed by the same letter do not differ by Tukey test (p<0.05). Figure 2. GAS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT
STRESS AND POTASSIUM FERTILIZATION A. S. DIAS et al. GAS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT
STRESS AND POTASSIUM FERTILIZATION Transpiration – E of grafted West Indian cherry as a function of irrigation water salinity – ECw (A) and
potassium doses (B), at 400 days after transplantation. Melo et al. (2014) state that K accumulation in plants
favors an osmotic gradient that facilitates water
movement, regulating stomatal opening and closure,
playing a fundamental role in cell turgidity, transport
of carbohydrates and transpiration. An increment in the potassium fertilization
caused a linear increase in the transpiration rate of
the West Indian cherry plants and, according to the
regression equation (Figure 1B), plants subjected to
125% of the recommendation by Musser obtained E
of 1.07 mmol H2O m-2 s-1, which is equivalent to a
77.32% increase compared to those which received
only 50% of the K2O recommendation. The increase
of leaf transpiration in the West Indian cherry plants
reflects the importance of K in stomatal regulation
and photosynthesis, since the CO2 assimilation rate
depends on the entry of this gas into the plant. Mendes et al. (2013) subjected eucalyptus
plants to water restriction, similar to what occurs in
plants under salt stress, and observed that plants
which received K fertilization showed better
physiological performance than unfertilized ones,
with a better stomatal control and consequently
higher values of A and E, and that E increased from 432 Rev. Caatinga, Mossoró, v. 32, n. 2, p. 429 – 439, abr. – jun., 2019 GAS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT
STRESS AND POTASSIUM FERTILIZATION A. S. DIAS et al. A. S. DIAS et al. subjected to the lowest level of water salinity
(Figure 2D). This result means that the CO2 in the
West Indian cherry plants grown using water with a
high salt concentration is being totally fixed when it
reaches the mesophyll cells, thus denoting that these
plants
have
undergone
possible
metabolic
restrictions
to
the
Calvin
cycle
(SOARES et al., 2013), a reduction in the synthesis
of
sugars
in
the
photosynthetic
process
(FREIRE et al., 2014), and a reduction in the
substrate for RuBisCO (SILVA et al., 2015). In
addition to these factors, as the stress becomes more
severe, the mesophyll cells dehydrate, the mesophyll
metabolism is damaged and, consequently, the
carboxylation
efficiency
is
compromised
(TAIZ; ZEIGER, 2013). water, compared to those under irrigation with a
salinity level of 0.8 dS m-1. The increase of Ci in
plants under salt stress is due to stomatal closure,
which leads to a reduction of the mesophyll’s
capacity to assimilate carbon, which is not being
fixed when it reaches the mesophyll cells (TAIZ;
ZEIGER, 2013). This situation indicates a possible
deterioration in the photosynthetic structure, since
the damage caused to the structures responsible for
CO2 fixation is not only due to stomatal factors, for
example, an accumulation of salts in the leaves
(HUSSAIN et al., 2012). These authors emphasize
that the accumulation of salts in the leaves negatively
affects processes that are essential for plants such as
A, gs and Fv/Fm. In relation to the instantaneous carboxylation
efficiency, this study makes it possible to ascertain
whether non-stomatal factors have an influence on
photosynthesis. According to Silva et al. (2015), this
variable depends on the CO2 availability in the leaf
mesophyll, the amount of light, temperature and
enzymatic activity. Thus, the CEi of the West Indian
cherry plants was also negatively influenced by the
use of water with high EC. Plants irrigated using
water of the lowest EC (0.8 dS m-1) showed a CEi of
0.043 [(µmol m-2 s-1) (μmol mol 1) -1], whereas those
subjected to 3.8 dS m-1 obtained a CEi of 0.019
[(µmol m-2 s-1) (μmol mol 1) -1], i.e., plants under the
highest ECw level showed a reduction of 55.81% in
their capacity to fix CO2 in comparison to those Determining
chlorophyll
a
fluorescence
signals is an important tool to assess photosynthetic
apparatus integrity and the possible influence of non-
stomatal factors on CEi. A. S. DIAS et al. In relation to variables
relative to chlorophyll a fluorescence (Fo, Fm, Fv,
Fv/Fm) and photosynthetic pigments (Chl a, Chl b
and carotenoids), the F test results (Table 2) revealed
a significant influence of the irrigation water salinity
on all the variables analysed. Potassium doses
significantly affected only the content of chlorophyll
b in the West Indian cherry leaves. Interaction
between the studied factors (SL x KD) significantly
influenced the maximum fluorescence, potential
quantum efficiency and Chl b content. Table 2. Summary of F test for initial fluorescence (Fo), maximum fluorescence (Fm), variable fluorescence (Fv), potential
quantum efficiency (Fv/Fm), chlorophyll a content (Chl a), chlorophyll b content (Chl b) and carotenoid (Car) content in
leaves of grafted West Indian cherry plants grown under water salinity and potassium doses, at 400 days after transplanting. Source of variation
Test F
Fo
Fm
Fv
Fv/Fm
Chl a
Chl b
Car
Saline levels (SL)
**
*
**
**
*
**
**
K dose (KD)
ns
ns
ns
ns
ns
**
ns
Interaction (SL x KD)
ns
*
ns
*
ns
**
ns
Blocks
ns
ns
ns
ns
ns
ns
ns
CV (%)
7.79
16.93
13.22
19.77
23.79
24.02
15.29
ns,**, * respectively not significant, significant at p < 0.01 and p < 0.05 probability levels. ns,**, * respectively not significant, significant at p < 0.01 and p < 0.05 probability levels. Figure 3A shows that irrigation using
3.8 dS m-1 water caused an initial fluorescence (Fo)
of 117.42 in the West Indian cherry leaves, whereas
in plants irrigated with ECw of 0.8 dS m-1 Fo was
equal to 144.75. Alterations in Fo may occur when
there is damage to the photosystem II reaction center
or due to a reduction in the transfer of excitation
energy from the light-harvesting system to the
reaction center (BAKER; ROSENQVIST, 2004). Thus, the increase of salt concentration in irrigation
water caused damage to the PSII reaction center or depletion in the transfer of excitation energy,
possibly due to the biochemical and physiological
changes caused in the plant by the stress, since Fo
emission
occurs
within
the
first
phase
of
fluorescence and represents the energy released by
chlorophyll a molecules from the photosystem II
antenna, before the electrons moved to the P680
reaction center (PSII), a minimum component of the
fluorescence signal (VIEIRA et al., 2010) being
emitted when all dark-adapted reaction centers are
open (GORBE; CALATAYUD, 2012). Rev. A. S. DIAS et al. Stomatal conductance - gs (A), CO2 assimilation rate - A (B), internal CO2 concentration – Ci (C) and
instantaneous carboxylation efficiency - CEi (D) of grafted West Indian cherry, as a function of irrigation water salinity –
ECw, at 400 days after transplanting. Figure 2. Stomatal conductance - gs (A), CO2 assimilation rate - A (B), internal CO2 concentration – Ci (C) and
instantaneous carboxylation efficiency - CEi (D) of grafted West Indian cherry, as a function of irrigation water salinity –
ECw, at 400 days after transplanting. affects net photosynthesis (SYVERTSEN; GARCÍA
-SÁNCHEZ, 2014), due to the stomatal effects
which limit the photosynthetic process (PRAZERES
et al., 2015; SOUSA et al., 2016). In agreement with the results obtained here,
Hussain et al. (2012) also observed a reduction of gs
in different citrus genotypes when subjected to
irrigation using water containing 75 mM of NaCl. These authors state that the primary effect of salinity
on citrus, as observed in the present study, is the
reduction in stomatal conductance. Studies evaluating the effect of salt stress on
the photosynthesis of cowpea (PRAZERES et al.,
2015) and citrus (SOUSA et al., 2016) have found
similar trends to those observed in the present study,
i.e., a reduction in the CO2 assimilation rate as a
result of increased irrigation water salinity. These
authors commented that such effect is due to the
reductions of gs and E, corroborating the idea that
transpiration directly influences the CO2 assimilation
rate. According to the means comparison test,
irrigation using water with an electrical conductivity
of 3.8 dS m-1 negatively affected the photosynthetic
process in the West Indian cherry plants (Figure 2B),
whose CO2 assimilation rate decreased by 3.53 μmol
m-2 s-1 in comparison to those subjected to the lowest
salinity level, a reduction corresponding to 49.13%. The decrease in stomatal conductance with the
consequent reduction in CO2 diffusion negatively Figure 2C shows an increase of 39.33 μmol
mol-1 in the Ci of plants irrigated with 3.8 dS m-1 433 Rev. Caatinga, Mossoró, v. 32, n. 2, p. 429 – 439, abr. – jun., 2019 GAS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT
STRESS AND POTASSIUM FERTILIZATION A. S. DIAS et al. Caatinga, Mossoró, v. 32, n. 2, p. 429 – 439, abr. – jun., 2019 434 GAS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT
STRESS AND POTASSIUM FERTILIZATION GAS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT
STRESS AND POTASSIUM FERTILIZATION GAS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT
STRESS AND POTASSIUM FERTILIZATION
A. S. DIAS et al. Means followed by the same letter do not differ by Tukey test (p<0.05). Figure 3. Initial fluorescence - Fo (A) and variable fluorescence - Fv (B) of grafted West Indian cherry, as a function of
irrigation water salinity – ECw, at 400 days after transplanting. AS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT
STRESS AND POTASSIUM FERTILIZATION Means followed by the same letter do not differ by Tukey test (p<0.05). Figure 3. Initial fluorescence - Fo (A) and variable fluorescence - Fv (B) of grafted West Indian cherry, as a function of
irrigation water salinity – ECw, at 400 days after transplanting. Figure 3. Initial fluorescence - Fo (A) and variable fluorescence - Fv (B) of grafted West Indian cherry, as a fu
irrigation water salinity – ECw, at 400 days after transplanting. With a similar trend to that of Fo, the variable
fluorescence of West Indian cherry was lower in
plants subjected to high salinity (3.8 dS m-1) in the
irrigation water (Figure 3B). Plants irrigated with
low-salinity water had Fv of 330.08, which was
39.57% higher than that found in plants subjected to
irrigation with 3.8 dS m-1. Such reduction of Fv in
West Indian cherry reflects losses of photochemical
activity in the leaves. For Baker (2008), the variable
fluorescence reflects the capacity of the plant to
transfer energy from electrons ejected from pigment
molecules to the formation of NADPH, ATP and
reduced ferredoxin (Fdr) and, consequently, a higher
capacity for CO2 assimilation in the biochemical
phase of photosynthesis. Freire et al. (2014) report
that the reduction in Fv indicates damage to the
photosynthetic apparatus due to the use of saline
water, consequently compromising PSII. negatively affected not only by the deleterious
effects of salinity, but also by the reduction in water
availability to the West Indian cherry plants caused
by the decrease in the osmotic potential of the soil
solution due to irrigation with saline water. A. S. DIAS et al. A. S. DIAS et al. The reduction of Fm observed in plants
irrigated with 0.8 dS m-1 and fertilized with the
highest
dose
of
K
(125%
of
the
K2O
recommendation) can be explained by the use of KCl
in the present study as a source of K, since it has a
high salt index (116.3) and, when associated with
salt stress, can induce a reduction in the water
availability to plants as a consequence of the
decrease in the osmotic potential of the soil solution. For Silva et al. (2015), the maximum fluorescence
represents
the
maximum
intensity
of
the
fluorescence emitted, when almost all the quinone is
reduced and the reaction centers reach their
maximum capacity for photochemical reactions, a
process which requires electrons from water. By
contrast, the increase of Fm in plants subjected to
salt stress reflects the benefits of K for plants, which
are associated with characteristics that lead to
efficient water use, such as regulation of turgidity,
stomatal opening and closure, and control of
transpiration. Fv/Fm values ranging from 0.75 to 0.85 indicate that
the photosynthetic apparatus is intact. However, the
Fv/Fm values presented in this study for plants
irrigated with 3.8 dS m-1 are below the proposed
interval, regardless of the K dose. Therefore, it can
be inferred that the K doses tested were not able to
mitigate the deleterious effects of the salt stress on
photosystem II. For Freire et al. (2013), one of the factors
linked to the photosynthetic efficiency of plants
(Fv/Fm) and, consequently, to their growth and
adaptability to adverse environments, is the content
of chlorophyll and carotenoids in their leaves. In the
present study, the lowest contents of chlorophyll a
(Figure 5A) and carotenoids (Figure 5B) were found
in
plants
irrigated
with
high-salinity
water
(3.8 dS m-1). Compared to plants irrigated using
water of the lowest ECw level (0.8 dS m-1), there
were reductions of 21.48 and 57.21% in the Chl a
and Car contents, respectively (Figure 5A and 5B). Reduction in the chlorophyll a content in
plants exposed to water salinity is probably due to
the increase of the enzyme chlorophyllase, which
degrades the molecules of this photosynthesizing
pigment, as observed by Freire et al. (2013). A. S. DIAS et al. On the
other hand, the decrease in carotenoid content may
be attributed, among other causes, to the fact that salt
stress leads to a reduction in the production of
photosynthetic pigments, inducing the degradation of
β-carotene, causing a decrease in the content of
carotenoids, which are integrated components of the
thylakoids, acting in the absorption and transfer of
light to chlorophyll (SILVA et al., 2016). As observed for the maximum fluorescence,
the
interaction
between
the
studied
factors
(SL x KD) also significantly influenced the potential
quantum efficiency of PSII. According to the
regression equations (Figure 4B), the Fv/Fm data of
the West Indian cherry plants irrigated with 0.8 and
3.8 dS m-1 fitted to a quadratic model. For plants
subjected to irrigation with 0.8 dS m-1 there was a
reduction in the quantum efficiency of those which
received 75% of the K2O recommendation compared
to plants fertilized with 50%. However, from this
dose on there was an increase of Fv/Fm as a function
of K fertilization and, according to the regression
equation,
the
maximum
value
of
Fv/Fm
(1.23) was estimated in the West Indian cherry plants
fertilized with 125% of K, representing a 16.24%
increase in quantum efficiency compared to those
which received a K dose of 50%. The increase in
photochemical efficiency as a function of K
fertilization is due to higher activity of ribulose-1,5-
bisphosphate carboxylase/oxygenase (RuBisCO) and
the photosynthetic rate caused by the increase of K
content in the leaves (MENDES et al., 2013). g
p y (
)
Moreover, these reductions can be considered
as the acclimation of the plants to the salt stress
imposed, in an attempt to conserve energy and,
consequently, capture less light energy, and so
reduce the flow of electrons to the electron transfer
chain, thus avoiding any photo-oxidative stresses
(SILVA et al., 2016). Tatagiba et al. (2014) explain
that the contents of chlorophyll and carotenoids
decrease under high salt concentrations and that the
significant reductions found in A, as observed in the
present study, may also be attributed to the loss of
these photosynthetic pigments because, according to
Taiz and Zeiger (2013), these pigments are an
integral part of the light-harvesting complex for the
photosynthetic process. For plants subjected to irrigation with 3.8 dS
m-1 water, the regression equation (Figure 4B) shows
that the maximum estimated value of Fv/Fm
(0.59) occurred at the dose of 83% of the K2O
recommendation. A. S. DIAS et al. The maximum fluorescence of the West
Indian cherry plants responded significantly to the
interaction between the salinity levels and potassium
doses (Figure 4A) and, according to the regression
equations, the data fitted to a quadratic model. The
regression equations (Figure 4A) showed that, in
plants irrigated using water of the lowest salinity
(0.8 dS m-1), the increment in K dose caused an
increase in Fm up to the estimated dose equivalent to
83%
of
the
recommendation
of
Musser
(409.78), from which this variable decreased. Under
irrigation using 3.8 dS m-1 water, there was an initial
reduction in Fm between the doses of 50 and 70% of
the K recommendation, with increments from this
point on, and the highest value for this variable (468)
was found in plants fertilized with 125% of the K2O
recommendation, which is equivalent to an increase
of 11.82% compared to those cultivated with a K
dose of 50% of the recommendation. Silva et al. (2015) observed that the highest
value of variable fluorescence (1842.13) occurred
with the application of 166% ETc, representing a
29% increment compared to the application of 33%
ETc, i.e., higher water availability led to an increase
in Fv, from which it can be inferred that Fv was Figure 4. Maximum fluorescence – Fm (A) and potential quantum efficiency - Fv/Fm (B) in grafted West Indian cherry, as
a function of the interaction between irrigation water salinity – ECw and potassium doses, at 400 days after transplanting. Figure 4. Maximum fluorescence – Fm (A) and potential quantum efficiency - Fv/Fm (B) in grafted West Indian cherry, as
a function of the interaction between irrigation water salinity – ECw and potassium doses, at 400 days after transplanting. Rev. Caatinga, Mossoró, v. 32, n. 2, p. 429 – 439, abr. – jun., 2019 Rev. Caatinga, Mossoró, v. 32, n. 2, p. 429 – 439, abr. – jun., 2019 435 A. S. DIAS et al. According to Silva et al. (2015), Rev. Caatinga, Mossoró, v. 32, n. 2, p. 429 – 439, abr. – jun., 2019 436 GAS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT
STRESS AND POTASSIUM FERTILIZATION AS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT
STRESS AND POTASSIUM FERTILIZATION GAS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT
STRESS AND POTASSIUM FERTILIZATION
A. S. DIAS et al. 1
2 Means followed by the same letter do not differ by Tukey test (p<0.05). Figure 5. Contents of chlorophyll a – Chl a (A) and carotenoids – Car (B) in grafted West Indian cherry, as a function of
irrigation water salinity – ECw and chlorophyll b content – Chl b (C), as a function of the interaction between ECw and
potassium doses. 1 Means followed by the same letter do not differ by Tukey test (p<0.05). Figure 5. Contents of chlorophyll a – Chl a (A) and carotenoids – Car (B) in grafted West Indian cherry, as a function of
irrigation water salinity – ECw and chlorophyll b content – Chl b (C), as a function of the interaction between ECw and
potassium doses Regarding the chlorophyll b of the West
Indian cherry plants, the interaction between the
studied factors (saline levels and potassium doses)
had a significant effect on its content in the leaves. According to Figure 5C, for plants irrigated using
water of the lowest salinity (0.8 dS m-1), the data
fitted best to a decreasing linear model and,
according to the regression equation (Figure 5C),
Chl b contents decreased by 19.26% between plants
fertilized with 125% of the K recommendation and
those subjected to 50%, which represents a reduction
of 0.14 mg g-1 FM. For the Chl b contents in plants
irrigated with 3.8 dS m-1 water, the data fitted to a
quadratic model and, according to the regression
equation, this variable decreased between K doses of
50 and 70% and increased from this point on,
reaching its highest value (1.94 mg g-1 FM) in plants
fertilized with 125% of the K2O recommendation. CONCLUSIONS CAETANO, P. K. Processamento tecnológico e
avaliação energética de geléia de acerola. 2010. 94
f. Dissertação (Mestrado em Agronomia: Área de
Energia na Agricultura) - Universidade Estadual
Paulista Julio de Mesquita Filho, Botucatu, 2010. The
gas
exchanges,
chlorophyll
a
fluorescence and photosynthetic pigments of West
Indian cherry are negatively affected by irrigation
using water with an electrical conductivity of 3.8 dS
m-1,
which
compromised
the
photosynthetic
apparatus, a situation evidenced by the reduction in
photosystem II quantum efficiency. CAVALCANTE, L. F. et al. Clorofila e carotenoides
em maracujazeiro-amarelo irrigado com águas
salinas no solo com biofertilizante bovino. Revista
Brasileira de Fruticultura, v. 33, sup., p. 699-705,
2011. Potassium doses led to increments in the
transpiration, chlorophyll a maximum fluorescence
and chlorophyll b content of West Indian cherry
grown under salt stress. Potassium doses of up to
125% of the recommendation do not attenuate the
negative effects of irrigation with 3.8 dS m-1 water
on the potential quantum efficiency of West Indian
cherry leaves. DONAGEMA, G. K. et al. Manual de métodos de
análise de solos. 2. ed. rev. Rio de Janeiro, RJ:
Embrapa Solos, 2011. 230 p. (Embrapa Solos. Documentos, 132). EMPRESA
BRASILEIRA
DE
PESQUISA
AGROPECUÁRIA - EMBRAPA. Cultivar acerola
BRS
366-Juburu. Fortaleza,
CE:
Embrapa
Agroindústria Tropical, 2012. Folder. ACKNOWLEDGMENTS To the National Program of Post-Doctorate
(PNPD/CAPES/UFCG), for granting the scholarship
to the second author and to the National Institute of
Science and Technology in Salinity - INCTSal, for
funding the project. FERREIRA, D. F. Sisvar: a guide for its bootstrap
procedures in multiple comparisons. Ciência e
Agrotecnologia, v. 38, n. 2, p. 109-112, 2014. FREIRE, J. L. O. et al. Rendimento quântico e trocas
gasosas em maracujazeiro amarelo sob salinidade
hídrica, biofertilização e cobertura morta. Revista
Ciência Agronômica, v. 45, n. 1, p. 82-91, 2014. GAS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT
STRESS AND POTASSIUM FERTILIZATION GAS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT
STRESS AND POTASSIUM FERTILIZATION A. S. DIAS et al. chlorophyll
fluorescence
can
improve
crop
production strategies: an examination of future
possibilities. Journal Experimental Botany, v. 55,
n. 403, p. 1607-1621, 2004. pigment, the production of amino acids in the
activation of enzymes, and the synthesis of proteins. Porto et al. (2013) observed an increase in SPAD
readings in plants when the K2O doses increased. These authors also claim that the obtained results
reflect better N use in plants under K supply, as well
as higher water use efficiency in these plants. BONFIM-SILVA, E. M. et al. Leguminosa híbrida
Java submetida à calagem em Latossolo Vermelho
do Cerrado. Enciclopédia Biosfera, v. 7, n. 13,
p. 1811-1820, 2011. A. S. DIAS et al. From the increase of Chl b contents in the leaves of
the West Indian cherry plants subjected to salinity in
response to K fertilization, it can be inferred that the
supplementation with this nutrient reduced the
deleterious effects of salt stress on this pigment, an
important fact because it is directly related to
photosynthesis and its increment may improve the
photosynthetic rate. (Figure 4A), the reduction in Chl b content can be
explained by the possible negative effects of high
doses of K on plants subjected to salt stress, which
has
already
been
reported
in
the
literature
(LACERDA et al., 2003; PRAZERES et al., 2015),
as well as by the decrease in Ca and Mg contents in
the plants, as a consequence of the effect of
competitive inhibition between these two ions so that
the increase in K+ induces the deficiency of Ca2+ and
Mg2+ (SILVA; TREVIZAM, 2015). Therefore, it is
related to the chlorophyll content, since Mg
participates in the structure of the chlorophyll
molecule, occupying a central position, and is also a
cofactor in ATP hydrolysis (BONFIM-SILVA et al.,
2011). This hypothesis is supported by Melo et al. (2014), who observed that Ca and Mg contents in the
leaves of “Prata-Anã” banana decreased as the K
doses applied by fertigation increased. The increase observed in the chlorophyll
content of plants irrigated with 3.8 dS m-1 in
response to K fertilization is associated with the role
played by this nutrient in the plant. Although K does
not participate in the structure of chlorophyll
molecules, or any organic compound, it plays an
important role in the N metabolism, which requires
adequate quantities of K in the cytoplasm (VIANA;
KIEHL, 2010), fundamental for the synthesis of this It is believed that, as observed for Fm Rev. Caatinga, Mossoró, v. 32, n. 2, p. 429 – 439, abr. – jun., 2019 437 GAS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT
STRESS AND POTASSIUM FERTILIZATION potássio. Revista Ciência Agronômica, v. 41, n. 1,
p. 18-28, 2010. gasosas de plantas de feijão-caupi sob irrigação
salina e doses de potássio, Revista Agroambiente,
v. 9, n. 2, p. 111-118, 2015. HEIDARI, M.; JAMSHID, P. Interaction between
salinity and potassium on grain yield, carbohydrate
content and nutrient uptake in pearl millet. ARPN
Journal of Agricultural and Biological Science,
v. 5, n. 6, p. 39-46, 2010. SÁ, F. V. S. et al. Water relations and gas exchanges
of West Indian cherry under salt stress and nitrogen
and phosphorus doses. Journal of Agricultural
Science, v. 9, n. 10, p.168-177, 2017. SILVA, A. R. A. et al. Pigmentos fotossintéticos e
potencial hídrico foliar em plantas jovens de
coqueiro sob estresse hídrico e salino, Revista
Agroambiente, v. 10, n. 4, p. 317-325, 2016. HUSSAIN, S. et al. Physiological analysis of salt
stress behavior of citrus species and genera: low
chloride accumulation as an indicator of salt
tolerance. South African Journal of Botany, v. 81,
s/n., p. 103-112, 2012. SILVA, F. G. et al. Trocas gasosas e fluorescência
da clorofila em plantas de berinjela sob laminas de
irrigação, Revista
Brasileira
de
Engenharia
Agrícola e Ambiental, v. 19, n. 10, p. 946-952,
2015. LACERDA, C. F. et al. Crescimento e acúmulo de
íons em folhas de sorgo forrageiro submetido a
soluções iso-osmóticas de sais (NaCl + KCl). Revista Ciência Agronômica, v. 34, n. 1, p. 1-6,
2003. SILVA, M. L. S.; TREVIZAM, A. R. Interações
iônicas e seus efeitos na nutrição das plantas. Jornal
Informações Agronômicas, v. 37, n. 149, p. 10-16,
2015. MACIEL, M. I. S. et al. Caracterização físico-
química de frutos de genótipos de aceroleira
(Malpighia emarginata D.C.), Ciência e Tecnologia
de Alimentos, v. 30, n. 4, p. 865-869, 2010. SOARES, L. A. A. et al. Troca de CO2 do feijão-
caupi irrigado com água salina e fertilização
nitrogenada, Agropecuária Científica no Semi-
Árido, v. 9, n. 3, p. 30-37, 2013. MEDEIROS, J. F. Qualidade da água de irrigação
utilizada nas propriedades assistidas pelo “GAT”
nos Estados do RN, PB, CE e avaliação da
salinidade dos solos. 1992. 173 f. Dissertação
(Mestrado em Engenharia Agrícola: Área de
Concentração Irrigação e Drenagem) - Universidade
Federal de Campina Grande, Campina Grande, 1992. SOUSA, J. R. M. et al. Impact of saline conditions
and nitrogen fertilization on citrus production and
gas exchanges, Revista Caatinga, v. 29, n. 2, p. 415
-424, 2016. MELO, A. S. et al. GAS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT
STRESS AND POTASSIUM FERTILIZATION GAS EXCHANGES, QUANTUM YIELD AND PHOTOSYNTHETIC PIGMENTS OF WEST INDIAN CHERRY UNDER SALT
STRESS AND POTASSIUM FERTILIZATION REFERENCES ADRIANO, E. et al. Qualidade de fruto da aceroleira
cv. Olivier em dois estádios de maturação. Revista
Brasileira Fruticultura, v. 33, sup., p. 541-545,
2011. FREIRE, J. L. O. et al. Teores de clorofila e
composição mineral foliar do maracujazeiro irrigado
com águas salinas e biofertilizante, Revista de
Ciências Agrárias, v. 36, n. 1, p. 57-70, 2013. ARAÚJO, H. S. et al. Doses de potássio em
cobertura
na
cultura
da
abóbora,
Pesquisa
Agropecuária Tropical, v. 42, n. 4, p. 469-475,
2012. GONÇALVES, E. R. et al. Trocas gasosas e
fluorescência da clorofila a em variedades de cana-
de-açúcar submetidas à deficiência hídrica. Revista
Brasileira de Engenharia Agrícola e Ambiental,
v. 14, n. 4, p. 378–386, 2010. ARNON, D. I. Copper enzymes in isolated
cloroplasts: polyphenoloxidases in Beta vulgaris. Annual Reviews Plant Physiology, v. 24, n. 1, p. 1-
15, 1949. GORBE, E.; CALATAYUD, A. Applications of
chlorophyll fluorescence imaging technique in
horticultural
research:
a
review. Scientia
Horticulturae, v. 138, s/n., p. 24-35, 2012. BAKER, N. R. Chlorophyll fluorescence: a probe of
photosynthesis in vivo. Annual Reviews of Plant
Biology, v. 59, s/n., p. 89-113, 2008. Rev. Caatinga, Mossoró, v. 32, n. 2, p. 429 – 439, abr. – jun., 2019
, s/n., p. 89-113, 2008. R.; ROSENQVIST, E. Applications of
GURGEL, M. T.; GHEYI, H. R.; OLIVEIRA, F. H. T. Acúmulo de matéria seca e nutrientes em
meloeiro produzido sob estresse salino e doses de 2, p. 429 – 439, abr. – jun., 2019
GURGEL, M. T.; GHEYI, H. R.; OLIVEIRA, F. H. T. Acúmulo de matéria seca e nutrientes em
meloeiro produzido sob estresse salino e doses de GURGEL, M. T.; GHEYI, H. R.; OLIVEIRA, F. H. T. Acúmulo de matéria seca e nutrientes em
meloeiro produzido sob estresse salino e doses de BAKER, N. R.; ROSENQVIST, E. Applications of 438 Rev. Caatinga, Mossoró, v. 32, n. 2, p. 429 – 439, abr. – jun., 2019 potássio. Revista Ciência Agronômica, v. 41, n. 1,
p. 18-28, 2010. Chlorophyll and macronutrients
content in leaf tissue of Musa sp ‘Prata-Anã’ under
fertigation, African
Journal
of
Agricultural
Research, v. 9, n. 22, p. 1714-1720, 2014. SYVERTSEN, J. P., GARCIA-SANCHEZ, F. Multiple abiotic stresses occurring with salinity
stress in citrus. Environmental and Experimental
Botany, v. 103, s/n., p. 128-137, 2014. MENDES, H. S. J. et al. Respostas fisiológicas de
genótipos de Eucalyptus grandis x E. urophylla à
disponibilidade
hídrica
e
adubação
potássica. Revista Cerne, v. 19, n. 4, p. 603-611, 2013. TAIZ, L.; ZEIGER, E. Fisiologia vegetal. 5. ed. Porto Alegre, RS: Artmed, 2013. 954 p. TATAGIBA, S. D. et al. Limitações fotossintéticas
em folhas de plantas de tomateiro submetidas a
crescentes
concentrações
salinas. Revista
Engenharia na Agricultura, v. 22, n. 2, p. 138-149,
2014. MUSSER, R. dos S. Tratos culturais na cultura da
acerola. In: SÃO JOSÉ, A. R.; ALVES, R. E. (Eds.). Acerola no Brasil: Produção e mercado. Vitória da
Conquista, BA: UESB, 1995. cap. 3, p. 47-52. VIANA, E. M.; KIEHL, J. C. Doses de nitrogênio e
potássio no crescimento do trigo. Bragantia, v. 69,
n. 4, p. 975-982, 2010. PÁDUA, L. S. et al. Produção de porta-enxerto de
goiabeira cultivado com águas de diferentes
salinidades e doses de nitrogênio, Revista Ciência
Agronômica, v. 48, n. 4, p. 596-604, 2017. VIEIRA, D. A. P. et al. Fluorescência e teores de
clorofilas em abacaxizeiro cv. Pérola submetido a
diferentes concentrações de sulfato de amônio. Revista Brasileira de Fruticultura, v. 32, n. 2,
p. 360, 2010. VIEIRA, D. A. P. et al. Fluorescência e teores de
clorofilas em abacaxizeiro cv. Pérola submetido a
diferentes concentrações de sulfato de amônio. PORTO, R. A. et al. Adubação potássica em plantas
de rúcula: produção e eficiência no uso da água. Revista Agroambiente, v. 7, n. 1, p. 28-35, 2013. Revista Brasileira de Fruticultura, v. 32, n. 2,
p. 360, 2010. PRAZERES, S. S. et al. Crescimento e trocas This work is licensed under a Creative Commons Attribution-CC-BY https://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution-CC-BY https://creativecommons.org/licenses/by/4.0/ 439 Rev. Caatinga, Mossoró, v. 32, n. 2, p. 429 – 439, abr. – jun., 2019 Rev. Caatinga, Mossoró, v. 32, n. 2, p. 429 – 439, abr. – jun., 2019
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T. J. Coles, Capitalism and Coronavirus: How Institutionalized Greed Turned a Crisis into a Catastrophe, reviewed by Raymond Michalowski
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State crime
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Reviewed by Raymond Michalowski In the opening pages of Capitalism and Coronavirus, author T. J. Coles sets out two
key goals. The first is to document causal linkages between the search for capital
accumulation, in Cole’s terms “enrichment of the few,” and the many and complex
ways this search has increased levels of morbidity, mortality, and social disruption
due to COVID-19 (SARS-COV-2). The second is to consider why the most extreme
capitalist countries in the world, according to Coles the UK and the US, fared worse
in dealing with COVID-19 than the capitalist nation-states of Germany, New Zealand,
Vietnam, and South Korea, and the semi-autonomous regions of Hong Kong, and the
state of Kerala in India. By placing the COVID-19 pandemic within a comparative framework, Coles lays
out a broad canvas for assessing the variable nature of the relationship between capi
talism and coronavirus as it manifests itself in different national experiences with the
disease. Capitalism and Coronavirus begins this project by listing seven key variables that
can shape the impact of COVID-19 on different national populations. These are: (1)
The age composition of a population, given that, at the time of the book’s writing,
COVID-19 appeared to be far more deadly for elderly as compared to young or middle-
aged groups. (2) The strength of a nation’s health care system. (3) The extent and char
acter of poverty within the country. (4) The size of a nation’s ethnic minority population,
insofar as ethnic minorities often experience multiple health risks such as poverty, inad
equate housing, limited access to health care, and overall poorer health. (5) Population
density, because of its positive relationship to rates of disease transmission. (6) Location. Here Cole’s identifies places like New York or London as relevant “locations” for ram
pant COVID-19. Although he does not explicitly identify these urban incubators of
pandemic as examples of the wider phenomenon of what are frequently termed “global
cities,” the implication is there. This suggests that another method of addressing the
question about why various capitalist countries responded differently to the pandemic
might be to compare the COVID-19 experience across global cities, rather than just
across nation-states. (7) The overall health of the population, which is to some extent a
consequence of the other six factors, along with additional characteristics of a nation’s
social structure and cultural practices. T. J. Coles, Capitalism and Coronavirus: How Institutionalized Greed
Turned a Crisis into a Catastrophe, published independently, 2020,
290pp, £15.99 (paperback). T. J. Coles, Capitalism and Coronavirus: How Institutionalized Greed
Turned a Crisis into a Catastrophe, published independently, 2020,
290pp, £15.99 (paperback). Reviewed by Raymond Michalowski By locating the problem of COVID-19 in a comparative context, Coles points
to a way toward analysing the variable relationship between capitalism and public
health. As one of the first books to focus explicitly on how global, neo-liberal Produced and distributed by Pluto Journals www.plutojournals.com/scj/ 161 161 BOOK REVIEWS T. J. Coles, Capitalism and Coronavirus: How Institutionalized Greed
Turned a Crisis into a Catastrophe, published independently, 2020,
290pp, £15.99 (paperback). Produced and distributed by Pluto Journals www.plutojournals.com/scj/ DOI:10.13169/statecrime.11.1.0161 DOI:10.13169/statecrime.11.1.0161 DOI:10.13169/statecrime.11.1.0161 DOI:10.13169/statecrime.11.1.0161 162 BOOK REVIEWS capitalism has shaped and been shaped by national experiences under the current
pandemic conditions, Capitalism and Coronavirus makes a valuable contribution
to the emergent literature on the political-economy of COVID-19. While Capitalism and Coronavirus is not explicitly grounded in state crime or state-
corporate crime analysis, it offers several elements of use to criminologists. First, by
presenting a compendium of over 500 references to news stories and reports about
COVID-19 gleaned from a global array of online sources, the book can serve as a refer
ence work of contemporaneous writings about the pandemic. The index and reference
section of Capitalism and Coronavirus alone constitutes 41 per cent of the book. I would
note, however, that few books on health, health policy, or state or corporate wrongdoing
show up among the referenced work. Whether this is, or is not, a positive step toward
relocating social analysis to the meta-verse is something for readers to decide. Second, in its exploration of the ways capitalism is either responsible for or
exacerbated COVID-19, which might have occurred under any circumstances (the
author vacillates between these positions), Capitalism and Coronavirus fore
grounds the importance of analysing the relationship between political-economic
arrangements and upstream determinants of health. Although Coles does not spe
cifically address this point, I would suggest that adverse, upstream health condi
tions created by capitalist social structures are a crucial form of wrongful state and
corporate social harm, but one that receives relatively little attention within crimi
nology, with the notable exception of analyses focused on environmental crimes
such as pollution, industrial disasters, or global climate change. Third, Capitalism and Coronavirus models the need to recognize social com
plexity in the analysis of health outcomes. Coles does a good job in considering
how cross-connections between multiple social forces such as government poli
cies, inequality, racism, sexism, and political and economic inequalities produce a
public health whole larger than the sum of any of its individual parts. Taken together, these qualities make Coronavirus and Capitalism a worthwhile
read. From the perspective of criminological analysis, however, the book is under
developed in three ways that limit its use in developing new theoretical frames for
studying problems of capitalism and public health. DOI:10.13169/statecrime.11.1.0161 First, the book’s framing of capitalism as the cause of all bad health outcomes
has the feel of the proverbial man who has only a hammer, and so sees every prob
lem as a nail. While this may be consistent with a base/superstructure approach to
Marxist analysis, it undervalues the important theoretical developments of struc
turalist, post-modernist, feminist, and critical race theorizing that have demon
strated, or at least offered powerful suggestions, that economy is not the only
independent variable shaping societies. Coles’s instrumental Marxist approach, with its focus on economy as the key
motive force, makes Capitalism and Coronavirus better suited to inviting political State Crime 11.1 2022 BOOK REVIEWS 163 outrage than suggesting pathways to deeper analyses of the problem. Given the
horrific levels of death, disease, and disruption caused by malfeasance, incompe
tence, corruption, and political manipulation in the face of a grave viral threat over
the last three years, outrage is appropriate. However, many times while reading the
book I kept hoping for more portals that would take me beneath the narrative of
wrongdoing to a deeper analytic understanding. Second, by laying much of the blame for the coronavirus disaster on the search by
capitalists for private enrichment, that is, greed, Capitalism and Coronavirus risks the
theoretical error of supplanting social structures with individual behaviour as the key
locus of the problem. Left-sympathetic explanations for the problems of capitalist
society that rest on the private flaws of capitalists fit well with Margaret Thatcher’s
articulation of the neo-liberal ideology that that there is no society; there are only
“individual men and women.”1 What is missing when we focus on capitalists and
their greed is a theoretical framework that explains why capitalists act the way they
do, one that reaches beyond concepts of outrage-inducing moral failing. Third, Capitalism and Coronavirus provides valuable information about how
differently organized capitalisms have resulted in differing levels of death, dis
ease, and disruption over the course of the pandemic. What is absent is a theoreti
cal framework that helps explain how and why different capitalist countries forged
different paths to capital accumulation, and in doing so generated different public
health outcomes. It is here where the comparative analysis between countries feels
thin. This may be a critique too far, since answering that question would require
significant historical, political-economic, and cultural analysis of each of the
countries being compared. DOI:10.13169/statecrime.11.1.0161 It might have been beneficial, however, if the author
had problematized those differences rather than presenting them as givens. Despite being somewhat theoretically underdeveloped, Capitalism and
Coronavirus is worth reading for anyone interested in public health wrongs as
state and/or state-corporate crimes. Raymond Michalowski, Arizona Regents’ Emeritus Professor of Criminology,
Northern Arizona University, Flagstaff, Arizona. Raymond Michalowski, Arizona Regents’ Emeritus Professor of Criminology,
Northern Arizona University, Flagstaff, Arizona. Produced and distributed by Pluto Journals www.plutojournals.com/scj/ Note 1. While Thatcher’s full statement was more nuanced than this, it nevertheless captured the essence of a
Hayekian understanding that social structures are the outcome of individual and collective experiences
and actions, not the cause of them (Steele, 2009, “There Is No Society,” New Statesmen. Available
online at: https://iea.org.uk/blog/there-is-no-such-thing-as-society (accessed 14 April 2022). Produced and distributed by Pluto Journals www.plutojournals.com/scj/
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Rockfall Hazard in an Old Abandoned Aggregate Quarry in the City of T&#233;touan, Morocco
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International journal of geosciences
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1. Introduction pedestrians and as an unofficial parking lot, which makes
it a source of great risk for these people even though the
site has been officially included in the urban area. The city of Tétouan has witnessed a very important
demographic growth in the last few decades. This growth
is related mostly to a phenomenon of rural exodus that
has been increasing due to various causes like job op-
portunities and better conditions of life but mostly be-
cause of the long period of drought in Morocco peaked in
the late 1980’s and early 1990’s. The total population has
gone from 206.556 in 1982 to 318.698 in 2004 (official
census data from the High Commission for Planning). This growth has put a big strain on the urban capacity of
the city resulting in the creation of various slum areas
around the city. International Journal of Geosciences, 2013, 4, 1228-1232
http://dx.doi.org/10.4236/ijg.2013.48116 Published Online October 2013 (http://www.scirp.org/journal/ijg) Hatim Dellero*, Younes El Kharim
Department of Geology, Faculty of Sciences of Tétouan, Abdelmalerk Essaadi University, Tétouan, Morocco
Email: *Hatim_dellero@hotmail.com Hatim Dellero*, Younes El Kharim
Department of Geology, Faculty of Sciences of Tétouan, Abdelmalerk Essaadi University, Tétouan, Morocco
Email: *Hatim_dellero@hotmail.com Received August 15, 2013; revised September 12, 2013; accepted September 29, 2013 Copyright © 2013 Hatim Dellero, Younes El Kharim. This is an open access article distributed under the Creative Commons Attribu-
tion License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited. ABSTRACT Quarries are places of high instability due to blastings, if left without rehabilitation they become sites of great risk of
rockfall, which are the case of the Carian quarry, a small abandoned aggregate quarry, which turned into a slum
neighborhood in the north western part of the city of Tétouan, and that has a long history of rockfall incidents since it
was shut down in the 60’s. Using rockmass characterization techniques, kinematic study and stability analysis, it was
confirmed that many instability mechanisms are found on the walls of the quarry and cause a high risk of rockfall dur-
ing the rain season, especially that the study area is inhabited. Rockfall trajectory modelling techniques allow the calcu-
lation of vertical distribution of rocks falling from the quarry’s walls along different profiles in the area and therefore
the determination of a safety perimeter from the quarry wall. Keywords: Abandoned Quarry; Rockfall; Safety Parameter; Tétouan *Corresponding author. Copyright © 2013 SciRes. 2. Description of the Quarry The subject of this study is a small scale abandoned ag-
gregate quarry exploiting a small hill in the north western
part of Tétouan at the following coordinates: 35˚34'32"N;
5˚23'30"W. The quarry stretches in NE-SW direction
with its face toward the south east and it extends ap-
proximately over 120 m (Figure 1). The face of the
quarry from 8 to 30 m high is highly irregular. From a geological point of view the exploited site is a
klippe that belongs to the Predorsalian nappe [1,2],
which is of limestone with marl interbeds of the Upper
Oligocene Age. One of these areas constitutes the subject of this study. It is an old abandoned quarry left without rehabilitation
since it was shut down in the 60s, which became a place
for unauthorized construction that has converted the site
into a neighborhood that conveniently took the name of
“CARIAN” which means “quarry” in the local dialect. Many houses are surrounding the quarry’s wall; others
are built on top of the quarry very close the edge of the
slope. On the wall of the quarry multiple caves are
founds and they can be frequented by people on a daily
basis and its ground is used as a frequent walkway for The face of the quarry is heavily fractured due to both
tectonics and to the use of explosives during its active
years. The joints in the rock mass vary from small scale
fractures to decametric faults that run vertically through
the whole rock mass; the fracturing becomes denser to-
wards the east as the rock benches narrow (Figure 2). The face of the quarry is highly weathered and unclean
due to human activity in which they use the slope as a
dump, at its foot we can find blocks of rocks of multiple IJG H. DELLERO, Y. EL KHARIM 1229 Figure 1. The plan of the quarry. situ), width and fill of the opening, weathering and the
existence of water. The measurements were then reported on the canvas of
Schmidt (Figure 4) with the help of DIPS (5.1) (Roc-
Sciences) [4] in which we were able to determine five
different join sets represented in Table 1 with their dif-
ferent parameters. 3. History of Rockfalls where:
n
is the normal stress;
r
is the residual
friction angle of the surface; JRC is the joint roughness
coefficient and JCS is the joint wall compressive
strength. There is no official registry of the previous incidents but
according to local residents in the neighborhood, they are
frequent and can happen every year with some major
ones that occurred in 1969, 1972 and 1977 during the
rainy season, resulting the fall of big blocks of rock
(Figure 3). Starting from a basic friction angle of
b
= 28˚ we
were able to estimate
r
for each join set using the
following equation:
20
r
b
r R
(2) In recent years, a major rockfall incident happened
January the 29th and the 31st of 2010, which followed a
week of rain (208 mm) and two days after the rainiest
day of the year (88 mm). Another one of a lesser scale
happened in mid-January 2012 resulting screes and
rockfalls, these two incidences have affected mostly the
eastern area of the quarry where it is most fractured and
caused it to recede noticeably. (2) where r is the Schmidt rebound number wet and weath-
ered fracture surfaces and R is the Schmidt rebound
number on dry unweathered sawn surfaces. Combining Equations (1) and (2) we were able to de-
termine the specific shear strength value for each family
set. 5.1. Geotechnical Parameters The rock have a density of 2631 kg/m3, GSI = 48,
n
= 148 MPa and mi = 8; and using the Hoek-Brown fail-
ure criterion we were able to determine the average co-
hesion; c = 0.337 MPa using RocData (Rocscience,
2004). 5. Stability Analysis sizes that varies from few centimeters to metric size. People from the area have confirmed that a lot of these
rocks did fell off the quarry face, that lay on top of
layers of harden overburden material, from the old min-
ing activity, which, presumably has been serving as a
catch bench for these rock falls. Multiple screes also can
be noticed on quarry’s wall; some of them look more
recent than others (Figure 2), we have also found multi-
ple caves of different sizes across the wall of the quarry,
some of which are at its foot and form a cantilever—like
structure. • Angle of friction Using Barton’s equation for estimating shear strength
in a rock mass [6]
tan
JRClog10 JCS
n
r
n
(1) (1) Quality of Rock Mass and Geomechanics
Classification After having established the geotechnical parameters of
the rock mass, it was possible to estimate its geomechan-
ics quality, using the RMR classification proposed by
Bienawski (1989) [5], as shown in Table 2. The value of
the RMR in the quarry was estimated at 61 which reflect
a fair to good rock quality. Figure 1. The plan of the quarry. Copyright © 2013 SciRes. 5.2. Kinematic Analysis In order to characterize the rock mass, a study of its dis-
continuities was conducted on the quarry’s wall using the
one proposed by the IRSM [3], in which we have real-
ized an inventory of discontinuities that appear across the
quarry’s wall in order to determinate these parameters:
orientation, persistence, spacing, roughness, strength (in The stereographic representation of the join sets with the
slope (Figure 5) allows the realization of a kinematic
analysis on the walls of the quarry divided in five do-
mains, following the variations of slope to identify the
different failure mechanisms that occur in the quarry
7,8]. [ IJG H. DELLERO, Y. EL KHARIM 1230 Figure 2. Fracturing and instability components showing on the face of the quarry. (1) Talus; (2) Caves; (3) Overburden; (4)
Unstable rocks attached to the face of the quarry; (5) Highly fractured area. Figure 2. Fracturing and instability components showing on the face of the quarry. (1) Talus; (2) Caves; (3) Overburden; (4)
Unstable rocks attached to the face of the quarry; (5) Highly fractured area. Figure 2. Fracturing and instability components showing on the face of the quarry. (1) Talus; (2) Caves; (3) Overburden; (4)
Unstable rocks attached to the face of the quarry; (5) Highly fractured area. Figure 3. Photo of the eastern part of the quarry showing
different blocks that have fallen off the slope during differ-
ent incidences. Figure 5. Stereographic representations of the join set and
slope in one of the positions. Figure 3. Photo of the eastern part of the quarry showing
different blocks that have fallen off the slope during differ-
ent incidences. Figure 5. Stereographic representations of the join set and
slope in one of the positions. Figure 4. Stereographic representation of joins and join sets
in the quarry. Copyright © 2013 SciRes. 5.3. Stability Analysis of Failure Mechanisms R.M.R = 61
Parameter
Index
Value
Resistance (Mpa)
>250 (15)
100 - 250 (12)
50 - 100 (7)
X
25 - 50 (4)
<25 (2)
7
RQD
90 - 100 (20) X
75 - 90 (17)
50 - 75 (13)
25 - 50 (8)
<25 (3)
20
spacing
>2 (20)
0.6 - 2 (15)
X
0.2 - 06 (10)
0.06 - 2.0 (8)
<0.06 (6)
15
Persistence
<1 m (6)
1- 3 m (4)
3 - 10 m (2)
X
10 - 20 m (1)
>20 m (0)
2
width
closed (6)
<0.1 mm(5)
0.1 - 1 mm(4)
1 - 5 mm (1)
X
>5 mm (0)
1
surface
very rough (6)
rough (5)
slightly rough (3)
X
smooth (1)
very smooth (0)
3
fill
clean (6)
hard < 5 mm (4)
hard < 5 mm (2)
soft < 5 mm (1)
soft > 5 mm (0) X
0
weathering
healthy (6)
slightly weathered (5)
moderately weathered (3)
X very weathered (2) decomposed (0)
3
water
dry (15)
humid (10)
X
wet (7)
dripping (4)
flowing (0)
10
Table 3. Results of stability analysis of the different found in each position of the quarry. Table 3. Results of stability analysis of the different found in each position of the quarry. Table 3. Results of stability analysis of the different found in each position of the quarry. Saturation en eau
0%
50%
100%
Position
Failure
FS
PF
FS
PF
FS
PF
wedge 1 - 3
108
0.00%
106
3.20%
0
34.70%
1
wedge 2 - 5
20.6
0.00%
19.84
4.60%
0
41.60%
wedge 4 - 5
16.4
0.00%
16.36
0.00 %
0.86
55%
2
planar 5
1.9
0.00%
1.21
39.70%
0
100%
wedge 1 - 3
7
0.00%
6.5
0.90%
0
11.50%
3
wedge 2 - 5
0
0.00%
17
1.30%
14
20.15%
wedge 1 - 3
12
0.00%
10
4.40%
0
41.60%
wedge 1 - 4
5.9
0.00%
5.7
0.00%
3.3
5.00%
wedge 2 - 5
15
0.00%
14.7
0.00%
13
2.00%
4
wedge 2 - 3
62
2.00%
0
86.00%
0
100%
wedge 1 - 3
102
0.00%
101
1.10%
12.6
15%
5
wedge 2 - 5
46.7
0.00%
44.5
5.70%
0
50%
5.4. Copyright © 2013 SciRes. 5.3. Stability Analysis of Failure Mechanisms Rockfall Simulation and Determination of
the Risk Area
Virtual simulation of rockfall phenomena was done on
13 profiles across the quarry’s face, using Rocfall (Roc-
Sciences, 2004) [4], allows the estimation of the possible
trajectories of a rockfall and calculate the horizontal dis-
tributions of its end points (Figure 8). These results enabled the determination of the distance
from the quarries wall where most rockfalls could occur. Reporting these distances on the quarry’s plan allows to
draw a high risk area and determinate a safe distance
from the wall of the quarry (Figure 9). 5.3. Stability Analysis of Failure Mechanisms The stability analysis was done using specialized pro-
grams like Swedge (4.0) [4] (Figures 6 and 7) for wedge
analysis and Roc-Plane [4] (2.0) for planar failures, these
two programs allow to estimate the probability of failure
(PF) for each case, taking into consideration the different
variables like orientation of the joints, orientation and
height of the slope, the rock’s specific weight, cohesion,
friction angle, water pressure in the joints and seismic
coefficient. Table 3 shows the results of these analyses with the
values of probability of failure associated to each case
with degrees of water fill 0%, 50% and 100%. The results show that in the actual orientation of the
quarry’s face, many wedges exist along its wall, and
while they are totally stable in dry conditions, they are at
risk of falling when the joints get filled with water. Figure 4. Stereographic representation of joins and join sets
in the quarry. IJG H. DELLERO, Y. EL KHARIM
1231
Table 1. Summary table characterizing the join sets found. Join set
J1
J2
J3
J4
J5
Dip direction (˚)
0
25
339
287
189
Dip (˚)
27
74
71
77
69
Persistence along Direction (m)
3_10
3_10; <1
1_3
3_10
3_10
Persistence along dip (m)
3_10
1_3; <1
1_3
1_3
3_10
spacing (m)
0.6 - 2
0.6 - 2
0.6 - 2
0.6 - 2
0.6 - 2
JRC
8
11
11
9
11
JCS (Mpa)
77
89
87
83
95
Width (cm)
1
3
1
0
3
Fill
Dry clay
Wet soil
Wet soil
-
Varies
Weathering
Light weathering
Moderate weathering
Light weathering
Light weathering
Moderate weathering
Effect of water
Dry
Humid
Humid
Dry
Dry
Table 2. Rock mass rating (RMR) index of Bienawsky 1989. 5.3. Stability Analysis of Failure Mechanisms R.M.R = 61
Parameter
Index
Value
Resistance (Mpa)
>250 (15)
100 - 250 (12)
50 - 100 (7)
X
25 - 50 (4)
<25 (2)
7
RQD
90 - 100 (20) X
75 - 90 (17)
50 - 75 (13)
25 - 50 (8)
<25 (3)
20
spacing
>2 (20)
0.6 - 2 (15)
X
0.2 - 06 (10)
0.06 - 2.0 (8)
<0.06 (6)
15
Persistence
<1 m (6)
1- 3 m (4)
3 - 10 m (2)
X
10 - 20 m (1)
>20 m (0)
2
width
closed (6)
<0.1 mm(5)
0.1 - 1 mm(4)
1 - 5 mm (1)
X
>5 mm (0)
1
surface
very rough (6)
rough (5)
slightly rough (3)
X
smooth (1)
very smooth (0)
3
fill
clean (6)
hard < 5 mm (4)
hard < 5 mm (2)
soft < 5 mm (1)
soft > 5 mm (0) X
0
weathering
healthy (6)
slightly weathered (5)
moderately weathered (3)
X very weathered (2) decomposed (0)
3
water
dry (15)
humid (10)
X
wet (7)
dripping (4)
flowing (0)
10
Table 3. Results of stability analysis of the different found in each position of the quarry. Saturation en eau
0%
50%
100%
Position
Failure
FS
PF
FS
PF
FS
PF
wedge 1 - 3
108
0.00%
106
3.20%
0
34.70%
1
wedge 2 - 5
20.6
0.00%
19.84
4.60%
0
41.60%
wedge 4 - 5
16.4
0.00%
16.36
0.00 %
0.86
55%
2
planar 5
1.9
0.00%
1.21
39.70%
0
100%
wedge 1 - 3
7
0.00%
6.5
0.90%
0
11.50%
3
wedge 2 - 5
0
0.00%
17
1.30%
14
20.15%
wedge 1 - 3
12
0.00%
10
4.40%
0
41.60%
wedge 1 - 4
5.9
0.00%
5.7
0.00%
3.3
5.00%
wedge 2 - 5
15
0.00%
14.7
0.00%
13
2.00%
4
wedge 2 - 3
62
2.00%
0
86.00%
0
100%
wedge 1 - 3
102
0.00%
101
1.10%
12.6
15%
5
wedge 2 - 5
46.7
0.00%
44.5
5.70%
0
50%
5.4. Rockfall Simulation and Determination of
the Risk Area
Virtual simulation of rockfall phenomena was done on
13 profiles across the quarry’s face, using Rocfall (Roc-
Sciences, 2004) [4], allows the estimation of the possible
trajectories of a rockfall and calculate the horizontal dis-
tributions of its end points (Figure 8). These results enabled the determination of the distance
from the quarries wall where most rockfalls could occur. 5.3. Stability Analysis of Failure Mechanisms Reporting these distances on the quarry’s plan allows to
draw a high risk area and determinate a safe distance
from the wall of the quarry (Figure 9). H. DELLERO, Y. EL KHARIM 1231 Table 1. Summary table characterizing the join sets found. Join set
J1
J2
J3
J4
J5
Dip direction (˚)
0
25
339
287
189
Dip (˚)
27
74
71
77
69
Persistence along Direction (m)
3_10
3_10; <1
1_3
3_10
3_10
Persistence along dip (m)
3_10
1_3; <1
1_3
1_3
3_10
spacing (m)
0.6 - 2
0.6 - 2
0.6 - 2
0.6 - 2
0.6 - 2
JRC
8
11
11
9
11
JCS (Mpa)
77
89
87
83
95
Width (cm)
1
3
1
0
3
Fill
Dry clay
Wet soil
Wet soil
-
Varies
Weathering
Light weathering
Moderate weathering
Light weathering
Light weathering
Moderate weathering
Effect of water
Dry
Humid
Humid
Dry
Dry
Table 2. Rock mass rating (RMR) index of Bienawsky 1989. R.M.R = 61
Parameter
Index
Value
Resistance (Mpa)
>250 (15)
100 - 250 (12)
50 - 100 (7)
X
25 - 50 (4)
<25 (2)
7
RQD
90 - 100 (20) X
75 - 90 (17)
50 - 75 (13)
25 - 50 (8)
<25 (3)
20
spacing
>2 (20)
0.6 - 2 (15)
X
0.2 - 06 (10)
0.06 - 2.0 (8)
<0.06 (6)
15
Persistence
<1 m (6)
1- 3 m (4)
3 - 10 m (2)
X
10 - 20 m (1)
>20 m (0)
2
width
closed (6)
<0.1 mm(5)
0.1 - 1 mm(4)
1 - 5 mm (1)
X
>5 mm (0)
1
surface
very rough (6)
rough (5)
slightly rough (3)
X
smooth (1)
very smooth (0)
3
fill
clean (6)
hard < 5 mm (4)
hard < 5 mm (2)
soft < 5 mm (1)
soft > 5 mm (0) X
0
weathering
healthy (6)
slightly weathered (5)
moderately weathered (3)
X very weathered (2) decomposed (0)
3
water
dry (15)
humid (10)
X
wet (7)
dripping (4)
flowing (0)
10
Table 3. Results of stability analysis of the different found in each position of the quarry. Table 1. Summary table characterizing the join sets found. Table 2. Rock mass rating (RMR) index of Bienawsky 1989. Table 2. Rock mass rating (RMR) index of Bienawsky 1989. http://dx.doi.org/10.1007/BF01261801 [7]
E. Hoek and J. Bray, “Rock Slope Engineering,” IMM,
London, 1974. [8]
J. A. Hudson and J. P. Harrison, “Engineering Rock Me-
chanics: An Introduction to the Principles,” Pergamon
Press, London, 1997, pp. 307-325. 5.4. Rockfall Simulation and Determination of
the Risk Area 5.4. Rockfall Simulation and Determination of
the Risk Area tributions of its end points (Figure 8). tributions of its end points (Figure 8). These results enabled the determination of the distance
from the quarries wall where most rockfalls could occur. These results enabled the determination of the distance
from the quarries wall where most rockfalls could occur. Reporting these distances on the quarry’s plan allows to
draw a high risk area and determinate a safe distance
from the wall of the quarry (Figure 9). Virtual simulation of rockfall phenomena was done on
13 profiles across the quarry’s face, using Rocfall (Roc-
Sciences, 2004) [4], allows the estimation of the possible
trajectories of a rockfall and calculate the horizontal dis- Copyright © 2013 SciRes. IJG H. DELLERO, Y. EL KHARIM 1232 Figure 9. Rockfall risk areas in the quarry. Figure 6. Virtual modeling of a wedge type failure in the
quarry done by swedge. Figure 6. Virtual modeling of a wedge type failure in the
quarry done by swedge. Figure 7. Stereographic representation of the same failure. Figure 8. Rock simulation and its vertical destribution in
one of the profiles done by Rocfall (4.0). Figure 6. Virtual modeling of a wedge type failure in the
quarry done by swedge. Figure 6. Virtual modeling of a wedge type failure in the
quarry done by swedge. Figure 7. Stereographic representation of the same failure. Figure 9. Rockfall risk areas in the quarry. Figure 9. Rockfall risk areas in the quarry. Figure 9. Rockfall risk areas in the quarry. Figure 9. Rockfall risk areas in the quarry. have left the Carian quarry at a genuine risk of rockfall
that can cause serious physical and/or propriety damages,
to the population of the area, passengers, and the houses
on top and cars at the ground. On the other hand these
instabilities cause a noticeable recede of face of the
quarry in some of its areas, which puts the houses on top
at a great risk. As these risks get even higher during the
raining season, it is advisable to keep a safe distance as
mentioned and get into rehabilitating the area as soon as
possible. REFERENCES Figure 7. Stereographic representation of the same failure. Figure 7. Stereographic representation of the same failure. [1]
M. Durand-Delga, “La Courbure de Gibraltar, Extrémité
Occidentale des Chaînes Alpines, unit l’Europe et l’Afri-
que,” Eclogae Geologicae Helvetiae, Vol. 65, No. 2, 1972,
pp. 267-278. Figure 8. Rock simulation and its vertical destribution in
one of the profiles done by Rocfall (4.0). Figure 8. Rock simulation and its vertical destribution in
one of the profiles done by Rocfall (4.0). [2]
Oliver Ph., “Evolution de la Limite Entre Zones Internes
et Zones Externes dans l’Arc de Gibraltar (Maroc-Es-
pagne),” Thèse Docteur es Science, Toulouse, 1984, p. 229. [3]
E. T. Brown, “Rock Characterization, Testing and Moni-
toring,” Pergamon Press, Oxford, 1981, p. 211. [4]
RocScience: “User’s Guide of Code ROCFALL, RO-
CLAB, SWEDGE, ROCPLANE & DIPS,” RocScience
Inc., Toronto, 2004. Figure 8. Rock simulation and its vertical destribution in
one of the profiles done by Rocfall (4.0). [5]
Z. T. Bieniawski, “Engineering Rock Mass Classifica-
tions—A Complete Manual for Engineers and Geologists
in Mining, Civil and Petroleum Engineering,” Wiley,
New York, 1989. The results of rockfall simulations show that in most
location within the quarry 80% - 99% of the rocks fall
only few meters away from its wall, which is due to
those levels of overburden that work as a catch bench,
still there is a risk that rocks can reach distances as far as
40 m from the wall in some places with a bounce level
that can reach over 1 m in height. [6]
N. Barton and V. Choubey, “The Shear Strength of Rock
Joints in Theory and Practice,” Rock Mechanics, Vol. 10,
No. 1-2, 1977, pp. 1-54. http://dx.doi.org/10.1007/BF01261801 6. Conclusion Years of poor practices of blasting and poor management IJG Copyright © 2013 SciRes.
|
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3D Interfacial and Spatiotemporal Regulation of Human Neuroepithelial Organoids
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10.1002/advs.202201106 Document status and date:
Published: 05/08/2022 Download date: 24. Oct. 2024 3D Interfacial and Spatiotemporal Regulation of Human
Neuroepithelial Organoids Chunling Tang, Xinhui Wang, Mirko D’Urso, Cas van der Putten,
and Nicholas A. Kurniawan* generation of different neuronal progenitor
cells and the development of the central ner-
vous system. Three-dimensional (3D) neu-
ral epithelial (NE) organoids derived from
pluripotent stem cells or embryonic stem
cells are ideal model system to study the
emergence of multicellular tissue complex-
ity, as they have been shown to recapitu-
late several key features of in vivo develop-
ment during neurulation stage.[4–6] In fact,
NT-like patterning with roof plate and floor
plate along the DV axis can be reassembled
in NE organoids. NE organoids are com-
monly generated through self-aggregation
of stem cells to form organized 3D archi-
tecture and functions in reconstituted extra-
cellular matrices.[4–7] While the role of in-
ductive and morphogenetic factors as well
as the biochemical signaling networks that
underlie the cell differentiation in this pro-
cess have been extensively studied,[6,7] how
the self-organization and cellular patterning
in NE organoids take place in the presence
of extracellular interfacial cues is still largely
unknown. This has hampered the ratio-
nal design of next-generation NE organoid
models with more complex cellular organi-
zations that can better mimic the complete
NT developmental process. Neuroepithelial (NE) organoids with dorsal–ventral patterning provide a
useful three-dimensional (3D) in vitro model to interrogate neural tube
formation during early development of the central nervous system. Understanding the fundamental processes behind the cellular
self-organization in NE organoids holds the key to the engineering of
organoids with higher, more in vivo-like complexity. However, little is known
about the cellular regulation driving the NE development, especially in the
presence of interfacial cues from the microenvironment. Here a simple 3D
culture system that allows generation and manipulation of NE organoids from
human-induced pluripotent stem cells (hiPSCs), displaying developmental
phases of hiPSC differentiation and self-aggregation, first into NE cysts with
lumen structure and then toward NE organoids with floor-plate patterning, is
established. Longitudinal inhibition reveals distinct and dynamic roles of
actomyosin contractility and yes-associated protein (YAP) signaling in
governing these phases. By growing NE organoids on culture chips
containing anisotropic surfaces or confining microniches, it is further
demonstrated that interfacial cues can sensitively exert dimension-dependent
influence on luminal cyst and organoid morphology, successful floor-plate
patterning, as well as cytoskeletal regulation and YAP activity. This study
therefore sheds new light on how organoid and tissue architecture can be
steered through intracellular and extracellular means. RESEARCH ARTICLE www.tue.nl/taverne Take down policy
If you believe that this document breaches copyright please contact us at:
openaccess@tue.nl
providing details and we will investigate your claim. Download date: 24. Oct. 2024 © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (1 of 16) 1. Introduction p
p
Exciting works in the last few years have started to explore
bioengineering approaches to present specific physical extracel-
lular cues for guiding organoid self-organization and cell-fate
determination.[7–10] In particular, substrate geometries were used
as artificial physical boundaries during cellular and tissue orga-
nization, enabling shape-guided organoid morphogenesis.[8–10]
For instance, 3D intestinal organoids grown in tubular mi-
croniches organize into crypt-like structures, following the
predefined shape.[8,10] Previous studies of NE organoids using
a poly(ethylene glycol) hydrogel-supported 3D culture system
reported that both the formation and the DV patterning of NE
organoids could be modulated by extracellular matrix rigidity.[6,7]
These studies not only point to the potential of using extra-
cellular cues to modulate the formation of NE organoids, but
also suggest the involvement of cell’s intracellular organization
and sensing of the physical environment. Indeed, significant
actin cytoskeletal rearrangement that stabilized into a central
actin ring has been detected during NE cyst formation.[6] Actin
and myosin assembled into contractile actomyosin bundle or
stress fibers in nonmuscle cells, whose dynamics are generally Cellular patterning of the neural tube (NT) along the dorsal–
ventral (DV) axis is a critical yet complex step during the neurula-
tion stage of early embryogenesis.[1–3] This patterning leads to the C. Tang, X. Wang, M. D’Urso, C. van der Putten, N. A. Kurniawan
Department of Biomedical Engineering
Eindhoven University of Technology
PO Box 513, Eindhoven 5600 MB, The Netherlands
E-mail: n.a.kurniawan@tue.nl
C. Tang, X. Wang, M. D’Urso, C. van der Putten, N. A. Kurniawan
Institute for Complex Molecular Systems
PO Box 513, Eindhoven 5600 MB, The Netherlands C. Tang, X. Wang, M. D’Urso, C. van der Putten, N. A. Kurniawan
Department of Biomedical Engineering
Eindhoven University of Technology
PO Box 513, Eindhoven 5600 MB, The Netherlands
E-mail: n.a.kurniawan@tue.nl
C. Tang, X. Wang, M. D’Urso, C. van der Putten, N. A. Kurniawan
Institute for Complex Molecular Systems
PO Box 513, Eindhoven 5600 MB, The Netherlands The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/advs.202201106 © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH. 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. www.advancedscience.com www.advancedscience.com luminal NE cysts and DV patterning of the NE organoids. More-
over, the culture configuration enables both microscopic moni-
toring and longitudinal manipulation of the organoid formation
process through cellular and extracellular cues. known to be important for a variety of cell behaviors, includ-
ing migration, aggregation, and construction of multicellular
structures.[11–13] Moreover, cytoskeletal organization and the
Hippo signaling pathway, which is important in determining
cell fate, are highly mechanosensitive and dependent on extra-
cellular cues.[14–17] Both cytoskeletal arrangement and activity of
yes-associated protein (YAP) respond to extracellular geometrical
cues, including surface topographies and confinement.[14–17]
In the context of NT development and NE organoid formation,
the roles of these intracellular regulation and extracellular cues
remain poorly understood. The out-of-plane morphogenesis in our approach is facili-
tated by the presence of Geltrex in the medium. We therefore
asked whether varying Geltrex concentration in the neural in-
duction medium can affect the self-organization of NE cysts and
organoids. This is particularly interesting in light of a recent
study that showed that higher arginine–glycine–aspartate (RGD)
ligand density in hiPSC culture results in a higher rate of lumen
formation.[18] We found that, under different Geltrex concentra-
tions in the medium (2%, 4%, and 8% v/v), single hiPSCs were
able to self-assemble and differentiate into NE cysts (Figure 1C)
and DV patterned NE organoids (Figure 1E; Figure S1A,B, Sup-
porting Information). These 3D multicellular structures kept
growing and increased in size throughout the complete culture
process from day 0 to day 18, with comparable developmental
progression (Figure S1C, Supporting Information). Interestingly,
increasing Geltrex concentration led to a non-monotonic effect in
cellular self-organization. At the initial phase of NE cyst forma-
tion, raising Geltrex concentration from 2% to 4% nearly doubled
the number of formed 3D NE cysts, without affecting cyst or lu-
men size (Figure 1D). However, a further increase to 8% Geltrex
concentration did not translate to a further increase in the rate
of NE cyst formation (Figure 1D). These effects carried forward
to the later DV patterning stage. Remarkably, 4% and 8% Geltrex
both decreased the NE organoid size (Figure 1F). Moreover, 8%
Geltrex resulted in significantly larger lumen-to-organoid size ra-
tio and lower patterning success of the NE organoids (Figure 1F). 2.1. Generation of NE Organoids from Human iPSC in a
Manipulatable Culture Environment To enable investigation into the relative roles of intracellular and
extracellular cues in NE organoids, an important first step is to
develop a 3D organoid culture system that allows simple, one-
pot manipulations of these cues. Previous studies have shown
successful production of DV patterned NE organoids in a 3D cul-
ture system by embedding pluripotent stem cells in hydrogels
or growing them on hydrogel beds.[4] Inspired by this approach,
we seeded single hiPSCs at 8000–10 000 cells cm−2 on 1% (v/v)
Geltrex-coated glass surface and further added 2% (v/v) Geltrex
to the neural induction medium to provide a biomimetic envi-
ronment for the 3D development of NE organoids (Figure 1A). Under this condition, single hiPSCs successfully self-aggregated
into 3D cystic structures with smooth outer surface and contain-
ing central lumen with apical–basal polarity on day 6, as evi-
denced by the apical enrichment of N-cadherin (Figure 1B). Con-
currently, the hiPSCs differentiated into neural epithelial cells,
indicated by the expression of early neuroectodermal marker
PAX6 (Figure 1B). After addition of AtRA and Shh from day 4
to day 9, FOXA2+ ventral NE cells started to be present in the NE
cysts on day 10 and continued to accumulate in the floor-plate re-
gion. At the same time, PAX3+ dorsal NE cells became detectable
in the opposite roof-plate region, displaying key architectural fea-
tures of a DV patterned NE organoid with lumen structure on
day 18 (Figure 1B). These data show that our simple culture sys-
tem can be used for producing NE organoids from single hiPSCs,
which undergo distinct developmental phases of the formation of DOI: 10.1002/advs.202201106 © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (1 of 16) Adv. Sci. 2022, 9, 2201106 www.advancedscience.com Altogether, our data indicate for the first time that the formation
and morphological features of NE organoids can be manipulated
through modulation of the 3D culture condition, by changing the
concentration of soluble Geltrex in the medium. In this study, we present a simple yet manipulatable 3D cul-
ture system to produce cystic NE organoids. With this approach,
human-induced pluripotent stem cells (hiPSCs) undergo self-
aggregation and differentiation, lumen formation, and polariza-
tion to form NE cyst, which were then patterned with roof plate
and floor plate along the DV axis to form NE organoids. We fur-
ther show that the organization of the actin cytoskeleton and the
activation of YAP are required and play differential roles in mod-
ulating different phases of NE organoid self-organization. More-
over, extracellular interfacial cues in the form of soluble ligands,
anisotropic topographies, and geometrical confinement in micro-
fabricated substrates and 3D microniches were found to sensi-
tively control the morphogenetic process and cellular patterning
in the NE organoids. Together, our study reveals how interfacial
cues spatiotemporally regulate and can be used to modulate 3D
cellular self-organization in human NT development. 2. Results 2.1. Generation of NE Organoids from Human iPSC in a
Manipulatable Culture Environment Adv. Sci. 2022, 9, 2201106 © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (2 of 16) www.advancedsciencenews.com 2.2. Actomyosin Contractility Regulates NE Cyst Formation and
Floor-Plate Patterning It is generally accepted that the actin cytoskeleton is crucial for
morphogenetic processes,[11,13,19] and actin filaments have been
shown to be mechanically responsive to external forces during
the formation of well-organized NE organoid structure.[6,7] How-
ever, the active role of actomyosin contractility in the different
cellular differentiation and self-organization phases during NE
organoid formation is still elusive. To investigate this, we per-
formed a systematic inhibition study, exploiting the possibility of
probing our organoids at different developmental phases. At the
NE cyst formation phase, single hiPSCs gradually differentiated,
self-aggregated, and formed polarized lumen structure after day
3, accompanied by the clear presence of distinct actin stress
fibers throughout the process until day 7 (Figure 2A; Figure
S2, Supporting Information). Inhibiting actin polymerization
by addition of cytochalasin D on day 1 resulted in fragmented
actin structures in the cells, while inhibition of nonmuscle
myosin II by blebbistatin as well as upstream Rho-associated
kinase (ROCK) by Y27632 prevented formation of actin stress
fibers (Figure 2A). In all cases with the inhibitor treatments,
the individual hiPSCs failed to self-aggregate into 3D cysts with © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (2 of 16) www.advancedsciencenews.com
www.advancedscience.com
Figure 1. Formation of 3D NE organoids from human iPSCs. A) Culture system for generating 3D DV patterned NE organoids from single hiPSCs. B) Developmental phases during the formation of NE organoids from single hiPSCs. Phase 1, hiPSCs were digested into single cells and seeded on
Geltrex-coated glass coverslip on day 0. Phase 2, hiPSCs differentiated into NE cells and self-organized into cystic structures with smooth outer surface
and containing central lumen with apical-basal polarity on day 6. Phase 3, development of NE cysts with FOXA2+ ventral NE cells and PAX3+ dorsal
NE cells forming DV patterned NE organoids Top panels show bright field images of hiPSCs NE cysts and NE organoids Bottom panels show Fi
1 F
i
f 3D NE
id f
h
iPSC
A) C l
f
i
3D DV
d NE
id f
i
l hiPS Figure 1. Formation of 3D NE organoids from human iPSCs. A) Culture system for generating 3D DV patterned NE organoids from single hiPSCs. B) Developmental phases during the formation of NE organoids from single hiPSCs. Phase 1, hiPSCs were digested into single cells and seeded on
Geltrex-coated glass coverslip on day 0. Adv. Sci. 2022, 9, 2201106 © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (3 of 16) www.advancedscience.com 2–3), but thereafter translocated to the cytoplasm (Figure 3A). Quantification of YAP intensity confirmed a gradual decrease of
the nuclear-to-cytoplasmic YAP ratio from day 2 to day 18, where
the ratio was close to 1 (1.17 ± 0.05) on day 6 (Figure 3B). This
suggests that YAP is involved in the regulation of luminal NE
cysts. To test this, we added specific YAP inhibitor, verteporfin, in
the early phase of NE cyst formation. Adding verteporfin at a low
concentration (1 × 10−6 m) on day 1 (at the stage of single hiPSC
culture) repressed YAP expression in the cells and led to dramatic
consequences, such as disruption of actin structure, failure of the
initiation of lumen formation, and sometimes loss of nuclear in-
tegrity in 3D NE cysts on day 2 and 3 (Figure S5, Supporting In-
formation). To specifically target the lumen polarization phase,
which was consistently observed on day 4, we added verteporfin
in the culture medium from day 2 until day 5. Addition of 1 ×
10−6 m verteporfin suppressed YAP expression without affecting
nuclear integrity nor the overall cystic structure, but abrogated
lumen formation and polarization in the NE cysts (Figure 3C). Increasing verteporfin concentration to 2 × 10−6 m resulted in
more pronounced effects, with punctate actin aggregates and fail-
ure to form 3D cystic structure with smooth surface (Figure 3C). Given this importance of YAP in the initial formation of 3D lumi-
nal NE cysts, we then asked whether YAP also plays a role in the
maintenance of cellular architecture and the subsequent floor-
plate patterning of NE organoids. When verteporfin was intro-
duced from day 14 to day 18, no observable difference was found
between the cellular organization, number density, and success
rate of floor-plate patterning of the NE organoids in the control
and verteporfin-treated samples (Figure 3D,E). This is consistent
with the cytoplasmic localization and therefore lack of transcrip-
tional activity of YAP in late-stage NE organoids. Therefore, our
data show that YAP is critical for lumen formation and polarity in
NE cysts, but is not involved in regulating the patterning process
of NE organoids. lumen structure, despite positive PAX6 expression (Figure 2A;
Figure S3A, Supporting Information). Subsequent washout of
inhibitors on day 3 allowed the cells to reaggregate into 3D cysts
structure with polarized lumen (Figure 2B; Figure S3B, Sup-
porting Information). 2.2. Actomyosin Contractility Regulates NE Cyst Formation and
Floor-Plate Patterning Phase 2, hiPSCs differentiated into NE cells and self-organized into cystic structures with smooth outer surface
and containing central lumen with apical-basal polarity on day 6. Phase 3, development of NE cysts with FOXA2+ ventral NE cells and PAX3+ dorsal
NE cells, forming DV patterned NE organoids. Top panels show bright-field images of hiPSCs, NE cysts, and NE organoids. Bottom panels show
immunofluorescence staining of pluripotency marker SOX2, F-actin (phalloidin), N-cadherin, early neuroectodermal marker PAX6, dorsal marker PAX3,
and ventral marker FOXA2 in corresponding phases during NE organoid formation. Scale bars: 100 μm. C) Representative bright-field (BF) images of NE
cysts on day 7 and the corresponding immunofluorescence staining of PAX6, N-cadherin, and nuclei in NE cysts formed in the presence of 2%, 4%, and
8% Geltrex in the neural induction medium. Scale bars: 100 μm. D) Quantification of the number density, area, and lumen-to-cyst size ratio of NE cysts
on day 7, n ≥5. E) Representative bright-field (BF) images (scale bars: 100 μm) and the corresponding immunofluorescence staining (scale bars: 50 μm) © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (3 of 16) Adv. Sci. 2022, 9, 2201106 www.advancedscience.com Given this importance of YAP in the initial formation of 3D lumi-
nal NE cysts, we then asked whether YAP also plays a role in the
maintenance of cellular architecture and the subsequent floor-
plate patterning of NE organoids. When verteporfin was intro-
duced from day 14 to day 18, no observable difference was found
between the cellular organization, number density, and success
rate of floor-plate patterning of the NE organoids in the control
and verteporfin-treated samples (Figure 3D,E). This is consistent
with the cytoplasmic localization and therefore lack of transcrip-
tional activity of YAP in late-stage NE organoids. Therefore, our
data show that YAP is critical for lumen formation and polarity in
NE cysts, but is not involved in regulating the patterning process
of NE organoids. We next asked whether actomyosin contractility is equally crit-
ical in the later phase of NE organoids formation and floor-plate
patterning. When the inhibitors were added on day 14, bright-
field imaging and immunofluorescence staining showed that NE
organoids in the inhibitor-treated samples maintained an overall
morphology comparable to the control samples on day 18 (Fig-
ure 2D). However, blebbistatin and Y27632 treatments led to nar-
row, elongated lumen (Figure 2D) and significantly reduced the
success rate in floor-plate patterning compared to the control
samples (Figure 2E). Interestingly, cytochalasin D-treated sam-
ples exhibited increased number density of NE organoids com-
pared to control group (Figure 2E), but 100% of the organoids lost
their lumen and the percentage of floor-plate patterned organoids
dropped to half of the percentage in the control samples (Fig-
ure 2D,E). Together, these data demonstrate the importance of
actin structures in both the NE cyst formation phase and the
floor-plate patterning phase of NE organoids. of F-actin (phalloidin), PAX6, FOXA2, and nuclei of NE organoids on day 18 formed in the presence of 2%, 4%, and 8% Geltrex in the neural induction
medium. F) Quantification of the number density, area, lumen-to-organoid size ratio, and the percentage of floor-plate patterned NE organoids on day
18, n ≥3. Error bars in (D) and (F) represent S.E.M. P-values of statistical significance were represented as: * P < 0.05, ** P < 0.01, *** P < 0.001, ****
P < 0.0001, ns (not significant) P > 0.05. Using one-way analysis of variance (ANOVA) followed by a Tukey’s multiple comparisons test. www.advancedscience.com Within 3 days after inhibitor washout, no
statistically significant difference in the area and number density
of 3D NE cysts was found between control, blebbistatin-treated,
and Y27632-treated samples. By contrast, the cytochalasin D-
treated samples presented higher number density but smaller
NE cysts (Figure 2C), and with less well-defined luminal surface
(Figure S3B, Supporting Information). These results indicate
that active actin dynamics are required for the morphogenetic
processes behind the 3D formation of luminal NE cysts. To test
whether actin dynamics are also required for the maintenance of
the 3D cellular architecture, we applied the inhibitors on day 5,
after the successful initial formation of the 3D luminal cysts. In-
deed, all inhibitors caused collapse of the polarized lumen in 3D
NE cysts (Figure S4A, Supporting Information) and disassembly
of the already formed cysts, resulting in significantly sparser NE
cysts (Figure S4B, Supporting Information). 2–3), but thereafter translocated to the cytoplasm (Figure 3A). Quantification of YAP intensity confirmed a gradual decrease of
the nuclear-to-cytoplasmic YAP ratio from day 2 to day 18, where
the ratio was close to 1 (1.17 ± 0.05) on day 6 (Figure 3B). This
suggests that YAP is involved in the regulation of luminal NE
cysts. To test this, we added specific YAP inhibitor, verteporfin, in
the early phase of NE cyst formation. Adding verteporfin at a low
concentration (1 × 10−6 m) on day 1 (at the stage of single hiPSC
culture) repressed YAP expression in the cells and led to dramatic
consequences, such as disruption of actin structure, failure of the
initiation of lumen formation, and sometimes loss of nuclear in-
tegrity in 3D NE cysts on day 2 and 3 (Figure S5, Supporting In-
formation). To specifically target the lumen polarization phase,
which was consistently observed on day 4, we added verteporfin
in the culture medium from day 2 until day 5. Addition of 1 ×
10−6 m verteporfin suppressed YAP expression without affecting
nuclear integrity nor the overall cystic structure, but abrogated
lumen formation and polarization in the NE cysts (Figure 3C). Increasing verteporfin concentration to 2 × 10−6 m resulted in
more pronounced effects, with punctate actin aggregates and fail-
ure to form 3D cystic structure with smooth surface (Figure 3C). www.advancedsciencenews.com 2.3. YAP Is Involved in NE Cyst Formation but not Floor-Plate
Patterning Cytoskeletal organization and YAP activity, which we have shown
to be important for organoid formation and patterning, are
known to be mechanosensitive and highly dependent on physical
cues in the extracellular environment.[11,14–17] Recent studies have The transcriptional regulator YAP is known to mediate the abil-
ity of cells to sense and respond to physical cues from the envi-
ronment, by shuttling between the nucleus (therefore function-
ally active) and the cytoplasm (inactive state).[20–23] Cellular pro-
cesses involved in NE organoid formation, such as cell contractil-
ity, substrate adhesion, and differentiation, have been found to be
associated with YAP transcriptional activity.[21,22] Moreover, YAP
has also recently been reported to regulate lumen formation in
hiPSC-derived cysts.[18] Thus, we next assessed the role of YAP in
the formation of luminal NE cysts and organoids. Temporal pro-
filing of the YAP expression indicated that YAP localized mostly
in the nuclei, where it is primed to be active, at the early stage (day also started to explore the exciting possibility of using external
geometrical effects to influence the self-organization process of
3D organoids.[8,9] Thus, we first sought to understand the impact
of substrate topography in modulating NE cyst formation. poly-
dimethylsiloxane (PDMS) chips were microfabricated with lin-
ear grooves of different dimensions using photolithography (see
the Experimental Section). Specifically, the PDMS chips contain
grooves and ridges with different lateral dimensions and spac-
ings of 5 μm (Chip 5 μm), 10 μm (Chip 10 μm), and 20 μm (Chip
20 μm), respectively, and a height of 6 μm (Figure S6, Support-
ing Information). After being coated with a thin layer of 1% (v/v) © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (4 of 16) Adv. Sci. 2022, 9, 2201106 dvancedsciencenews.com
www.advancedscience.com 2.5. 3D Microniches Shape NE Cyst Formation and Affect
Floor-Plate Patterning of NE Organoids p g
p y
g
y
As topography sensing and the subsequent cellular con-
tact guidance have been shown to be mediated by dynamic
actomyosin,[24–26] we hypothesized that the effects of the
anisotropic substrate on NE cyst formation can be targeted by pre-
treating hiPSCs with ML7, a myosin light chain kinase inhibitor. Addition of ML7 (10 × 10−6 m) for the first 48 h indeed suppressed
the formation of multicellular structures with anisotropic orien-
tation and the formation of NE cysts were increased dramatically,
interestingly for both anisotropic and flat (control) PDMS sur-
faces (Figure S8, Supporting Information). Consistent with this,
the reduced NE cyst size and number density that were observed
on anisotropic PDMS surfaces compared to on flat PDMS were
largely erased in ML7 pretreated samples (Figure S8A,B, Sup-
porting Information). Given the impact of anisotropic surface on
actomyosin contractility and NE cysts formation, we also exam-
ined the actin organization and YAP expression in the NE cysts
generated on different PDMS topographies. Total mean fluores-
cence intensity (tMFI) of both F-actin (phalloidin) and YAP was
significantly lower in NE cysts growing on all anisotropic PDMS
surfaces on day 5, compared to on flat PDMS (Figure 4F). This
decrease was still observed for YAP on day 7, but, interestingly,
it diminished with time and significant difference in actin MFI
was only detected between NE cysts on Chip 5 μm and on flat
PDMS on day 7 (Figure 4E,G), suggesting that the 3D, out-of-
plane growth of the NE cysts over time can attenuate the initial
strong effect of substrate topography. Taken together, these re-
sults showed the capability of topographical features of the sub-
strate in controlling NE cyst formation through modulation of
cytoskeletal organization and YAP expression. Finally, we hypothesized that geometric confinement in the form
of 3D anisotropic microniches could have an impact on shaping
the self-assembly of NE cysts and organoids from single hiPSCs. To test this hypothesis, we seeded single hiPSCs on PDMS chips
with linear grooves of either 50 μm (Chip 50 μm) and 100 μm
(Chip 100 μm) in width and 90 μm in height (Figure S6, Sup-
porting Information; Figure 6A), which were chosen to be suffi-
ciently large to geometrically confine the growth of the organoids. As expected, the morphology of the multicellular structures self-
aggregated from hiPSCs followed the shape of these microniches
(Figure 6B). www.advancedscience.com Notably, compared to NE organoids grown on flat PDMS
surface, higher number density of NE organoids was obtained on
all anisotropic PDMS surfaces on day 14, but these organoids ex-
hibited significantly lower lumen-to-organoid size ratio, smaller
size, and decreased percentage of patterned organoids (Fig-
ure 5C). Strikingly, these topography dependences became pro-
gressively weaker with longer culture times (Figure 5D,E). Simi-
larly, F-actin (phalloidin) fluorescence intensity was significantly
lower for NE organoids generated on the anisotropic topogra-
phies compared to on flat PDMS surface on day 14 (Figure 5F),
but these differences were no longer detectable on day 16 and 18
(Figure 5G,H). These results indicate that substrate topograph-
ical cues can affect the early formation and patterning of NE
organoids, but their relevance become increasingly diminished
as the organoids mature. www.advancedscience.com www.advancedsciencenews.com w.advancedsciencenews.com
www.advancedscienc Figure 2. Actomyosin contractility is crucial for 3D NE organoid construction and patterning. A) Immunofluorescence staining of F-actin (phalloidin) in
NE cysts from day 2 to day 7 during NE cyst formation. Y276329 (10 × 10−6 m), (±)-Blebbistatin (10 × 10−6 m), or cytochalasin D (0.2 × 10−6 m) was added
from day 1 to day 7. Scale bars: 10 μm. B) Bright-field images of NE cysts (scale bars: 100 μm) and the corresponding immunofluorescence staining of N-
cadherin and PAX6 in NE cysts (scale bars: 50 μm) on day 7. Inhibitors were added on day 1 and washed out on day 3. C) Quantification of number density © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (5 of 16) © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (5 of 16) Adv. Sci. 2022, 9, 2201106 and area of NE cysts on day 7 after inhibitor washout on day 3, n ≥7. D. Bright-field images of NE organoids and the corresponding immunofluorescence
staining of F-actin (phalloidin) and FOXA2 in NE organoids on day 18, showing representative examples of successful and unsuccessful floor plate
patterning in the organoids. Inhibitors were added from day 14 to day 18 during NE organoid culture. Scale bars: 50 μm. E) Quantification of number
density of NE organoids and the percentage of floor plate patterned NE organoids on day 18 under different treatments, n ≥5. Error bars in (C) and (E)
represent S.E.M. P-values of statistical significance were represented as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns (not significant)
P > 0.05, using one-way analysis of variance (ANOVA) followed by Dunnett multiple comparisons test. www.advancedscience.com www.advancedscience.com unsuccessful floor-plate patterning, were observed on the PDMS
substrates from day 14 (Figure 5A,B; Figure S9, Supporting Infor-
mation). Notably, compared to NE organoids grown on flat PDMS
surface, higher number density of NE organoids was obtained on
all anisotropic PDMS surfaces on day 14, but these organoids ex-
hibited significantly lower lumen-to-organoid size ratio, smaller
size, and decreased percentage of patterned organoids (Fig-
ure 5C). Strikingly, these topography dependences became pro-
gressively weaker with longer culture times (Figure 5D,E). Simi-
larly, F-actin (phalloidin) fluorescence intensity was significantly
lower for NE organoids generated on the anisotropic topogra-
phies compared to on flat PDMS surface on day 14 (Figure 5F),
but these differences were no longer detectable on day 16 and 18
(Figure 5G,H). These results indicate that substrate topograph-
ical cues can affect the early formation and patterning of NE
organoids, but their relevance become increasingly diminished
as the organoids mature. Geltrex, the chips were used for growing hiPSCs (Figure 4A). As shown by immunofluorescence staining, hiPSCs growing on
these PDMS surface were able to self-organize into NE cysts,
which were defined with polarized lumen structure and which
expressed neural epithelial cell marker PAX6 as well as apical N-
cadherin (Figure 4A,B). Compared to the number density of NE
cysts obtained on control substrates (flat PDMS), lower NE cyst
number density was observed on Chip 20 μm on day 5 (Figure 4C)
and on both Chip 20 μm and Chip 10 μm on day 7 (Figure 4D). In
addition, the size of the NE cysts and the lumen-to-cyst size ratio
of the NE cysts derived from all anisotropic PDMS surfaces were
significantly decreased on day 5 and day 7 compared to control
(Figure 4C,D). Notably, we observed that hiPSCs were also able
to self-assemble along the orientation of anisotropic PDMS sur-
face and form multicellular structures that exhibited polarized
lumen structure but with jagged surface (Figure S7, Supporting
Information). This suggests that the cells were able to sense and
respond orientationally to the anisotropic substrate and that this
topography sensing interferes with the NE cyst formation. unsuccessful floor-plate patterning, were observed on the PDMS
substrates from day 14 (Figure 5A,B; Figure S9, Supporting Infor-
mation). Adv. Sci. 2022, 9, 2201106 © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (6 of 16) www.advancedsciencenews.com 2.5. 3D Microniches Shape NE Cyst Formation and Affect
Floor-Plate Patterning of NE Organoids Immunofluorescence staining showed that these
multicellular structures expressed PAX6 and apical N-cadherin
and contained polarized central lumens, confirming a similar
cystic structure as NE cysts growing on flat PDMS (Figure 6B). Despite this, the spatial confinement in the microniches resulted
in significantly lower lumen-to-cyst size ratio on both day 5 and
day 7 compared to those for the NE cysts formed on flat PDMS
(Figure 6C,D). Interestingly, the effect is more pronounced with
the 50 μm microniches than with the 100 μm microniches, high-
lighting the stronger confinement effect in the former. This is
furthermore mirrored by a similar trend in the intracellular reg-
ulation of actin and YAP (Figure 6E–G), where both total MFI of
F-actin (phalloidin) and YAP were found to be significantly lower
in NE cysts from the 50 μm microniches, but not the 100 μm mi-
croniches, compared to on flat PDMS after 7 days of culture (Fig-
ure 6G). To further examine the effect of microniches on floor-
plate patterning of NE organoids, the NE cysts were kept grow-
ing on the PDMS chips. The morphology of the NE organoids
maintained similar shape as 3D microniches and both successful Next, we examined the late-stage NE organoid formation on
the PDMS topographies. NE organoids with smooth surface and
well-defined central lumen structures, with both successful and © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (6 of 16) Adv. Sci. 2022, 9, 2201106 www.advancedsciencenews.com
www.advancedscience.com www.advancedsciencenews.com www.advancedscience.com Figure 3. The dynamic role of YAP in the NE cyst formation and NE organoid patterning. A) Immunofluorescence staining of YAP in NE organoids from
day 2 to day 18 during NE organoid formation. Scale bars: 25 μm. B) Quantification of nuclear-to-cytoplasmic YAP intensity ratio on day 2, day 6, and
2201106 (7 f 16) Figure 3. The dynamic role of YAP in the NE cyst formation and NE organoid patterning. A) Immunofluorescence staining of YAP in NE organoids from
day 2 to day 18 during NE organoid formation. Scale bars: 25 μm. B) Quantification of nuclear-to-cytoplasmic YAP intensity ratio on day 2, day 6, and Figure 3. The dynamic role of YAP in the NE cyst formation and NE organoid patterning. A) Immunofluorescence staining of YAP in NE organoids from
day 2 to day 18 during NE organoid formation. Scale bars: 25 μm. www.advancedscience.com At the same time, these setups present different ligand density,
availability, and distribution to the cells, and a recent study indeed
demonstrated that higher RGD density, but not matrix stiffness,
is associated with lumen formation in hiPSC clusters.[18] Consis-
tent with this, our results indicate that increasing soluble Geltrex
concentration in the medium leads to higher lumen-to-organoid
size ratio. Moreover, we show that higher percentage of soluble
Geltrex promotes the formation of NE cysts and NE organoids on
Geltrex precoated surfaces under neural induction environment,
providing a simple experimental handle to control NE organoid
formation. At the same time, these setups present different ligand density,
availability, and distribution to the cells, and a recent study indeed
demonstrated that higher RGD density, but not matrix stiffness,
is associated with lumen formation in hiPSC clusters.[18] Consis-
tent with this, our results indicate that increasing soluble Geltrex
concentration in the medium leads to higher lumen-to-organoid
size ratio. Moreover, we show that higher percentage of soluble
Geltrex promotes the formation of NE cysts and NE organoids on
Geltrex precoated surfaces under neural induction environment,
providing a simple experimental handle to control NE organoid
formation. and unsuccessful floor-plate patterning were observed in the lu-
minal NE organoids (Figure 7A,B; Figure S10, Supporting Infor-
mation). However, NE organoids growing in the 50 and 100 μm
microniches were generally characterized with lower lumen-to-
organoid size ratio, smaller size, and reduced expression of actin
from day 14 to day 18 (Figure 7C–H). Strikingly, all NE organoids
in the 50 μm microniches failed to achieve floor-plate pattern-
ing on day 14 and the percentage of patterned NE organoids in
these microniches was generally very low compared to in other
conditions (Figure 7C–E). These results suggest that geometrical
confinement can have a strong effect on the late-stage patterning
phase of NE organoids. NE organoid morphogenesis includes apical–basal polarity of
lumen and cell-fate determination of DV patterning. Previous
studies have demonstrated that cytoskeletal organization is
crucial for constructing NE organoids from single hiPSC under
neural induction conditions.[7] However, the role of cytoskeletal
remodeling in the different phases of NE organoids morphogen-
esis was still unclear. Our study reveals that, during the initiation
phase from single hiPSCs, actin polymerization inhibition using
cytochalasin D, nonmuscle myosin II inhibition by blebbistatin,
as well as ROCK inhibition by Y27632 all prevented single hiPSCs
self-aggregate into 3D cystic structures. day 18, n ≥3. C) Immunofluorescence staining of F-actin (phalloidin), YAP, N-cadherin, and PAX6 in NE cysts on day 5. Verteporfin (0, 1, or 2 × 10−6 m)
was added from day 2 to day 5 during NE cyst culture. Scale bars: 50 μm. D) Immunofluorescence staining of F-actin (phalloidin) and FOXA2 in NE
organoids on day 18. Verteporfin (1 × 10−6 m) was added from day 14 to day 18 during 3D NE organoid culture. Scale bars: 50 μm. E) Quantification of
number density of NE organoids and the percentage of floor plate patterned NE organoids on day 18 under different treatments, n ≥5. Error bars in (B)
and (E) represent S.E.M. P-values of statistical significance were represented as: * P < 0.05, ** P < 0.01, ns (not significant) P > 0.05, using one-way
analysis of variance (ANOVA) followed by Dunnett multiple comparisons test (B) and Student’s t-test (E). www.advancedscience.com This effect is reversible,
as washout of these inhibitions at an early stage enables recon-
struction of well-organized 3D cystic structures from cell clusters. By contrast, during the later stage of NE cysts formation, inhi-
bition of F-actin filament formation and polymerization causes
loss of apical–basal polarity or even the complete lumen struc-
ture. Moreover, during the late-stage floor-plate patterning, these
inhibitions significantly repressed the cellular patterning but led
to distinct effects on the lumen architecture of the NE organoids. ROCK and myosin II inhibition resulted in NE organoids with
elongated lumen, whereas under cytochalasin D treatment the
NE organoids completely lost the lumen structure, suggesting
that NE organoids may have a contractility-independent com-
pensatory mechanism for the maintenance of lumen. These
results illustrate a dynamic function of cytoskeleton organization
in controlling cellular self-aggregation, lumen formation and
polarization, as well as floor-plate patterning during different
stages of NE organoid formation (Figure 8). Recent study with
hiPSC-derived cystic structures also described the involvement of
the transcriptional regulator YAP in lumen formation.[18] It was
shown that, in hiPSC clusters, YAP is initially localized in the nu-
cleus, indicative of YAP being active, and as the lumens form over
time, YAP translocates to the cytoplasm, where it is inactive.[18] In
line with this observation, we found that, under neural induction
conditions, YAP is also localized in the nucleus at the early stage
of NE cyst formation and gradually translocates to the cytoplasm
toward the end of NE organoids formation (Figure 8). Further-
more, our YAP inhibition studies at different developmental
stages reveals that YAP is especially crucial for maintain-
ing apical–basal polarity of lumen during NE cyst formation
stage 2.5. 3D Microniches Shape NE Cyst Formation and Affect
Floor-Plate Patterning of NE Organoids B) Quantification of nuclear-to-cytoplasmic YAP intensity ratio on day 2, day 6, and Figure 3. The dynamic role of YAP in the NE cyst formation and NE organoid patterning. A) Immunofluorescence staining of YAP in NE organoids from
day 2 to day 18 during NE organoid formation. Scale bars: 25 μm. B) Quantification of nuclear-to-cytoplasmic YAP intensity ratio on day 2, day 6, and 201106
© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (7 of 16) © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (7 of 16) Adv. Sci. 2022, 9, 2201106 © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (8 of 16) Adv. Sci. 2022, 9, 2201106 www.advancedsciencenews.com www.advancedsciencenews.com
www.advancedscience.com
Figure 4. Anisotropic substrate topographies affect NE cyst formation. A) Schematic of growing hiPSC on PDMS chips with linear grooves and control
flat PDMS chip to generate NE cysts. B) NE cysts growing on PDMS chip with linear grooves of different dimensions (Chip 5 μm, Chip 10 μm, Chip 20 μm)
and control PDMS chip with flat surface. Bright-field images and immunofluorescence staining of N-cadherin and PAX6 in NE cysts on day 7. Scale bars:
50 μm. C,D) Quantification of number density, area, and lumen-to-cyst size ratio of NE cysts on (C) day 5 and (D) day 7, n ≥3. E) Immunofluorescence
staining of F-actin (phalloidin) and YAP in NE cysts growing on PDMS chip with linear grooves of different dimensions (Chip 5 μm, Chip 10 μm, Chip
20 μm) and control PDMS chip with flat surface on day 7. Scale bars: 20 μm. F,G) Quantification of F-actin (phalloidin) and YAP intensity in NE cysts on
(F) day 5 and (G) day 7, n ≥3. Total mean fluorescence intensity (tMFI) of F-actin (phalloidin) and YAP were shown as dot plots and the corresponding
MFI of each slide along the Z-stack were indicated as curve graphs. Error bars in (C), (D), (F), and (G) represent S.E.M. P-values of statistical significance
were represented as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, using one-way analysis of variance (ANOVA) followed by Dunnett multiple
comparisons test. Previous studies on 2D substrates by our group and others
has only started to be explored.[8–10] For instance, a recent study www.advancedsciencenews.com www.advancedscience.com Figure 4. Anisotropic substrate topographies affect NE cyst formation. A) Schematic of growing hiPSC on PDMS chips with linear grooves and control
flat PDMS chip to generate NE cysts. B) NE cysts growing on PDMS chip with linear grooves of different dimensions (Chip 5 μm, Chip 10 μm, Chip 20 μm)
and control PDMS chip with flat surface. Bright-field images and immunofluorescence staining of N-cadherin and PAX6 in NE cysts on day 7. Scale bars:
50 μm. C,D) Quantification of number density, area, and lumen-to-cyst size ratio of NE cysts on (C) day 5 and (D) day 7, n ≥3. Adv. Sci. 2022, 9, 2201106 © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (9 of 16) 3. Discussion 3D organoid models have emerged as unique in vitro models for
studying organ development, function, and disease.[27,28] Con-
ventional organoid generation relies on the encapsulation and
growth of organoids in drops of solidified reconstituted extra-
cellular matrices.[29–33] Studying how stem cells sense different
extracellular cues during organoid formation is technically chal-
lenging using this approach, as the organoids are fully embed-
ded in a 3D microenvironment with local differences of both
physical parameters and growth factor accessibility.[31,32] Recent
studies have put forward alternative strategies of providing sol-
uble extracellular matrix or phasing the reconstitution of the ex-
tracellular matrix to support 3D organoid formation, which can
allow one to subject the cells to external physical cues during
organoid formation.[4,34–36] In this work, we established a simple
culture system to generate 3D NE organoids by growing hiPSCs
on Geltrex-precoated surface with soluble Geltrex in the medium,
which furthermore enables easy manipulation of the biochem-
ical and physical culture conditions of the organoids. Consis-
tent with the features of both human and mouse NE organoids
from previous studies,[4,5,25] this system allows hiPSCs to self-
organize and differentiate into 3D spherical NE cysts with cen-
tral lumen structure, which then develop into dorsal–ventral pat-
terned NE organoids (Figure 8). Note that there is an overall vari-
ation of NE organoid size and phenotype heterogeneity within
each tested culture condition, although we did not observe any
size-dependent trend in NE organoid patterning. As we focused
on the floor-plate patterning process, which was detectable af-
ter 18 days of culture in this system, we have not gone for pe-
riods of culture longer than 18 days to further follow the growth
of the NE organoids in size. Our strategy of providing both Gel-
trex substrate coating and soluble Geltrex in the medium to sup-
port 3D growth appears to circumvent a previous observation that
hPSCs growing on Geltrex precoated surface self-assembled into
multicellular structures with jagged outside surface, whereas NE
organoids with smooth surface were obtained only on Geltrex
bed, which was attributed to differences in the matrix rigidity.[4] © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (8 of 16) Adv. Sci. 2022, 9, 2201106 Adv. Sci. 2022, 9, 2201106 www.advancedsciencenews.com
www.advancedscience.com
Figure 5. Anisotropic substrate topographies affect floor-plate patterning of NE organoids. A,B) NE organoids growing on PDMS chip with linear grooves
of different dimensions (Chip 5 μm, Chip 10 μm, Chip 20 μm) and control PDMS chip with flat surface. Bright-field images (Scale bars: 100 μm) and
immunofluorescence staining (Scale bars: 50 μm) of F-actin (phalloidin) and FOXA2 in NE organoids on A) day 14 and B) day 18, showing representative
examples of successful floor-plate patterning in NE organoids. C–E) Quantification of area, lumen-to-organoid size ratio, number density of NE organoids
and percentage of floor-plate patterned NE organoids on (C) day 14, (D) day 16, and (E) day 18, n ≥3. F–H) Quantification of F-actin (phalloidin) intensity
in NE organoids on (F) day 14, (G) day 16, and (H) day 18, n ≥3. Total mean fluorescence intensity (MFI) of F-actin (phalloidin) were shown as dot plots
(left) and the corresponding MFI of each slide along the Z-stack were indicated as curve graphs (right). Error bars in (C–H) represent S.E.M. P-values
of statistical significance were represented as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, using one-way analysis of variance (ANOVA)
followed by Dunnett multiple comparisons test. Adv. Sci. 2022, 9, 2201106
© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (10 of 16) E) Immunofluorescence
staining of F-actin (phalloidin) and YAP in NE cysts growing on PDMS chip with linear grooves of different dimensions (Chip 5 μm, Chip 10 μm, Chip
20 μm) and control PDMS chip with flat surface on day 7. Scale bars: 20 μm. F,G) Quantification of F-actin (phalloidin) and YAP intensity in NE cysts on
(F) day 5 and (G) day 7, n ≥3. Total mean fluorescence intensity (tMFI) of F-actin (phalloidin) and YAP were shown as dot plots and the corresponding
MFI of each slide along the Z-stack were indicated as curve graphs. Error bars in (C), (D), (F), and (G) represent S.E.M. P-values of statistical significance
were represented as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, using one-way analysis of variance (ANOVA) followed by Dunnett multiple
comparisons test. has only started to be explored.[8–10] For instance, a recent study
presents a chip-based culture system to control the patterning
and morphogenesis of intestinal organoids via the physical
properties.[10] In our study, when hiPSCs were seeded on
anisotropic surface, the self-aggregated multicellular morphol-
ogy develops along the orientation of linear grooves, indicating Previous studies on 2D substrates by our group and others
have demonstrated that cytoskeletal organization and YAP
activity can be sensitively tuned by biophysical cues in the
cellular microenvironments, such as topography, geometry, and
confinement.[14,37–40] However, the role of these biophysical cues
on in the context of 3D cellular self-organization and organoids © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (9 of 16) © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (10 of 16) F,G) Quantification of F-actin (phalloidin) and YAP intensity in NE cysts on (F) day
5 and (G) day 7, n ≥4. Total mean fluorescence intensity (MFI) of F-actin (phalloidin) and YAP were shown as dot plots (left) and the corresponding
MFI of each slide along the Z-stack were indicated as curve graphs (right). Error bars in (C), (D), (F), and (G) represent S.E.M. P-values of statistical Figure 6. 3D microniches guide NE cyst formation. A) Schematic of growing hiPSC on PDMS chips with linear grooves microniche and control flat PDMS
chip to generate NE cysts. B) NE cysts growing on PDMS chip with linear grooves microniche of either 50 μm (Chip 50 μm) and 100 μm (Chip 100 μm) in
width or 90 μm in height. Flat PDMS chip was used as control. Bright-field images and immunofluorescence staining of N-cadherin and PAX6 in NE cysts
on day 7. Scale bars: 50 μm. C,D) Quantification lumen-to-cyst size ratio of NE cysts on (C) day 5 and (D) day 7, n ≥15. E) Immunofluorescence staining
of F-actin (phalloidin) and YAP in NE cysts growing on PDMS chip with linear grooves microniche of different dimensions (Chip 50 μm, Chip 100 μm)
and control PDMS chip with flat surface on day 7. Scale bars: 50 μm. F,G) Quantification of F-actin (phalloidin) and YAP intensity in NE cysts on (F) day
5 and (G) day 7, n ≥4. Total mean fluorescence intensity (MFI) of F-actin (phalloidin) and YAP were shown as dot plots (left) and the corresponding
MFI of each slide along the Z-stack were indicated as curve graphs (right). Error bars in (C), (D), (F), and (G) represent S.E.M. P-values of statistical
significance were represented as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, using one-way analysis of variance (ANOVA) followed by
Dunnett multiple comparisons test. by systematically examining the 3D spatial distribution of cell
phenotype in a large number of patterned NE organoids. Tem-
poral quantification of actin and YAP expressions indicate a
repression of actin filaments formation and YAP activity in NE
cysts forming on anisotropic substrates and in 3D microniches. Moreover, decreased actin filaments formation is also detected in
NE organoids induced by geometrical cues. Thus, cytoskeleton
and YAP in NE cysts and organoids maintain mechanosensitive
capability to response to different extracellular interfacial cues. © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (11 of 16) Adv. Sci. 2022, 9, 2201106 Figure 6. 3D microniches guide NE cyst formation. A) Schematic of growing hiPSC on PDMS chips with linear grooves microniche and control flat PDMS
chip to generate NE cysts. B) NE cysts growing on PDMS chip with linear grooves microniche of either 50 μm (Chip 50 μm) and 100 μm (Chip 100 μm) in
width or 90 μm in height. Flat PDMS chip was used as control. Bright-field images and immunofluorescence staining of N-cadherin and PAX6 in NE cysts
on day 7. Scale bars: 50 μm. C,D) Quantification lumen-to-cyst size ratio of NE cysts on (C) day 5 and (D) day 7, n ≥15. E) Immunofluorescence staining
of F-actin (phalloidin) and YAP in NE cysts growing on PDMS chip with linear grooves microniche of different dimensions (Chip 50 μm, Chip 100 μm)
and control PDMS chip with flat surface on day 7. Scale bars: 50 μm. F,G) Quantification of F-actin (phalloidin) and YAP intensity in NE cysts on (F) day
5 and (G) day 7, n ≥4. Total mean fluorescence intensity (MFI) of F-actin (phalloidin) and YAP were shown as dot plots (left) and the corresponding
MFI of each slide along the Z-stack were indicated as curve graphs (right). Error bars in (C), (D), (F), and (G) represent S.E.M. P-values of statistical
significance were represented as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, using one-way analysis of variance (ANOVA) followed by
Dunnett multiple comparisons test. Figure 6. 3D microniches guide NE cyst formation. A) Schematic of growing hiPSC on PDMS chips with linear grooves microniche and control flat PDMS
chip to generate NE cysts. B) NE cysts growing on PDMS chip with linear grooves microniche of either 50 μm (Chip 50 μm) and 100 μm (Chip 100 μm) in
width or 90 μm in height. Flat PDMS chip was used as control. Bright-field images and immunofluorescence staining of N-cadherin and PAX6 in NE cysts
on day 7. Scale bars: 50 μm. C,D) Quantification lumen-to-cyst size ratio of NE cysts on (C) day 5 and (D) day 7, n ≥15. E) Immunofluorescence staining
of F-actin (phalloidin) and YAP in NE cysts growing on PDMS chip with linear grooves microniche of different dimensions (Chip 50 μm, Chip 100 μm)
and control PDMS chip with flat surface on day 7. Scale bars: 50 μm. www.advancedscience.com www.advancedsciencenews.com www.advancedscience.com Figure 5. Anisotropic substrate topographies affect floor-plate patterning of NE organoids. A,B) NE organoids growing on PDMS chip with linear grooves
of different dimensions (Chip 5 μm, Chip 10 μm, Chip 20 μm) and control PDMS chip with flat surface. Bright-field images (Scale bars: 100 μm) and
immunofluorescence staining (Scale bars: 50 μm) of F-actin (phalloidin) and FOXA2 in NE organoids on A) day 14 and B) day 18, showing representative
examples of successful floor-plate patterning in NE organoids. C–E) Quantification of area, lumen-to-organoid size ratio, number density of NE organoids
and percentage of floor-plate patterned NE organoids on (C) day 14, (D) day 16, and (E) day 18, n ≥3. F–H) Quantification of F-actin (phalloidin) intensity
in NE organoids on (F) day 14, (G) day 16, and (H) day 18, n ≥3. Total mean fluorescence intensity (MFI) of F-actin (phalloidin) were shown as dot plots
(left) and the corresponding MFI of each slide along the Z stack were indicated as curve graphs (right) Error bars in (C H) represent S E M P values Figure 5. Anisotropic substrate topographies affect floor-plate patterning of NE organoids. A,B) NE organoids growing on PDMS chip with linear grooves
of different dimensions (Chip 5 μm, Chip 10 μm, Chip 20 μm) and control PDMS chip with flat surface. Bright-field images (Scale bars: 100 μm) and
immunofluorescence staining (Scale bars: 50 μm) of F-actin (phalloidin) and FOXA2 in NE organoids on A) day 14 and B) day 18, showing representative
examples of successful floor-plate patterning in NE organoids. C–E) Quantification of area, lumen-to-organoid size ratio, number density of NE organoids
and percentage of floor-plate patterned NE organoids on (C) day 14, (D) day 16, and (E) day 18, n ≥3. F–H) Quantification of F-actin (phalloidin) intensity
in NE organoids on (F) day 14, (G) day 16, and (H) day 18, n ≥3. Total mean fluorescence intensity (MFI) of F-actin (phalloidin) were shown as dot plots
(left) and the corresponding MFI of each slide along the Z-stack were indicated as curve graphs (right). Error bars in (C–H) represent S.E.M. P-values
of statistical significance were represented as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, using one-way analysis of variance (ANOVA)
followed by Dunnett multiple comparisons test. © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (10 of 16) Adv. Sci. 2022, 9, 2201106 In
other words, our study provides evidence that external interfacial
cues can potentially be exploited to modulate or even steer NE
organoid development through mechanosensitive intracellular
regulation. Consistent with this idea, previous studies of grow-
ing NE organoids in a poly(ethylene glycol) hydrogel-supported
3D culture system have also started to explore how matrix
rigidity can affect NE organoid formation and patterning.[6,7] In
addition, our results also show varying Geltrex concentration in that cells are able to sense and respond orientationally to the
anisotropic substrate. This is followed by decreased formation
density, area, and lumen-to-cyst size ratio of the NE cysts growing
on anisotropic surface. This impact of interfacial anisotropy on
NE cysts further extends to the NE formation, as the lumen-to-
organoid size ratio, organoid area, and percentage of patterned
organoid are significantly reduced (Figure 8). When the surface
geometrical cues are large enough to confine the NE organoids in
microniches, the NE cysts and organoids are micropatterned with
similar morphology as the 3D microniches, exhibiting decreased
lumen-to-cyst size ratio (for NE cysts) and smaller lumen-to-
organoid size ratio (for NE organoids), as well as lower percentage
of patterned organoids (Figure 8). These observations indicate
that both approaches can strongly influence NE cyst and organoid
formation process. Future studies can further explore the ex-
citing possibility to manipulate NE organoid self-organization,
such as controlling the localization of floor-plate formation, © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (11 of 16) www.advancedsciencenews.com
www.advancedscience.com
Figure 7. 3D microniches affect floor-plate patterning of NE organoids. A,B) NE organoids growing on PDMS chip with linear grooves microniche of
different dimensions (Chip 50 μm, Chip 100 μm) and flat PDMS was used as a control. Bright-field images (Scale bars: 100 μm) and immunofluorescence
staining (Scale bars: 50 μm) of F-actin (phalloidin) and FOXA2 in NE organoids on (A) day 14 and (B) day 18, showing representative examples of
successful and unsuccessful floor-plate patterning in NE organoids. C–E) Quantification of area, lumen-to-organoid size ratio of NE organoids and
percentage of floor-plate patterned NE organoids on (C) day 14, (D) day 16, and (E) day 18, n ≥3. F–H) Quantification of F-actin (phalloidin) intensity
in NE organoids on (F) day 14, (G) day 16, and (H) day 18, n ≥3. Adv. Sci. 2022, 9, 2201106 © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (12 of 16) Adv. Sci. 2022, 9, 2201106 www.advancedsciencenews.com Figure 8. Schematic illustration of the interfacial and intracellular regulation of NE organoid formation and development. Single hiPSCs self-organize
into floor-plate patterned NE organoids under neural induction condition during culture of 18 days, during which the role of YAP gradually decreases
(middle). Disruption of the cytoskeletal organization and actomyosin contractility severely impacts different stages in the process, resulting in failure to
form cysts, lumen structures, and floor-plate patterning (top). Furthermore, different interfacial cues presented on the substrate modulate the cellular
self-assembly process, the organoid morphology, and the cell organization within the organoids (bottom). the neural induction medium can affect the self-organization
of NE organoids, indicating the potential effect of extracellular
biochemical signal on shaping NE organoid formation. may be involved through different mechanisms in the sensing of
interfacial cues in NE cysts and NE organoids. Particularly, not
only the dynamic of actin cytoskeleton, but other different mod-
ulators are able to independently regulate YAP activity triggered
by extracellular biophysical cues.[21,22] Coupled with the differen-
tial roles of actin and YAP in regulating NE organoid formation at
different developmental stages, the response of actin and YAP to
different extracellular geometry suggests their dynamic interplay
in the cellular self-organization process of NE organoids occur-
ring on anisotropic substrates and 3D microniche, which should
be further explored. Given the mechanoresponsiveness of both
F-actin and YAP to interfacial geometrical cues, we speculate
that NE organoid formation and patterning can also be modu-
lated by other mechanical factors via these pathways. In line with
this hypothesis for NE organoids, a recent study on intestinal The mechanosensitive regulation in NE organoids conferred
by interfacial cues appears to be both space- and time-dependent. For instance, the dimensions of the anisotropic linear grooves,
the size of the 3D microniches, as well as the growing time-
point of NE organoids can all influence the interfacial geomet-
ric effects. In addition, the downregulation of YAP in NE cysts
on the groove topographies starts later (from day 7) than in the
3D microniches (from day 5). On other hand, the downregula-
tion of actin on the groove topographies starts earlier (from day
5 for NE cysts and from day 14 for NE organoids) than in the 3D
microniches (from day 7 for NE cysts and from day 18 for NE
organoids). These data suggest that actin cytoskeleton and YAP © 2022 The Authors. Total mean fluorescence intensity (MFI) of F-actin (phalloidin) was shown as dot
plots (left) and corresponding MFI of each slide along the Z-stack were indicated as curve graphs (right). Error bars in (C–H) represent S.E.M. P-values
of statistical significance were represented as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, using one-way analysis of variance (ANOVA)
followed by Dunnett multiple comparisons test. Figure 7. 3D microniches affect floor-plate patterning of NE organoids. A,B) NE organoids growing on PDMS chip with linear grooves microniche of
different dimensions (Chip 50 μm, Chip 100 μm) and flat PDMS was used as a control. Bright-field images (Scale bars: 100 μm) and immunofluorescence
staining (Scale bars: 50 μm) of F-actin (phalloidin) and FOXA2 in NE organoids on (A) day 14 and (B) day 18, showing representative examples of
successful and unsuccessful floor-plate patterning in NE organoids. C–E) Quantification of area, lumen-to-organoid size ratio of NE organoids and
percentage of floor-plate patterned NE organoids on (C) day 14, (D) day 16, and (E) day 18, n ≥3. F–H) Quantification of F-actin (phalloidin) intensity
in NE organoids on (F) day 14, (G) day 16, and (H) day 18, n ≥3. Total mean fluorescence intensity (MFI) of F-actin (phalloidin) was shown as dot
plots (left) and corresponding MFI of each slide along the Z-stack were indicated as curve graphs (right). Error bars in (C–H) represent S.E.M. P-values
of statistical significance were represented as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, using one-way analysis of variance (ANOVA)
followed by Dunnett multiple comparisons test. © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (12 of 16) Adv. Sci. 2022, 9, 2201106 www.advancedsciencenews.com
www.advancedscience.com
Figure 8. Schematic illustration of the interfacial and intracellular regulation of NE organoid formation and development. Single hiPSCs self-organize
into floor-plate patterned NE organoids under neural induction condition during culture of 18 days, during which the role of YAP gradually decreases
(middle). Disruption of the cytoskeletal organization and actomyosin contractility severely impacts different stages in the process, resulting in failure to
form cysts, lumen structures, and floor-plate patterning (top). Furthermore, different interfacial cues presented on the substrate modulate the cellular
self-assembly process, the organoid morphology, and the cell organization within the organoids (bottom). © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (13 of 16) 5. Experimental Section Reagents and Antibodies:
The following reagents were purchased and
used: mTeSR1 medium (Stem Cell Tech, #5850), ReLeSR (Stem Cell
Tech, #5872), DMEM/F12 medium (Invitrogen, #11330-032), neurobasal
medium (Invitrogen, #21103-049), advanced DMEM-F12 (Thermo Fisher,
#12634010), N2 supplement 50× (Thermo Fisher, #17502001), B27 sup-
plement 100× (Invitrogen, #17504044), MEM non-essential amino acids
(MEM-NEAA) 100× (Thermo Fisher, #11140-050); l-glutamine (Gibco,
#25030149), Geltrex LDEV-free, hESC-qualified, RGF-BM matrix (Thermo
Fisher, #A1413302); 𝛽-mercaptoethanol (Sigma-Aldrich, #M3148); accu-
tase (Sigma-Aldrich, #A6964-500ML); SB431542 (Abcam, #ab120163),
LDN193189 dihydrochloride (Sigma-Aldrich, SML0559-5MG), Y27632
(C14H21N3O·2HCl) (Stem Cell Tech, # 72304), all-trans retinoic acid
(AtRA) (Stem Cell Tech, #72262), recombinant human sonic hedgehog
(Shh) (Peprotech, #100-45), (±)-Blebbistatin (Sigma-Aldrich, #203390-
5MG), cytochalasin D (Sigma-Aldrich, #C8273-1MG), ML7 (Sigma-
Aldrich, # I2764-5MG), Verteporfin (Sigma-Aldrich, # SML0534-5MG),
paraformaldehyde, 16% w/v (Fisher Scientific, #11400580); Triton X-100
(Sigma-Aldrich, # 086031000), Sylgard 184 Silicone Elastomer Kit (Dow
Corning, # 1317318), phosphate-buffered saline (PBS) (Sigma-Aldrich,
#P4417), Hoechst 33342 (Invitrogen, #H3570), Alexa Fluor 647 phal-
loidin, 1/300 dilution (Thermo Fisher, #A22287), Alexa Fluor 488 phal-
loidin, 1/300 dilution (Thermo Fisher, # A12379). The following primary
antibodies were used: YAP antibody, mouse monoclonal IgG2a, 1/80di-
lution (Santa Cruz, # SC101199); PAX6 antibody, polyclonal Mouse IgG,
1/300 dilution (Invitrogen, # MA1-109); Ncadherin (CD325) antibody,
polyclonal rabbit IgG, 1/300 dilution (Abcam, #ab18203); FOXA2 an-
tibody, monoclonal Mouse IgG2a (1C7),1/200 dilution (Sigma-Aldrich
#SAB1403929); FOXA2 antibody, monoclonal Rabbit IgG (D56D6), 1/400
dilution (Cell Signaling Tech #8186); PAX3 antibody, polyclonal Rabbit IgG,
1/250 dilution (Invitrogen #38-1801). The following secondary antibodies
were used: Alexa Fluor 488 conjugated donkey antirabbit IgG (H+L),1/250
dilution, (Invitrogen, #A-21206); Alexa Fluor 555 conjugated donkey an-
tirabbit IgG (H+L), 1/250 dilution, (Invitrogen, #A-31572); Alexa Fluor 647
conjugated donkey anti-rabbit IgG (H+L), 1/250 dilution, (Invitrogen, #A-
31573); Alexa Fluor 488 conjugated donkey antimouse IgG (H+L),1/250
dilution, (Invitrogen, # A-21202); Alexa Fluor 647 conjugated donkey anti-
mouse IgG (H+L), 1/250 dilution (Invitrogen, # A-31571). Immunofluorescence Staining:
3D NE cysts and organoids were fixed
with 4% paraformaldehyde at room temperature for 15–20 min and there-
after washed with PBS. For immunofluorescence staining, 3D cysts and
organoids were incubated in blocking buffer (5% donkey serum in 0.3%
Triton X-100) for 60–90 min at room temperature. Subsequent primary
antibodies were diluted in blocking buffer and used for staining at 4 °C
overnight. Advanced Science published by Wiley-VCH GmbH
2201106 (13 of 16) 4. Conclusion The result presented in this study highlight the importance
of both intracellular regulation and interfacial cues in mod-
ulating NE organoid formation at different stages. A better
understanding of how interfacial cues determine cell fate and
tissue spatial organization in 3D organoids will pave the way
for next-generation methods for establishing and engineering
organoids with higher complexity in terms of both cellular
composition and tissue architecture. )
( / )
PDMS Chip Fabrication and Characterization:
PDMS chips containing
micropatterned substrates were fabricated using photolithography as de-
scribed previously.[25,42] Briefly, the features were produced on a silicon
master wafer by deep reactive-ion etching (Philips Innovation Services,
Eindhoven, the Netherlands) from a chromium photomask (Toppan Pho-
tomask, Corbeil Essonnes, France). Solution of silicon elastomer (PDMS,
Sylgard 184, Dow Corning) and curing agent with a weight ratio of 10:1
was mixed and degassed, which was subsequently poured onto the sili-
con master that was passivated using fluorosilane and cured in the oven
for 20–30 min at 110 °C. Cured PDMS chips with desired features (Fig-
ure S6, Supporting Information) were then peeled offfrom the master,
followed by cleaning with 70% ethanol and drying with compressed air, re-
spectively. The chips were coated with 1% Geltrex before cell seeding. For
verifying the dimension of PDMS chips after fabrication, cross-sections of
the PDMS chips were prepared using a sharp blade and images were cap-
tured using an optical microscope (Leica dmi8) with 10×/0.32 objective
lens (Figure S6, Supporting Information). www.advancedscience.com in DMEM/F12 medium with a v/v ratio of 1:90 was used for coating cell
culture flasks at 37 °C for at least 45 min before seeding hiPSCs. Medium
was changed daily and cells were passaged every 4–6 days either by using
ReLeSR or Accutase, and 10 × 10−6 m ROCK inhibitor Y27632 was added
to the medium for the first 24 h during passaging. organoids similarly suggests that geometrical cues can affect in-
testinal organoids through a mechanoresponsive machinery.[10]
Altogether, our study demonstrates that modulation of the self-
organization process of NE organoids could be achieved through
interfacial cues. Understanding how such cues in the niche reg-
ulate stem cell function and fate determination contributes to a
better understanding of how cells develop distinctive morpholo-
gies, providing guidance for the design of 3D organoid genera-
tion system. g p
g g
3D Neural Epithelial Organoid Culture:
Generation of 3D neural ep-
ithelial organoid were performed following previous protocol.[4] Briefly,
after hiPSC colonies were treated with Accutase for 4–6 min at 37 °C, sin-
gle hiPSC cell suspension was prepared in mTeSR1 medium with Y27632
(10 × 10−6 m). hiPSCs at an initial cell density of 8000–10 000 cells cm−2
were plated on substrate with 1% Geltrex coating and cultured overnight. For differentiating hiPSC to neural epithelial cells, medium was switched
to neural induction medium (NIM) from the following day (day 1) and 3D
NE cysts were maintained in NIM for 7 days. For inducing patterned NE
organoids: NIM was also used from the following day (day 1) to day 4, sub-
sequently culture medium was switched to patterning medium (PM) from
day 4 to day 9, and then NIM was used from day 9 to day 18. The details of
medium are described below, N2B27 medium: Advance DMEM/F12 (50%
v/v), neurobasal medium (50% v/v), N2 (0.5×), B27 (0.5×), MEM-NEAA
(1×), l-glutamine (2 × 10−3 m), and 𝛽-mercaptoethanol (0.1 × 10−3 m). NIM: N2B27 medium was supplemented with SB431542 (10 × 10−6 m),
LDN193189 (0.1 × 10−6 m), and 2% Geltrex (v/v). PM: SB431542 (10 ×
10−6 m), LDN193189 (0.1 × 10−6 m), Shh (10 × 10−9 m), AtRA (1 × 10−6
m), and 2% Geltrex (v/v). © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (14 of 16) www.advancedsciencenews.com Adv. Sci. 2022, 9, 2201106 Keywords organoid, respectively; each dot shown in the graph represents one mea-
sured NE cyst or organoid. For quantitative analysis of the fluorescence
intensity, images were taken by confocal microscope with identical set-
ting between different samples. MFI of each slide along the Z-stack of
a 3D cyst or organoid sample was profiled in Fiji.[43,44] tMFI was calcu-
lated by adding the MFI value from all slices of a corresponding cyst or
organoid sample together; each dot shown in the graph represents one
measured NE cyst or organoid. For quantifying the YAP fluorescence in-
tensity ratio between cytoplasm and nuclei, the Z-stacks were first decon-
volved through express deconvolution in Huygens professional (Scientific
Volume Imaging B.V.) for segmentation of YAP fluorescence signal from
the cytoplasm and nuclei, and then the YAP fluorescence intensity ratio
was calculated by dividing the MFI of YAP from the cytoplasm to that from
the nuclei. The percentage of patterned organoids was calculated by divid-
ing the number of floor-plate patterned organoids by the number of total
organoids from the same randomly chosen field of view; each dot shown
in the graphs represents a randomly chosen field of view where the per-
centage of patterned organoids was quantified. cellular self-organization, dorsal–ventral patterning, interfacial cues, neu-
ral tube, neuroepithelial organoid Received: February 24, 2022
Revised: April 20, 2022
Published online: June 6, 2022 [1] L. Wilson, M. Maden, Dev. Biol. 2005, 282, 1. [2] G. Le Dréau, E. Martí, Dev. Neurobiol. 2012, 72, 1471. [3] E. Nikolopoulou, G. L. Galea, A. Rolo, N. D. E. Greene, A. J. Copp,
Develompent 2017, 144, 552. [4] Y. Zheng, X. Xue, A. M. Resto-Irizarry, Z. Li, Y. Shao, Y. Zheng, G. Zhao, J. Fu, Sci. Adv. 2019, 5, 5933. Statistical Analysis:
All statistical analyses were conducted using
Graphpad Prism 8. A Student’s t-test was performed to compare the
statistical difference between two datasets. One-way analysis of variance
(ANOVA) followed by a Dunnett multiple comparisons test or Tukey’s mul-
tiple comparisons test was performed when more than two datasets were
compared. P-values of statistical significance were represented as: * P
< 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns (not significant)
P > 0.05. [5] A. Meinhardt, D. Eberle, A. Tazaki, A. Ranga, M. Niesche, M. Wilsch- [5] A. Meinhardt, D. Eberle, A. Tazaki, A. Ranga, M. Niesche, M. Wilsch-
Bräuninger, A. Stec, G. Schackert, M. Author Contributions [21] G. Nardone, J. Oliver-De La Cruz, J. Vrbsky, C. Martini, J. Pribyl, P. Skládal, M. Pešl, G. Caluori, S. Pagliari, F. Martino, Z. Maceckova, M. Hajduch, A. Sanz-Garcia, N. M. Pugno, G. B. Stokin, G. Forte, Nat. Commun. 2017, 8, 15321. C.T. and N.A.K. conceived and designed the project. C.T. developed exper-
imental approach, performed experiments, analyzed data, and prepared
all figures. X.W. contributed to immunofluorescence staining experiments. M.D. helped with fluorescence intensity analysis. C.v.d.P. characterized the
dimensions of the PDMS chips after fabrication. N.A.K. supported and
supervised the project. C.T. and N.A.K. wrote the manuscript. All authors
discussed and approved the manuscript. [22] S. Pagliari, V. Vinarsky, F. Martino, A. R. Perestrelo, J. Oliver De La
Cruz, G. Caluori, J. Vrbsky, P. Mozetic, A. Pompeiano, A. Zancla, S. G. Ranjani, P. Skladal, D. Kytyr, Z. Zdráhal, G. Grassi, M. Sampaolesi, A. Rainer, G. Forte, Cell Death Differ. 2021, 28, 1193. [23] Y. Sun, K. M. A. Yong, L. G. Villa-Diaz, X. Zhang, W. Chen, R. Phil-
son, S. Weng, H. Xu, P. H. Krebsbach, J. Fu, Nat. Mater. 2014, 13,
599. Conflict of Interest [18] D. Indana, P. Agarwal, N. Bhutani, O. Chaudhuri, Adv. Mater. 2021,
33, 2101966. The authors declare no conflict of interest. [19] L. Luo, Annu. Rev. Cell Dev. Biol. 2002, 18, 601. [20] G. Brusatin, T. Panciera, A. Gandin, A. Citron, S. Piccolo, Nat. Mater. 2018, 17, 1063. Keywords Lutolf, E. M. Tanaka, Stem Cell
Rep. 2014, 3, 987. Bräuninger, A. Stec, G. Schackert, M. Lutolf, E. M. Tanaka, Stem Cell
Rep. 2014, 3, 987. [6] A. Ranga, M. Girgin, A. Meinhardt, D. Eberle, M. Caiazzo, E. M
Tanaka, M. P. Lutolf, Proc. Natl. Acad. Sci. USA 2016, 113, E6831. [7] A. R. Abdel Fattah, B. Daza, G. Rustandi, M. Á. Berrocal-Rubio, B. Gorissen, S. Poovathingal, K. Davie, J. Barrasa-Fano, M. Cóndor, X. Cao, D. H. Rosenzweig, Y. Lei, R. Finnell, C. Verfaillie, M. Sampaolesi,
P. Dedecker, H. Van Oosterwyck, S. Aerts, A. Ranga, Nat. Commun. 2021, 12, 3192. Acknowledgements The authors gratefully acknowledge Dr. Jos Broers and Dr. Florence van
Tienen (Maastricht University) for sharing hiPSC cell line, and Dr. Gitta
Buskermolen and Dylan Mostert for assistance with the design of the
microfabricated PDMS chips. The project leading to this application re-
ceived funding from the European Research Council (ERC) under the
European Union’s Horizon 2020 research and innovation programme
(Grant Agreement No. 851960), the Dutch Research Council (Grant No. OCENW.XS2.017), Health-Holland (grant LSHI18002). X.W. is a recipient
of a China Scholarship Council scholarship (Grant No. 202009370078). C.T. is a recipient of a short-term fellowship from the Federation of the
European Biochemical Societies (FEBS). [10] N. Gjorevski, M. Nikolaev, T. E. Brown, O. Mitrofanova, N. Branden-
berg, F. W. DelRio, F. M. Yavitt, P. Liberali, K. S. Anseth, M. P. Lutolf,
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Biol. 2017, 18, 758. [16] F. Martino, A. R. Perestrelo, V. Vinarský, S. Pagliari, G. Forte, Front. Physiol. 2018, 9, 824. [17] A. R. Harris, P. Jreij, D. A. Fletcher, Annu. Rev. Biophys. 2018, 47, 617. Supporting Information [8] M. Nikolaev, O. Mitrofanova, N. Broguiere, S. Geraldo, D. Dutta, Y. Tabata, B. Elci, N. Brandenberg, I. Kolotuev, N. Gjorevski, H. Clevers,
M. P. Lutolf, Nature 2020, 585, 574. Supporting Information is available from the Wiley Online Library or from
the author. [9] E. Karzbrun, A. H. Khankhel, H. C. Megale, S. M. K. Glasauer, Y. Wyle,
G. Britton, A. Warmflash, K. S. Kosik, E. D. Siggia, B. I. Shraiman, S. J. Streichan, Nature 2021, 599, 268. 5. Experimental Section After three times of PBS wash in the following day, secondary
antibodies and/or phalloidin diluted in PBS were applied to 3D cysts and
organoids for incubating 60 min at room temperature. Nuclei were stained
and visualized by using Hoechst 33342 (1/2000 dilution) with incubation
at room temperature for 20 min, followed by three PBS washes. Samples
were mounted to glass slide with mounting solution and stored at 4 °C were mounted to glass slide with mounting solution and stored at 4 °C. Microscopy Imaging and Image Analysis:
Bright-field images were cap-
tured by using Leica DMi8 microscope (Leica) equipped with 10×/0.32
objective lens and immunofluorescent images were acquired in Leica TCS
SP8X confocal microscope (Leica) equipped with 20×/0.75 objective lens. All acquired images were processed by Fiji.[43,44] Confocal images were
taken with Z-stacks and the distance between slices in each Z-stack was
the same (2 μm) for all samples. The number density of NE cysts and
organoids was defined by counting the number of NE cysts and organoids
per mm2; each dot shown in the graph represents a randomly chosen
field of view where NE cysts or organoids was quantified. The size of NE
cysts and organoids was quantified by measuring the projected surface
area of NE cysts and organoids; each dot shown in the graph represents
one measured NE cyst or organoid. Lumen-to-cyst size ratio or lumen-to-
organoid size ratio was calculated by dividing the projected surface area
of the lumen by the projected surface area of the corresponding NE cyst or Human iPSC Culture:
hiPSCs were generated and cultured as de-
scribed previously.[41] Briefly, hiPSCs were maintained in mTeSR1 medium
and were cultured at 37 °C in 5% CO2 humidified incubator. Geltrex diluted © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (14 of 16) Adv. Sci. 2022, 9, 2201106 www.advancedsciencenews.com www.advancedsciencenews.com © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
2201106 (15 of 16) Data Availability Statement The data that support the findings of this study are available from the cor-
responding author upon reasonable request. [24] Y. Zhu, M. Carido, A. Meinhardt, T. Kurth, M. O. Karl, M. Ader, E. M. Tanaka, PLoS One 2013, 8, 54552. © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH
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2201106 (16 of 16) Adv. Sci. 2022, 9, 2201106
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Assessment of Ambient Air Quality in Primary Schools in Various Areas of Lahore
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International Journal For Multidisciplinary Research
| 2,023
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cc-by-sa
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International Journal for Multidisciplinary Research (IJFMR)
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com International Journal for Multidisciplinary Research (IJFMR)
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com Keywords: Ambient air Quality, Pollutant, Government and Private schools Keywords: Ambient air Quality, Pollutant, Government and Private schools Keywords: Ambient air Quality, Pollutant, Government and Private schools ABSTRACT Indoor air pollutants assessment in 54 primary schools of both government and private sectors in 9 different
towns of Lahore were carried out for the purpose of measuring concentration of Particulate matter (PM2.5,
PM10), Carbon monoxide(CO), Carbon dioxide (CO2),Nitric oxide(NO), Nitrogen dioxide (NO2),Nitrous
Oxide (NOx),Sulphur dioxide (SO2), Hydrogen sulphide (H2S), Ground level Ozone (O3)and Volatile
Organic Compounds (VOCs). After analysisthe level of CO2 is lower in both schools. The government
schools has more concentration than private schools. After analysis the level of COis lower in both schools. The government schools has more concentration than private schools. The level of O3 is lower in both
schools. Both government and private schools has almost same concentration. The concenteration of NOis
higher in both schools as compared to standard. The private schools has more concentration than
government schools. The level of NO2 is higher in both schools as compared to standard. The private
schools has more concentration than government schools. The concenteration of SO2 is so much higher in
both schools as compared to standard value. The government schools has more concentration than private
schools. The concenteration of H2Sis higher in both schools as compared to reference value. The
government schools has more concentration than private schools. The concenteration of VOC’sis low in
both schools as compared to standard value. Both schools have almost the same concentration.The
concenteration of PM10 is so much higher in both schools as compared to reference value. The government
schools has more concentration than private schools. The concenteration of PM2.5 is so much higher in both
schools as compared to standard value. The government schools has more concentration than private
schools.The standard value of the pollutants are CO2 (1000ppm), CO (10mg/m3), O3 (130µg/m3), NO
(40µg/m3), NO2 (80µg/m3), SO2 (120µg/m3), H2S (0.47ppb), VOC’s (25ppb), PM10 (150µg/m3), PM2.5
(15µg/m3). International Journal for Multidisciplinary Research (IJFMR)
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com towards the industrialization without taking natural strategies about topography and meteorological
conditions. The population has become double today as compare to past some decades (Baldasano et al. 2003). Population increase is directly proportional to: increase variety of mobile facilities for transportation
of products, increase needs of space, material, energy, food and comfort. These demands lead towards the
utilization of natural resources by deforestation. A number of mobile sources like road automobile and
stationary sources like mining, power plant, industry, commercial activities, trade centers and space heating
etc are responsible for the maximum level of pollutants in ambient air of cities. It is a difficult task to meet
the international standards of pollutant and control their emissions in developing countries because they
have low economies (Ozden et al. 2008). towards the industrialization without taking natural strategies about topography and meteorological
conditions. The population has become double today as compare to past some decades (Baldasano et al. 2003). Population increase is directly proportional to: increase variety of mobile facilities for transportation
of products, increase needs of space, material, energy, food and comfort. These demands lead towards the
utilization of natural resources by deforestation. A number of mobile sources like road automobile and
stationary sources like mining, power plant, industry, commercial activities, trade centers and space heating
etc are responsible for the maximum level of pollutants in ambient air of cities. It is a difficult task to meet
the international standards of pollutant and control their emissions in developing countries because they
have low economies (Ozden et al. 2008). A number of pollutants are responsible for pollution in air that alter the composition of air and damaged the
living population. Atmosphere has the ability to absorb the pollutant emitted from the different source of
origin but the concentration of these pollutants is depending upon the emission source. The air pollution is
not fixed in ratio but change its concentration temporarily or spatially. This change occurs due to
meteorological conditions and topography of an area. Air pollutants such as CO, NO, NO2, SO2, H2S, NH3,
ground level ozone (O3), Volatile organic compounds (VOCS), polycyclic aromatic hydrocarbons (PAHS)
has toxic effects on health of humans (Brunekreef and Holgate, 2002). As pollutants emit from their
sources, they dispersed, transport from source and are combine to form variety of secondary pollutants. This
phenomenon occurs in atmosphere. Introduction Air pollution is becoming a serious issue about public health worldwide. It is creating long term and worse
impacts on human’s health, plants, equipment, ecosystem and short term effect on visibility (Fenger, 1999;
Riga-Karandinos, 2005). Air quality has become a challenge in big cities because people are exposed to
high risk by pollutants. In developed countries, 75% population resides in urban areas whereas the urban
ratio in developing countries is more than 35% causing negative impacts on air quality (Wolf, 2002;
Agrawal et al. 2003; Vargas, 2003; Brajer et al.2006; Oudient et al. 2006). Air pollution in cities has
become more overwhelming due to increasing population. An abandoned increase in population direct Volume 5, Issue 1, January-February 2023 IJFMR23011625 1 Materials and Methods The research work was conducted in the Department of Environmental Sciences, University of Veterinary
and Animal Sciences Lahore (UVAS). In the current study, an assessment was carried out for 11 air
pollutants in the government and private schools in 9 towns of Lahore. The main objective of this study was
to assess the ambient air quality in private as well as public primary schools in all the 9 towns of Lahore. International Journal for Multidisciplinary Research (IJFMR)
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com plants, and industrial activities due to industrialization which produces variety of pollutants in atmosphere
causing pollution in Pakistan. As compare to United States, the emissions from Pakistani automobiles
contains 3.5 times more SO2 levels and 20 times higher CO2 and CO levels (Barber, 2008). Transport sector
is mainly dependent on diesel fuel which is the root cause of PM2.5 concentration in air (Shyamsundar et al. 2001). According to an estimation, in Pakistan, particulate matter pollution impacts on health costs 65
billion with 700 deaths of children and 22000 among adults (World Bank, 2006). Pakistan EPA and World
Bank estimate the total value of health impact caused by ambient air pollution in Pakistan. The health
impacts measured in Pakistan due to ambient air pollution with their annual costs are: Premature Mortality
rate is 21,791 and annual cost ranges 58-61 in billion rupees. There are 658 cases reported of mortality in
children with annual 0.83 billion costs. Chronic bronchitis caused 7,825 cases with 0.06 billion cost
annually. Respiratory symptoms leads to Lower respiratory illness have been seen in 706,808,732 and
4,924,148 cases with total cost of 0.84 billion respectively (EPA and World Bank, 2006). Pakistan Environmental Protection Agency has drafted the National Environmental Quality Standards for
Ambient Air. In pursuance of the statutory requirements under clause (e) of Sub-section (1) of section (6) of
the Pakistan Environmental Protection Act 1997, the drafted standards are being published. National
Ambient Air Quality Standards of Pakistan (NAAQS) are 35µg/m3, 80µg/m3, 40µg/m3, 120µg/m3, 5µg/m3
and 130µg/m3 for PM2.5, NO2, NO, SO2, CO and O3 respectively. Pakistan EPA and JICA 2001,
investigates the average pollutants level in ambient air of Lahore. The pollutant concentration of PM2.5, SO2,
NOx, CO and O3 was found to be so high as 895.0µg/m3, 44.60ppb, 156.60ppb, 2.82ppm and 8.50ppb
respectively as compare to permissible limits of WHO guidelines.Primary school going children are more
susceptible to the pollutant exposure because they inhale an air loaded with pollutant is larger than their
body weight. Children are growing; their respiratory airways are weak and tiny in size for hazardous
pollutants to pass through, so their respiratory system damaged earlier than adults (Aziz et al. 2014). International Journal for Multidisciplinary Research (IJFMR)
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com Ground level ozone forms when Oxides of nitrogen combines with the
VOCS in atmosphere (Fenger, 1999). The combination of primary or secondary pollutant with oxides of
sulphur leads to formation of secondary particulate matter (Atkinson, 2000). The oxidation of NO2 forms
HNO3 and then changed into NO3
-. These secondary pollutants are the main cause of reduced visibility,
acidification and has deleterious impacts on humans (Riga-Karandinos and Saitanis, 2005). WHO (World
Health Organization), EPA (Environment Protection Agency), World Bank and European Union Air
Quality Framework set up the standards of these pollutants for the safety measurements (Lim et al. 2005). WHO air quality guideline provides guidance about threshold level of air pollutants. Values for particulate
matter in air are: annual mean of PM2.5 is 10µg/m3& 24-hour mean of PM2.5 is 25µg/m3 and annual mean of
PM10 is 20µg/m3 & 24-hour mean of PM10is 50µg/m3. Value for 8-hour mean of O3 is 100µg/m3. A value for
annual mean of NO2 is40µg/m3 and 1-hour mean of NO2 is 200µg/m3. A value for 24-hour mean of SO2 is
20µg/m3 and 10 minutes mean of SO2 is 500µg/m3(WHO, 2005).According to EEA study, it was expected
that the urban cities of Europe will face elevated amount of air pollutants in 2010 (EEA, 2003). The
concentration of CO, NOx and NO2 above permissible limit in ambient air can cause heart coronary diseases
(Anne, 2006). Particulate matter has negative effects on health like cardiovascular, lung diseases and premature death in
children (Pope et al. 2002; NARSTO, 2004; Silva et al. 2013).Volatile organic compounds are ubiquitous
and are the major air pollutant of indoor ambient air (Wolkoff and Nielsen, 2001). It has been found that
very trace amount of VOC’s even than the standard concentrations are responsible for odor (Peng et al. 2009) and affect the human sensory system due to which people feels annoyance and creates health
problems (Nicell, 2009). Pakistan is becoming an urbanized country with total 180 million populations
(Government of Pakistan, 2012). Air pollution is increasing continuously by rising vehicle emissions, power Volume 5, Issue 1, January-February 2023 2 IJFMR23011625 Analysis of Ambient Air Ambient air pollutants (PM2.5, PM10, CO, CO2, NO, NO2, SO2, H2S, NOx, O3 and VOCs) were analyzed by
using HAZ-SCANNER Model HIM-6000 Ambient Air Quality Monitoring System. The air monitoring was
done by this system from February to March. Ambient air pollutants (PM2.5, PM10, CO, CO2, NO, NO2, SO2, H2S, NOx, O3 and VOCs) were analyzed by
using HAZ-SCANNER Model HIM-6000 Ambient Air Quality Monitoring System. The air monitoring was
done by this system from February to March. International Journal for Multidisciplinary Research (IJFMR)
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com 5. Wagha Town
6. Iqbal Town
7. Gulberg Town
8. Aziz Bhatti Town
9. Nishtar Town 9. Nishtar Town Statistical Analysis T-test was applied to detect if different groups of government and private schools produced statistically
significant difference to permissible values for air pollutants. Sample Collection The data was collected from 6 schools in each town (3 Private and 3 Government) and 54 in total. One point
was taken inside and one outside of each school. The 3 Government schools were categorize as (A, B & C)
while the 3 Private schools were categorized as (D, E & F). The nine towns are: 1. Data Gang Baksh Town 2. Ravi Town 3. Samnabad Town 4. Shalimar Town IJFMR23011625 3 Volume 5, Issue 1, January-February 2023 International Journal for Multidisciplinary Research (IJFMR)
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com
5. Wagha Town International Journal for Multidisciplinary Research (IJFMR)
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com Results Outcomes of this research provided mean concentration of pollutants found in ambient air of schools in
Lahore. It shows comparison of ten pollutants concentration (PM2.5(µg/m3), PM10 (µg/m3), CO (ppm),
CO2(ppm), NO (ppb), NO2(ppb), SO2 (ppb), H2S (ppb), O3(ppb)and VOCs (ppb)) between government and
private school groups of nine towns of Lahore and also compared them with standards. C02
Figure No 1:Concentration of CO2
0
100
200
300
400
500DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
Co2 Conc. CO2 Inside
CO2 Outside C02
Figure No 1:Concentration of CO2
0
100
200
300
400
500DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
Co2 Conc. CO2 Inside
CO2 Outside C02
Figure No 1:Concentration of CO2
Figure 1 shows a comparison of CO2 concentration between school groups of different towns. Results were
compared inside and outside of the schools. Results shows that CO2 concentration was high in Samnabad
town and low in Iqbal Town inside the school. CO2 concentration was high in Shalimar Town and low in
Gulberg outside the schools.Every town has an increasing trend of CO2 but still it value was under the set
standards. The set value of CO2 is 1000 ppm. 0
100
200
300
400
500DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
Co2 Conc. CO2 Inside
CO2 Outside Figure No 1:Concentration of CO2 Figure 1 shows a comparison of CO2 concentration between school groups of different towns. Results were
compared inside and outside of the schools. Results shows that CO2 concentration was high in Samnabad
town and low in Iqbal Town inside the school. CO2 concentration was high in Shalimar Town and low in
Gulberg outside the schools.Every town has an increasing trend of CO2 but still it value was under the set
standards. The set value of CO2 is 1000 ppm. Figure 1 shows a comparison of CO2 concentration between school groups of different towns. Results were
compared inside and outside of the schools. Results shows that CO2 concentration was high in Samnabad
town and low in Iqbal Town inside the school. CO2 concentration was high in Shalimar Town and low in
Gulberg outside the schools.Every town has an increasing trend of CO2 but still it value was under the set
standards. The set value of CO2 is 1000 ppm. Volume 5, Issue 1, January-February 2023 IJFMR23011625 4 International Journal for Multidisciplinary Research (IJFMR)
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com
CO
Figure No 2 Concentration of CO
Figure 2 shows a comparison of COconcentration between school groups of different towns. Results Results were
compared inside and outside of the schools. Results show that CO concentration was high in Ravi town and
low in Iqbal Town inside the school. CO concentration was high in Data Gang Baksh Town and low in
Iqbal Town outside the schools.CO concentration is under the set standards of NEQS. Set value is 10mg/m3. H2S
0
0.2
0.4
0.6
0.8
1
1.2
1.4DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
CO Inside
CO Outside International Journal for Multidisciplinary Research (IJFMR)
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com
CO
Figure No 2 Concentration of CO
Figure 2 shows a comparison of COconcentration between school groups of different towns. Results were
compared inside and outside of the schools. Results show that CO concentration was high in Ravi town and
low in Iqbal Town inside the school. CO concentration was high in Data Gang Baksh Town and low in
Iqbal Town outside the schools.CO concentration is under the set standards of NEQS. Set value is 10mg/m3. H2S
0
0.2
0.4
0.6
0.8
1
1.2
1.4DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
CO Inside
CO Outside
0
5
10
15
20
25
30DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
H2S Inside
H2S Outside CO 0
0.2
0.4
0.6
0.8
1
1.2
1.4DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
CO Inside
CO Outside Figure No 2 Concentration of CO Figure 2 shows a comparison of COconcentration between school groups of different towns. Results were
compared inside and outside of the schools. Results show that CO concentration was high in Ravi town and
low in Iqbal Town inside the school. CO concentration was high in Data Gang Baksh Town and low in
Iqbal Town outside the schools.CO concentration is under the set standards of NEQS. Set value is 10mg/m3. Figure No 3 Concentration of H2S
0
5
10
15
20
25
30DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
H2S Inside
H2S Outside Figure No 3 Concentration of H2S
Figure 3 shows a comparison of H2S concentration between school groups of different towns. Results were
compared inside and outside of the schools. Results show that H2S concentration was high in Iqbal Town
and low in Shalimar Town inside the school. H2S concentration was high in Ravi Town and low in Wagha
outside the schools. Results 0
5
10
15
20
25
30DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
H2S Inside
H2S Outside Figure No 3 Concentration of H2S
0
5
10
15
20
25
30DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
H2S Inside
H2S Outside Figure No 3 Concentration of H2S Figure No 3 Concentration of H2S Figure 3 shows a comparison of H2S concentration between school groups of different towns. Results were
compared inside and outside of the schools. Results show that H2S concentration was high in Iqbal Town
and low in Shalimar Town inside the school. H2S concentration was high in Ravi Town and low in Wagha
outside the schools. Figure 3 shows a comparison of H2S concentration between school groups of different towns. Results were
compared inside and outside of the schools. Results show that H2S concentration was high in Iqbal Town
and low in Shalimar Town inside the school. H2S concentration was high in Ravi Town and low in Wagha
outside the schools. Figure 3 shows a comparison of H2S concentration between school groups of different towns. Results were
compared inside and outside of the schools. Results show that H2S concentration was high in Iqbal Town
and low in Shalimar Town inside the school. H2S concentration was high in Ravi Town and low in Wagha
outside the schools. Volume 5, Issue 1, January-February 2023 5 IJFMR23011625 International Journal for Multidisciplinary Research (IJFMR)
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com
NO
0
10
20
30
40
50
60DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
NO Inside
NO Outside International Journal for Multidisciplinary Research (IJFMR)
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com
NO
Figure No 4 Concentration of NO
Figure 4 shows a comparison of NO concentration between school groups of different towns. Results were
compared inside and outside of the schools. Results show that NO concentration was high in Shalimar
Town and low in Data Gang Baksh Town inside the school. NO concentration was high in Ravi Town and
low in Iqbal Town outside the schools. NO values are very high than standards. Standard value for NO is
40µg/m3. NO
0
10
20
30
40
50
60DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
NO Inside
NO Outside International Journal for Multidisciplinary Research (IJFMR)
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com
NO
0
10
20
30
40
50
60DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
NO Inside
NO Outside 0
10
20
30
40
50
60DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
NO Inside
NO Outside Figure No 4 Concentration of NO Figure No 4 Concentration of NO Figure 4 shows a comparison of NO concentration between school groups of different towns. Results were
compared inside and outside of the schools. Results show that NO concentration was high in Shalimar
Town and low in Data Gang Baksh Town inside the school. NO concentration was high in Ravi Town and
low in Iqbal Town outside the schools. NO values are very high than standards. Standard value for NO is
40µg/m3. NO2
Figure No 5 Concentration of NO2
Figure 5 shows a comparison of NO2 concentration between school groups of different towns. Results were
compared inside and outside of the schools. Results show that NO2 concentration was high in Gulberg and
low in Data Gang Baksh Town inside the school. NO2 concentration was high in Ravi Town and low in
Aziz Bhatti Town outside the schools. 0
5
10
15
20
25DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
NO2 Inside
NO2 Outside NO2
Fi
N 5 C
t
ti
f NO
0
5
10
15
20
25DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
NO2 Inside
NO2 Outside NO2
0
5
10
15
20
25DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
NO2 Inside
NO2 Outside Figure No 5 Concentration of NO2 Figure 5 shows a comparison of NO2 concentration between school groups of different towns. Results were
compared inside and outside of the schools. Results show that NO2 concentration was high in Gulberg and
low in Data Gang Baksh Town inside the school. NO2 concentration was high in Ravi Town and low in
Aziz Bhatti Town outside the schools. Volume 5, Issue 1, January-February 2023 6 IJFMR23011625 International Journal for Multidisciplinary R
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Ema
NOx
Figure No 6 Concentration of NOx
0
20
40
60
80DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
Nox Inside
Nox Outside International Journal for Multidisciplinary Research (IJFMR)
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com
NOx
Figure No 6 Concentration of NOx
Figure 6 shows a comparison of NOx concentration between school groups of different towns. Results were
compared inside and outside of the schools. Results show that NOx concentration was high in Data Gang
Baksh Town and Wagha inside the school. NOx concentration was high in Ravi Town and low in Wagha
outside the schools. 0
20
40
60
80DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
Nox Inside
Nox Outside Nox Inside
Nox Outside Figure No 6 Concentration of NOx Figure 6 shows a comparison of NOx concentration between school groups of different towns. Figure No 4 Concentration of NO Results were
compared inside and outside of the schools. Results show that NOx concentration was high in Data Gang
Baksh Town and Wagha inside the school. NOx concentration was high in Ravi Town and low in Wagha
outside the schools. Figure 6 shows a comparison of NOx concentration between school groups of different towns. Results were
compared inside and outside of the schools. Results show that NOx concentration was high in Data Gang
Baksh Town and Wagha inside the school. NOx concentration was high in Ravi Town and low in Wagha
outside the schools. 0
2
4
6
8
10
12
14
16
O3 Inside
O3 Outside
O3
Figure No 7 Concentration of O3 O3 0
2
4
6
8
10
12
14
16
O3 Inside
O3 Outside
O3
Figure No 7 Concentration of O3 0
2
4
6
8
10
12
14
16
O3 Inside
O3 Outside
O3
Figure No 7 Concentration of O3
Figure 7 shows a comparison of O3 concentration between school groups of different towns. Results were
compared inside and outside of the schools. Results show that O3 concentration was high in RaviTown and
low in Data Gang Baksh Town inside the school. O3 concentration was high in Ravi Town and low in Data
Gang Baksh Town outside the schools. Ozone concentration is under the permissible values. Standard value
is 130µg/m3. Figure No 7 Concentration of O3 Figure 7 shows a comparison of O3 concentration between school groups of different towns. Results were
compared inside and outside of the schools. Results show that O3 concentration was high in RaviTown and
low in Data Gang Baksh Town inside the school. O3 concentration was high in Ravi Town and low in Data
Gang Baksh Town outside the schools. Ozone concentration is under the permissible values. Standard value
is 130µg/m3. Figure 7 shows a comparison of O3 concentration between school groups of different towns. Results were
compared inside and outside of the schools. Results show that O3 concentration was high in RaviTown and
low in Data Gang Baksh Town inside the school. O3 concentration was high in Ravi Town and low in Data
Gang Baksh Town outside the schools. Ozone concentration is under the permissible values. Standard value
is 130µg/m3. Figure No 4 Concentration of NO Volume 5, Issue 1, January-February 2023 IJFMR23011625 7 International Journal for Multidisciplinary Resea
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor
PM10
Figure No 8 Concentration of PM10
0
100
200
300
400
500
600DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
PM10 Inside
PM10 Outside International Journal for Multidisciplinary Research (IJFMR)
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com
PM10
Figure No 8 Concentration of PM10
Figure 8 shows a comparison of PM10 concentration between school groups of different towns. Results were
compared inside and outside of the schools. Results show that PM10 concentration was high in Ravi Town
and low in Aziz Bhatti Town inside the school. PM10 concentration was high in Ravi Town and low in Aziz
Bhatti Town outside the school. The value of PM10 is higher than the standards. The set value of PM10 is
150 µg/m3. Each town is significantly high value of this particulate matter. 0
100
200
300
400
500
600DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
PM10 Inside
PM10 Outside Figure No 8 Concentration of PM10 Figure 8 shows a comparison of PM10 concentration between school groups of different towns. Results were
compared inside and outside of the schools. Results show that PM10 concentration was high in Ravi Town
and low in Aziz Bhatti Town inside the school. PM10 concentration was high in Ravi Town and low in Aziz
Bhatti Town outside the school. The value of PM10 is higher than the standards. The set value of PM10 is
150 µg/m3. Each town is significantly high value of this particulate matter. PM2.5
Figure No 9 Concentration of PM2.5
0
50
100
150
200
250
300
350
400
450
500DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
PM2.5 Inside
PM2.5 Outside PM2.5 Figure No 9 Concentration of PM2.5 Volume 5, Issue 1, January-February 2023 Volume 5, Issue 1, January-February 2023 IJFMR23011625 8 SO2 2
Figure No 10 Concentration of SO2
Figure 10 shows a comparison of SO2 concentration between school groups of different towns. Results were
compared inside and outside of the schools. Results show that SO2 concentration was high in Ravi Town
and low in Aziz Bhatti inside the school. SO2 concentration was high in Ravi Town and low in Aziz Bhatti
outside the schools. 0
50
100
150
200
250
300
350
400
450DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
SO2 Inside
SO2 Outside 2
0
50
100
150
200
250
300
350
400
450DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
SO2 Inside
SO2 Outside Figure No 10 Concentration of SO2 Figure 10 shows a comparison of SO2 concentration between school groups of different towns. Results were
compared inside and outside of the schools. Results show that SO2 concentration was high in Ravi Town
and low in Aziz Bhatti inside the school. SO2 concentration was high in Ravi Town and low in Aziz Bhatti
outside the schools. International Journal for Multidisciplinary Research (IJFMR)
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com Figure 9 shows a comparison of PM2.5 concentration between school groups of different towns. Results
were compared inside and outside of the schools. Results show that PM2.5 concentration was high in Ravi
Town and low in Wagha inside the school. PM2.5 concentration was high in Ravi Town and low in Wagha
outside the schools. The converted value of P.M2.5 was very much high than the standards set by NEQS. The standard value is 15µg/m3 but the data collected from towns elaborates its very much high value. VOCs IJFMR23011625
Volume 5, Issue 1, January-February 2023
9
VOCs
Figure No 11 Concentration of VOCS
0
2
4
6
8
10
12
14
16DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
VOCs Inside
VOCs Outside VOCs
Figure No 11 Concentration of VOCS
0
2
4
6
8
10
12
14
16DGBTRaviSamnabadShalimarWaghaIqbalGulbergAziz BhattiNishtar
VOCs Inside
VOCs Outside Figure No 11 Concentration of VOCS Volume 5, Issue 1, January-February 2023 Volume 5, Issue 1, January-February 2023 IJFMR23011625 9 International Journal for Multidisciplinary Research (IJFMR)
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com Figure 11 shows a comparison of VOCs concentration between school groups of different towns. Results
were compared inside and outside of the schools. Results show that VOCs concentration was high in Data
Gang Baksh Town and low in Iqbal Town inside the school. VOCs concentration was high in Data Gang
Baksh Town and low in Aziz Bhatti outside the schools. Mean Pollutants Value Mean Pollutants Value
Figure No 12 Mean Concentrations of Pollutants in School Mean Pollutants Value Mean Pollutants Value Mean Pollutants Value
Figure No 12 Mean Concentrations of Pollutants in School
Pollutants PM2.5, PM10, CO, CO2, NO, NO2, SO2, H2S, O3 and VOCs found in air through many sources. The
concentration of these pollutants measured in volumetric units in air have been converted to compare them
with standard values of these pollutants given by National Environmental Quality Standards (NEQS). The
standard value of the pollutants are CO2 (1000ppm), CO (10mg/m3), O3 (130µg/m3), NO (40µg/m3), NO2
(80µg/m3), SO2 (120µg/m3), H2S (0.47ppb), VOC’s (25ppb), PM10 (150µg/m3), PM2.5 (15µg/m3). Values
are converted by this formula: Figure No 12 Mean Concentrations of Pollutants in School Pollutants PM2.5, PM10, CO, CO2, NO, NO2, SO2, H2S, O3 and VOCs found in air through many sources. The
concentration of these pollutants measured in volumetric units in air have been converted to compare them
with standard values of these pollutants given by National Environmental Quality Standards (NEQS). The
standard value of the pollutants are CO2 (1000ppm), CO (10mg/m3), O3 (130µg/m3), NO (40µg/m3), NO2
(80µg/m3), SO2 (120µg/m3), H2S (0.47ppb), VOC’s (25ppb), PM10 (150µg/m3), PM2.5 (15µg/m3). Values
are converted by this formula: Pollutants PM2.5, PM10, CO, CO2, NO, NO2, SO2, H2S, O3 and VOCs found in air through many sources. The
concentration of these pollutants measured in volumetric units in air have been converted to compare them
with standard values of these pollutants given by National Environmental Quality Standards (NEQS). The
standard value of the pollutants are CO2 (1000ppm), CO (10mg/m3), O3 (130µg/m3), NO (40µg/m3), NO2
(80µg/m3), SO2 (120µg/m3), H2S (0.47ppb), VOC’s (25ppb), PM10 (150µg/m3), PM2.5 (15µg/m3). Values
are converted by this formula: International Journal for Multidisciplinary Research (IJFMR)
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com lead towards the utilization of natural resources by deforestation. A number of mobile sources like road
automobile and stationary sources like mining, power plant, industry, commercial activities, trade centers
and space heating etc are responsible for the maximum level of pollutants in ambient air of cities. It is a
difficult task to meet the international standards of pollutant and control their emissions in developing
countries because they have low economies (Ozden et al. 2008). A number of pollutants are responsible for pollution in air that alter the composition of air and damaged the
living population. Atmosphere has the ability to absorb the pollutant emitted from the different source of
origin but the concentration of these pollutants is depending upon the emission source. The air pollution is
not fixed in ratio but change its concentration temporarily or spatially. This change occurs due to
meteorological conditions and topography of an area. Air pollutants such as Carbon-mono-oxide (CO),
Nitrous oxides (NO, NO2), Sulfur oxides(SO2), Hydrogen Sulfide (H2S), Ammonia( NH3), ground level
ozone (O3), Volatile organic compounds (VOCS), polycyclic aromatic hydrocarbons (PAHS) has harmful
effects on health of humans (Brunekreef and Holgate, 2002). As pollutants emit from their sources, they
dispersed, transport from source and are combine to form variety of secondary pollutants. This phenomenon
occurs in atmosphere. Ground level ozone forms when Oxides of nitrogen combines with the VOCS in
atmosphere (Fenger, 1999). The combination of primary or secondary pollutant with oxides of sulphur leads
to creation of secondary particulate matter (Atkinson, 2000). The oxidation of NO2 forms HNO3 and then
changed into NO3
-. These secondary pollutants are the main cause of reduced visibility, acidification and
has deleterious impacts on humans (Riga-Karandinos and Saitanis, 2005) Air pollution has become an alarming situation in Pakistan. The pollutants in air such as PM2.5, PM10, CO,
CO2, NO, NO2, SO2, H2S, O3 and VOCs have been reached to their maximum level in metropolitan cities. The maximum concentration of these pollutants is found about 200m near to the roadways and heavy loaded
roads (Brugge et al. 2007). Thus, the schools and buildings in this vicinity has more exposure to pollutants. This study was conducted to estimate the concentration of air pollutants in public and private schools of
Lahore. It was found that the level of CO2 was increased as compare to standard value 1000ppm. Discussion Air pollution is becoming a sensitive issue about public health worldwide. It is creating acute, chronic and
worse impacts on human’s health, plants, equipment, ecosystem and short term effect on visibility (Fenger,
1999; Riga-Karandinos, 2005). Air quality has become a challenge in big cities because people are exposed
to high risk by pollutants. In developed countries, 75% population resides in urban areas whereas the urban
ratio in developing countries is more than 35% causing negative impacts on air quality (Wolf, 2002;
Agrawal et al. 2003; Vargas, 2003; Brajer et al. 2006; Oudient et al. 2006). Air pollution in cities has become more overwhelming due to increasing population. An abandoned increase
in population direct towards the industrialization without taking natural strategies about topography and
meteorological conditions. The population has become double today as compare to past some decades
(Baldasano et al. 2003). Population increase is directly proportional to: increase variety of mobile facilities
for transportation of products, increase needs of space, material, energy, food and comfort. These demands Volume 5, Issue 1, January-February 2023 IJFMR23011625 10 International Journal for Multidisciplinary Research (IJFMR)
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com which means the null hypothesis is rejected. The mean concentration of NOin government schools is 35.85
and in private schools are 29.70. The significance value in case of NO is less than 0.05 which means the
null hypothesis is rejected.The mean concentration of NOx in government schools is 36.29 and in private
schools are 32.92. The significance value in case of NOx is less than 0.05 which means the null hypothesis
is rejected.The mean concentration of O3 in government schools is 12.6 and in private schools are 8.5. The
significance value in case of O3 is less than 0.05 which means the null hypothesis is rejected.The mean
concentration of PM10 in government schools is 374.21 and in private schools are 275.40. The significance
value in case of PM10 is less than 0.05 which means the null hypothesis is rejected.The mean concentration
of PM2.5 in government schools is 298.12 and in private schools are 198.79. The significance value in case
of PM2.5 is less than 0.05 which means the null hypothesis is rejected. The mean concentration of VOCsin
government schools is 19.10 and in private schools are 8.4. The significance value in case of VOCs is less
than 0.05 which means the null hypothesis is rejected. Conclusion The air quality condition of government school is dangerous as compared to private school. The mean
concentrations of all the parameters were above the permissible values (PEQS) in both the private and
government schools. There should be proper check and balance and rules and regulations as these
conditions are severe, not healthy for the children studying there. Acknowledgement: The authors thank to University of Veterinary and Animal Sciences Lahore, Pakistan
for financial assistance for this study. International Journal for Multidisciplinary Research (IJFMR)
E-ISSN: 2582-2160 ● Website: www.ijfmr.com ● Email: editor@ijfmr.com The value
of CO2 was higher in government school as compare to the private schools. Inadequate ventilation system is
the cause of high concentration of CO2 in classrooms. Through opening and closing of windows repeatedly,
an inadequate air passage was occur caused high CO2 value. The other factor is no of pupils present in the
classrooms. (Pegas et al 2011) was found that the high concentration of CO2 is responsible for high
concentration of other indoor air pollutants in classrooms. The mean concentration of CO2 in government
schools is 438.31 and in private schools are 349.80. The significance value in case of CO2 is less than 0.05
which means the null hypothesis is rejected. The concentration is higher in government school than private
school.The mean concentration of NO2 in government schools is 19.11 and in private schools are 17.11. The
significance value in case of NO2 is less than 0.05 which means the null hypothesis is rejected. The mean
concentration of SO2 in government schools is 326.33 and in private schools are 278.36. The significance
value in case of SO2 is less than 0.05 which means the null hypothesis is rejected.The mean concentration of
COin government schools is 0.98 and in private schools are 0.76. The significance value in case of CO is
less than 0.05 which means the null hypothesis is rejected. The mean concentration of H2Sin government
schools is 15.81 and in private schools are 12.6. The significance value in case of H2S is less than 0.05 Volume 5, Issue 1, January-February 2023 11 IJFMR23011625 International Journal for Multidisciplinary Research (IJFMR) Baldasano JM, Valera E, Jimenez P. 2003. Air quality data from large cities.Sci Total Environ. 307: 141–
65. Barber N. 2008. World in Focus-Focus on Pakistan, World Almanac Library, Stamford. Barber N. 2008. World in Focus-Focus on Pakistan, World Almanac Library, Stamf Brajer V, Mead RW, Xiao F. 2006. Valuing the health impacts of air pollution in Hong Kong.J Asian Econ. 17: 85–102. Branis M, Rezacova P, Domasova M. 2005. The effect of outdoor air and indoor human activity on mass
concentrations of PM10, PM2.5, and PM1 in a classroom. Environ Res. 99: 143-149. Brunekreef B. Holtage ST. 2002. Air pollution and health. The Lancet.360: 1233. Brunekreef B. Holtage ST. 2002. Air pollution and health. The Lancet.360: 1233. Carrier M, Apparicio P, Se´guin AM, Crouse D. 2014. Ambient air pollution concentration in Montreal and
environmentalequity: Are children at risk at school? Case Studies on Transport Policy. 2: 61–69. Choo CP, Jalaludin J, Hamedon TR, Adam NM. 2015. Preschools’ indoor air quality and respiratory health
symptoms among preschoolers in Selangor. Procedia Environ Sci. 30: 303-308. Daisey JM, Angell WJ. 1999. Indoor air quality, ventilation and health sympto schools: an analysis of
existing information. Proceedings of the 8th In Conference on Indoor Air Quality and Climate –
Indoor Air. 99: 1-6. EEA, 2003. Exposure of population to exceedances of EU air quality standards. Fenger J. 1999. Urban air quality. Atmos Environ. 33: 4877–4900. Fenger J. 1999. Urban air quality. Atmos Environ. 33: 4877–4900. Fromme H, Heitmann D, Dietrich S, Schierl R, Körner W, Kiranoglu M, Zapf A, Twardella D. 2008. Air
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environments. Atmos Pollution Res. 6(6). 1113-1122. IJFMR23011625 Volume 5, Issue 1, January-February 2023 15
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https://zenodo.org/record/8196052/files/2023%20Kjerpeseth%20Metformin%20Versus%20Insulin%20and%20Risk%20of%20Major%20Congenital%20Malformations%20in%20Pregnancies%20With%20Type%202%20Diabetes.pdf
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Metformin Versus Insulin and Risk of Major Congenital Malformations in Pregnancies With Type 2 Diabetes – A Nordic Register-Based Cohort Study
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Metformin Versus Insulin and Risk of Major Congenital
Malformations in Pregnancies With Type 2 Diabetes: A Nordic
Register-Based Cohort Study Metformin Versus Insulin and Risk of Major Congenital
Malformations in Pregnancies With Type 2 Diabetes: A Nordic
Register-Based Cohort Study
Lars J. Kjerpeseth, Carolyn E. Cesta, Kari Furu, Anders Engeland, Mika Gissler, Hanne L. Gulseth, Øystein Karlsta ;
( )
p
g
ARTICLE HIGHLIGHTS
• Unlike insulin, metformin crosses the placenta, raising concern about its fetal safety. • This cohort study assesses the risk of major congenital malformations with metformin versus insulin in pregnan-
cies with type 2 diabetes since trials have been too small to give precise estimates of this risk. • In the study comprising four Nordic countries, evidence of an increased risk of any or cardiac malformations was
not found for early pregnancy exposure to metformin (alone or in addition to insulin) versus insulin alone. • The findings can guide prescribers and patients who are considering metformin treatment for type 2 diabetes in
pregnancy. Downloaded from http://diabetesjournals.org/care/article-pdf/46/8/1556/729552/dc230256.pdf by guest on 29 July 2023 ded from http://diabetesjournals.org/care/article-pdf/46/8/1556/729552/dc230256.pdf by guest on 29 July 2023 Metformin Versus Insulin and Risk of Major Congenital
Malformations in Pregnancies With Type 2 Diabetes: A Nordic
Register-Based Cohort Study
Lars J. Kjerpeseth, Carolyn E. Cesta, Kari Furu, Anders Engeland, Mika Gissler, Hanne L. Gulseth, Øystein Karlstad,
Maarit K. Leinonen, Laura Pazzagli, Helga Zoega, and Jacqueline M. Cohen
Diabetes Care 2023;46(8):1556–1564 | https://doi.org/10.2337/dc23-0256
ARTICLE HIGHLIGHTS
• Unlike insulin, metformin crosses the placenta, raising concern about its fetal safety.
• This cohort study assesses the risk of major congenital malformations with metformin versus insulin in pregnan-
cies with type 2 diabetes since trials have been too small to give precise estimates of this risk.
• In the study comprising four Nordic countries, evidence of an increased risk of any or cardiac malformations was
not found for early pregnancy exposure to metformin (alone or in addition to insulin) versus insulin alone.
Th
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2 di b t
i Metformin Versus Insulin and Risk of Major Congenital
Malformations in Pregnancies With Type 2 Diabetes: A Nordic
Register-Based Cohort Study
Lars J. Kjerpeseth, Carolyn E. Cesta, Kari Furu, Anders Engeland, Mika Gissler, Hanne L. Gulseth, Øystein Karlstad,
Maarit K. Leinonen, Laura Pazzagli, Helga Zoega, and Jacqueline M. Cohen
Diabetes Care 2023;46(8):1556–1564 | https://doi.org/10.2337/dc23-0256
ARTICLE HIGHLIGHTS
• Unlike insulin, metformin crosses the placenta, raising concern about its fetal safety. • This cohort study assesses the risk of major congenital malformations with metformin versus insulin in pregnan-
cies with type 2 diabetes since trials have been too small to give precise estimates of this risk. • In the study comprising four Nordic countries, evidence of an increased risk of any or cardiac malformations was
not found for early pregnancy exposure to metformin (alone or in addition to insulin) versus insulin alone. Th
fidi
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2 di b t
i OBJECTIVE To assess the risk of major congenital malformations with metformin versus insu-
lin in pregnancies with type 2 diabetes. 1Department of Chronic Diseases, Norwegian
Institute of Public Health, Oslo, Norway
2Centre for Pharmacoepidemiology, Karolinska
Institutet, Stockholm, Sweden
3Centre for Fertility and Health, Norwegian
Institute of Public Health, Oslo, Norway
4Department of Global Public Health and Primary
Care, University of Bergen, Bergen, Norway
5Department of Knowledge Brokers, Finnish
Institute for Health and Welfare, Helsinki, Finland
6Academic Primary Health Care Centre, Region
Stockholm, Stockholm, Sweden
7Department of Molecular Medicine and Surgery,
Karolinska Institutet, Stockholm, Sweden
8Research Centre for Child Psychiatry, University
of Turku,Turku, Finland
9Division of Mental and Physical Health, Norwegian
Institute of Public Health, Oslo, Norway
10School of Population Health, Faculty of Medicine
and Health, UNSW Sydney, Sydney, Australia
11Centre of Public Health Sciences, Faculty of
Medicine, University of Iceland, Reykjavık, Iceland
Corresponding author: Lars J. Kjerpeseth, lars. kjerpeseth@fhi.no Metformin Versus Insulin and
Risk of Major Congenital
Malformations in Pregnancies
With Type 2 Diabetes: A Nordic
Register-Based Cohort Study
Diabetes Care 2023;46:1556–1564 | https://doi.org/10.2337/dc23-0256 Lars J. Kjerpeseth,1 Carolyn E. Cesta,2
Kari Furu,1,3 Anders Engeland,1,4
Mika Gissler,5,6,7,8 Hanne L. Gulseth,9
Øystein Karlstad,1 Maarit K. Leinonen,5
Laura Pazzagli,2 Helga Zoega,10,11 and
Jacqueline M. Cohen1,3 Lars J. Kjerpeseth,1 Carolyn E. Cesta,2
Kari Furu,1,3 Anders Engeland,1,4
Mika Gissler,5,6,7,8 Hanne L. Gulseth,9
Øystein Karlstad,1 Maarit K. Leinonen,5
Laura Pazzagli,2 Helga Zoega,10,11 and
Jacqueline M. Cohen1,3 ORIGINAL ARTICLE RESULTS Of 3,734,125 infants in the source population, 25,956 were exposed to metformin
or insulin in the first trimester, and 4,023 singleton infants were included. A mal-
formation was diagnosed in 147 (4.7%) of 3,145 infants with exposure to any
metformin (alone or in addition to insulin) and 50 (5.7%) of 878 infants with ex-
posure to insulin alone (RR 0.84, 95% CI 0.46–1.54). Among 2,852 infants exposed
to metformin alone and 293 infants exposed to metformin in addition to insulin
127 (4.4%) and 20 (6.8%), respectively, had a malformation. The adjusted risk
was not increased for either metformin alone (0.83, 0.44–1.58) or both metfor-
min and insulin (0.98, 0.56–1.69) versus insulin alone. Corresponding RRs for car-
diac malformations were 1.01 (0.55–1.84) for any metformin, 0.92 (0.47–1.81) for
metformin alone, and 1.72 (0.76–3.91) for both metformin and insulin. 11Centre of Public Health Sciences, Faculty of
Medicine, University of Iceland, Reykjavık, Iceland Corresponding author: Lars J. Kjerpeseth, lars. kjerpeseth@fhi.no Corresponding author: Lars J. Kjerpeseth, lars. kjerpeseth@fhi.no Received 12 February 2023 and accepted 27 May
2023 Received 12 February 2023 and accepted 27 May
2023 RESEARCH DESIGN AND METHODS This cohort study used four Nordic countries’ nationwide registers of live and
stillborn infants exposed to metformin or insulin during first trimester organo-
genesis. Main exclusion criteria were type 1 diabetes, polycystic ovary syndrome,
fertility treatment, and exposure to other diabetes drugs. Adjusted risk ratios
(RRs) and 95% CIs were estimated for any and cardiac malformations. CONCLUSIONS No evidence of an increased malformation risk with metformin versus insulin in
the first trimester was found. Results should be interpreted with caution since in-
formation on glycemic control was missing. ARTICLE HIGHLIGHTS • Unlike insulin, metformin crosses the placenta, raising concern about its fetal safety. • Unlike insulin, metformin crosses the placenta, raising concern about its fetal safety. • This cohort study assesses the risk of major congenital malformations with metformin versus insulin in pregnan-
cies with type 2 diabetes since trials have been too small to give precise estimates of this risk. cies with type 2 diabetes since trials have been too small to give precise estimates of this risk. • In the study comprising four Nordic countries, evidence of an increased risk of any or cardiac malformations was
not found for early pregnancy exposure to metformin (alone or in addition to insulin) versus insulin alone. • The findings can guide prescribers and patients who are considering metformin treatment for type 2 diabetes in • In the study comprising four Nordic countries, evidence of an increased risk of any or cardiac malformations was
not found for early pregnancy exposure to metformin (alone or in addition to insulin) versus insulin alone. Th
fidi
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i
h
id
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2 di b
i • In the study comprising four Nordic countries, evidence of an increased risk of any or cardiac malformations was
not found for early pregnancy exposure to metformin (alone or in addition to insulin) versus insulin alone. Th
fidi
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2 di b t
i y p
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(
)
• The findings can guide prescribers and patients who are considering metformin treatment for type 2 diabetes in
pregnancy. • The findings can guide prescribers and patients who are considering metformin treatment for type 2 diabetes in
pregnancy. 1556 Diabetes Care Volume 46, August 2023 1Department of Chronic Diseases, Norwegian
Institute of Public Health, Oslo, Norway
2Centre for Pharmacoepidemiology, Karolinska
Institutet, Stockholm, Sweden
3Centre for Fertility and Health, Norwegian
Institute of Public Health, Oslo, Norway
4Department of Global Public Health and Primary
Care, University of Bergen, Bergen, Norway
5Department of Knowledge Brokers, Finnish
Institute for Health and Welfare, Helsinki, Finland
6Academic Primary Health Care Centre, Region
Stockholm, Stockholm, Sweden
7Department of Molecular Medicine and Surgery,
Karolinska Institutet, Stockholm, Sweden
8Research Centre for Child Psychiatry, University
of Turku,Turku, Finland
9Division of Mental and Physical Health, Norwegian
Institute of Public Health, Oslo, Norway
10School of Population Health, Faculty of Medicine
and Health, UNSW Sydney, Sydney, Australia
11Centre of Public Health Sciences, Faculty of
Medicine, University of Iceland, Reykjavık, Iceland
Corresponding author: Lars J. Kjerpeseth, lars.
kjerpeseth@fhi.no This article contains supplementary material online
at https://doi.org/10.2337/figshare.23264624. Insulin has traditionally been the recom-
mended glucose-lowering drug in preg-
nancy since it has well-established efficacy
and safety (6). However, insulin is costly, is
cumbersome to administer, and requires
frequent glucose measurements. Metfor-
min, the first-line drug for type 2 diabetes
outside of pregnancy, is increasingly being
used during pregnancy (7). Since the intent of the study was to esti-
mate the effect of metformin on major
congenital malformations using insulin as
an active comparator, we focused on in-
fants born to mothers with type 2 diabe-
tes, thereby reducing the influence of
confounding by indication. Because of
underrecording of the diagnosis in the
register data, we defined type 2 diabetes
in mothers as the absence of type 1 diabe-
tes or other chronic diabetes than type 2
diabetes, polycystic ovary syndrome, and
assisted reproductive treatment, condi-
tions for which only one drug is indicated. Thus, infants were excluded if their
mother had been dispensed insulin be-
tween 90 days before last menstrual pe-
riod and end of first trimester and had a
recorded diagnosis of type 1 diabetes in
specialist health care or in the Norwegian
medical birth register before delivery. Similarly, infants born to mothers with
other types of chronic diabetes (except
type 2 diabetes), polycystic ovary syndrome,
or assisted reproductive treatment were ex-
cluded using diagnoses recorded in special-
ist (mainly) and primary health care before
delivery. Assisted reproductive treatment
was additionally identified from the medical
birth registers, diagnoses or procedures from
90 days before last menstrual period to deliv-
ery, or the dispensing of all three of the fol-
lowing drug classes from 90 days before last
menstrual period and end of first trimester:
gonadotropin-releasing hormone analogs,
gonadotropins, and human chorionic
gonadotropin. Unlike insulin, metformin crosses the
placenta, raising concerns of potential ter-
atogenicity (6,8). Randomized controlled
trials (RCTs) have not been able to ade-
quately investigate this risk because of
small sample sizes and because randomi-
zation to metformin typically occurs after
the first trimester, when major organs are
formed and are most sensitive to the de-
velopment of congenital malformations
(6,9). Observational studies on metformin
exposure in early pregnancy have been re-
assuring but scarce, especially for type 2
diabetes (10,11). The evidence on spe-
cific malformations is even more limited
because of small study sizes (11). Study Population y
p
We included singleton, live-born or still-
born infants with prenatal exposure to
insulin or metformin in first trimester
(Fig. 1). Exposure was defined as at least
one recorded dispensing, meaning a filled
prescription at the pharmacy, of metfor-
min (Anatomical Therapeutic Chemical
code A10BA02) or insulin (Anatomical
Therapeutic Chemical codes starting with
A10A) during the period. First trimester
was defined as the start date of last men-
strual period before pregnancy to 97 days
after last menstrual period. It was calcu-
lated by subtracting the gestational age
(as recorded in the medical birth regis-
ters, assessed primarily by ultrasound)
from the delivery date. Infants were not
included if the recorded gestational age
of the pregnancy was missing, <22 weeks,
>44 weeks, or implausible based on birth
weight (sex-specific birth weight z score
>4 SDs and gestational age <35 weeks)
because of uncertainty regarding the tim-
ing of the first trimester (13).Terminations
were excluded since we only had informa-
tion on induced abortions from 12 weeks
onward for Norway. To generate evidence, we pooled individ-
ually linked register data from four Nordic
countries. The nationwide registers in Nor-
dic countries provide complete coverage of
live births and stillbirths with accurate mea-
surement of gestational age, dispensed pre-
scription drugs, and recorded diagnoses in
mothers and infants (12). We compared
the risk of any and cardiac major congenital
malformations with prenatal exposure to
metformin (alone or in addition to insulin)
versus insulin alone in the first trimester in
pregnant women with type 2 diabetes. We
excluded those with other indications, such
as polycystic ovary syndrome and assisted
reproductive treatment, to minimize con-
founding by indication. This article contains supplementary material online
at https://doi.org/10.2337/figshare.23264624. While
the American Diabetes Association guide-
lines recommend insulin before metformin
for type 2 diabetes in pregnancy (8), the
prescribing advice is not consistent among
guidelines, product labels, and other drug
information sources (8).Thus, larger studies
are needed to explore the safety of first tri-
mester metformin exposure in pregnant
women with type 2 diabetes and particu-
larly to assess specific concerns, such as car-
diac malformations, the organ system most
often affected by malformations (6,11). This article contains supplementary material online
at https://doi.org/10.2337/figshare.23264624. This article contains supplementary material online
at https://doi.org/10.2337/figshare.23264624. This article contains supplementary material online
at https://doi.org/10.2337/figshare.23264624. This article contains supplementary material online
at https://doi.org/10.2337/figshare.23264624. This article is featured in a podcast available at
diabetesjournals.org/care/pages/diabetes_care_
on_air. The prevalence of type 2 diabetes in reproductive-age women and, consequently,
during pregnancy has grown rapidly in past decades (1–3). Type 2 diabetes is associ-
ated with several adverse birth outcomes, including an up to threefold increased risk
of nonchromosomal major congenital malformations (4). This risk, however, can be
substantially mitigated by appropriate glycemic control (2,4,5). © 2023 by the American Diabetes Association. Readers may use this article as long as the
work is properly cited, the use is educational
and not for profit, and the work is not altered. More information is available at https://www
.diabetesjournals.org/journals/pages/license. diabetesjournals.org/care Kjerpeseth and Associates 1557 Finland (1996–2016), Iceland (2004–2017),
Norway (2005–2020), and Sweden (July
2006–2019). Using personal identity num-
bers unique to all residents, mother and
infant pairs were linked to nationwide
registers on filled prescriptions and spe-
cialist health care in all countries. We also
linked to registers on cause of death (all
countries except for Finland and mothers
in Iceland) and educational attainment (all
countries except Finland), to primary care
data registers in Norway and Finland, and
to the Finnish Register of Congenital
Anomalies. In the Nordic health registers,
the sex assigned at birth is reported, while
gender identity is not available. Thus, in
this article, we define women as human
females of any gender identity. Further
details of the registers are available in
the Supplementary Material. Finland (1996–2016), Iceland (2004–2017),
Norway (2005–2020), and Sweden (July
2006–2019). Using personal identity num-
bers unique to all residents, mother and
infant pairs were linked to nationwide
registers on filled prescriptions and spe-
cialist health care in all countries. We also
linked to registers on cause of death (all
countries except for Finland and mothers
in Iceland) and educational attainment (all
countries except Finland), to primary care
data registers in Norway and Finland, and
to the Finnish Register of Congenital
Anomalies. In the Nordic health registers,
the sex assigned at birth is reported, while
gender identity is not available. Thus, in
this article, we define women as human
females of any gender identity. Further
details of the registers are available in
the Supplementary Material. Definition of Outcomes Definition of Outcomes
The outcome was major congenital mal-
formations in the infant, diagnosed within
1 year of birth and recorded in medical
birth, patient, malformation, or cause of
death registers. The definition was aligned
as closely as possible with the classification Definition of Exposure and
Comparison Groups We compared infants with prenatal ex-
posure to metformin in the first trimes-
ter with those exposed to insulin alone. Infants with prenatal exposure to both
metformin and insulin during the first
trimester were allocated to the metfor-
min group, which was then divided into
three exposure groups compared with
insulin alone in the primary analyses:
exposure to any metformin (alone or in
addition to insulin), metformin in addi-
tion to insulin, and metformin alone. To focus on outcomes associated with
metformin or insulin, we excluded infants
diagnosed with a teratogenic infection
(i.e., rubella, cytomegalovirus, toxoplas-
mosis), chromosomal anomaly, microdele-
tion, or genetic syndrome within 1 year of
birth. We also excluded infants with po-
tential exposure to other glucose-lowering
drugs or known teratogenic drugs in the
first trimester (Supplementary Table 1). Statistical Analyses Statistical Analyses
Data from the four countries were har-
monized in a common data model and
individually pooled into one cohort be-
fore the analyses were performed (15). To adjust for differences in baseline co-
variates between comparison groups,
we used propensity score fine stratifica-
tion with up to 50 strata and at least 3
exposed and nonexposed in each stra-
tum. After stratification, Mantel-Haenszel
pooling was used to estimate relative
risks (risk ratios [RRs]) with 95% CIs for
prenatal exposure to metformin versus
insulin. This method performs better
than traditional propensity score meth-
ods when the prevalence of the expo-
sure is low (16). All covariates previously
listed were included in the propensity
score model. Maternal country of birth,
cohabitation with a partner, and BMI had
missing values (0.6%, 4.3%, and 24.0%, re-
spectively) and were imputed 100 times
using predicted mean matching and RESEARCH DESIGN AND METHODS
Study Setting and Data Sources RESEARCH DESIGN AND METHODS
Study Setting and Data Sources This cohort study was based on data from
the nationwide medical birth registers of 1558
Malformation Risk With Metformin in Pregnancy Diabetes Care Volume 46, August 2023 Figure 1—Selection of the study population of infants from nationwide medical birth registers of Finland (1996–2016), Iceland (2004–2017), Nor-
way (2005–2020), and Sweden (July 2006–2019). Exclusions are sequential in the order listed. The metformin group included infants with prenatal
exposure to metformin alone or in addition to insulin. MCM, major congenital malformation. Figure 1—Selection of the study population of infants from nationwide medical birth registers of Finland (1996–2016), Iceland (2004–2017), Nor-
way (2005–2020), and Sweden (July 2006–2019). Exclusions are sequential in the order listed. The metformin group included infants with prenatal
exposure to metformin alone or in addition to insulin. MCM, major congenital malformation. included in a propensity score analysis
that was conducted in each imputed data
set. The estimates were then combined
using Rubin’s rules (17). We used Stata SE
17 for Windows statistical software (Sta-
taCorp, College Station, TX) to analyze the
data. diagnosis codes recorded from 1 year
before last menstrual period to end of
pregnancy, except for skin and vaginal
infections, which were identified from
1 year before last menstrual period to
end of first trimester. For Norway and
Finland, comorbidities were also defined
using drug reimbursement indication co-
des within the same time window. See
Supplementary Table 3 for the definitions
of the covariates. in the European Commission’s network of
population-based registries for the epidemi-
ological surveillance of congenital anomalies
(EUROCAT Guide 1.4) (14) (Supplementary
Table 2).We considered any major congeni-
tal malformation as the primary outcome
and the subgroup of major cardiac mal-
formations as the secondary outcome. For Finland, we only considered validated
diagnoses from the Finnish Register of
Congenital Malformations. To increase di-
agnostic validity for Iceland, Norway, and
Sweden, we required at least two diagno-
sis codes from the same subgroup to be
recorded on separate visit dates if the mal-
formation was only diagnosed in outpa-
tient specialist care. Sensitivity Analyses We conducted several sensitivity analyses,
each for both the primary and secondary
outcomes. First, a new-user design was
implemented by including only infants
born to mothers with no dispensing of
metformin or insulin from 90 days before
last menstrual period to end of gestational
week 6 (18).The aim of this approach was
to further exclude infants born to mothers
dispensed metformin for polycystic ovary
syndrome or assisted reproductive treat-
ment or insulin for type 1 diabetes.The re-
sulting study population thus included
infants born to mothers with type 2 diabe-
tes who did not receive pharmacological
treatment until after gestational week 6
or if type 2 diabetes in the mother was
first diagnosed during prenatal care. Sec-
ond, the study population was restricted
to women with at least one diagnosis of
type 2 diabetes recorded at any time in
the available look-back period before or
on the date of birth. Third, we conducted
a sensitivity analysis requiring at least two
dispensations of the drugs of interest to
reduce the impact of potential exposure
misclassification, since infants of mothers RESULTS In total, the source population included
3,734,125 infants. We identified 25,956
singleton, live-born or stillborn infants
with prenatal exposure to metformin or
insulin in the first trimester. After exclu-
sions, 4,023 infants remained in the fi-
nal study population: 878 (21.8%) were
exposed to insulin alone, while 3,145
(78.2%) pregnancies were exposed to any
metformin, either alone (2,852 infants) or
in addition to insulin (293 infants). The
most prevalent reasons for exclusion were
polycystic ovary syndrome and assisted re-
productive treatment in the mother among
those exposed to metformin and maternal
type 1 diabetes among those exposed to in-
sulin alone. Of note, Finland was the only
country contributing to the study popula-
tion before 2004 (152 infants). For both any and cardiac malforma-
tions, the sensitivity analyses were mostly
in line with the primary analyses (Table
3). In infants of mothers with polycystic
ovary syndrome, the risk of any (RR 1.12,
95% CI 0.92–1.36) and cardiac (1.12,
0.82–1.53) malformations did not differ
significantly with and without metformin
exposure. However, among infants of
mothers with a recorded type 2 diabetes
diagnosis, metformin (alone or in addition
to insulin) was associated with an increased
risk for cardiac malformations versus insulin
alone. Again, the estimate was uncertain
(2.03, 0.89–4.62). Mothers of infants with prenatal ex-
posure to insulin were generally older,
more often multiparous, and more of-
ten born in a non-Nordic country than
mothers of infants with prenatal expo-
sure to metformin (Table 1). All maternal
comorbidities and comedications were
generally more common in the insulin
group, especially chronic hypertension. BMI $30 kg/m2, lower education, and
no use of folic acid before or during preg-
nancy were also more prevalent in the
insulin group. Information on BMI, edu-
cation, and folate use, as well as smok-
ing, was missing for many mothers. To elucidate the two results suggesting
a potential increased risk of cardiac mal-
formations with exposure to metformin,
we performed a supplementary explor-
atory analysis. The distribution of sub-
groups of cardiac malformations among
infants exposed to insulin alone and
those exposed to any metformin was in-
vestigated. Right ventricular outflow ob-
struction defects were relatively more
common in the latter group, but this
may be explained by a lower number of
cases overall in the insulin group be-
cause of its smaller size compared with
metformin (Supplementary Fig. 1). Covariates
d To reduce confounding, we adjusted for
country and year of birth and maternal
characteristics including age at delivery,
country of birth (Nordic/non-Nordic or
Finnish/non-Finnish citizenship), and co-
habitation with a partner. Potential expo-
sure in the first trimester to suspected
teratogenic drugs, glucocorticoids, lipid-
modifying drugs, and antihypertensive
drugs was also adjusted for. Furthermore,
we adjusted for maternal BMI at the start
of pregnancy; maternal comorbidities;
potential complications of type 2 diabe-
tes, such as chronic hypertension, car-
diovascular disease, and other diabetes
complications; epilepsy; and severe mental
illness. Comorbidities were defined by Kjerpeseth and Associates 1559 diabetesjournals.org/care these data, which were used under li-
cense for the current study and, there-
fore, are not publicly available. cardiac malformations occurred in 63
(2.0%) and 18 (2.1%) infants exposed to
any metformin and insulin, respectively. After adjustment, neither any metformin
(RR 1.01, 95% CI 0.55–1.84) nor metfor-
min alone (0.92, 0.47–1.81) was associ-
ated with an increased risk of cardiac
malformations compared with insulin
alone (Table 2). In a corresponding anal-
ysis, an increased risk was observed for
exposure to both metformin and insulin;
however, the estimate was uncertain
(1.72, 0.76–3.91). with only a single dispensing of metformin
and/or insulin in first trimester may have
had limited or no prenatal exposure. Fourth,
a complete case analysis was conducted to
check the consistency with the results ob-
tained from the multiple imputation ap-
proach used in the primary analyses. Fifth,
smoking in early pregnancy, folic acid use
before and during pregnancy, and maternal
education were not included in the primary
analyses, since information on each variable
was unavailable for at least one of the in-
cluded countries. In a sensitivity analysis,
missing values of these variables were im-
puted 100 times using the same approach
as in the primary analyses. Sixth, high-
dimensional propensity score analyses
were undertaken to identify potential
proxies for unmeasured confounders,
such as glycemic control in mothers, and
to explore the impact of these on the ob-
served associations (19). The model in-
cluded the top 100 empirically selected
covariates and the following predefined
covariates: country and year of birth,
maternal age, country of birth, cohabita-
tion with a partner, and BMI.The empiri-
cally selected covariates were identified
from inpatient and outpatient diagnosis
codes, inpatient and outpatient proce-
dure codes, primary care codes, and
drug codes recorded before pregnancy. RESULTS A major congenital malformation oc-
curred in 147 (4.7%) infants with prenatal
exposure to metformin (alone or in addi-
tion to insulin) and 50 (5.7%) infants with
prenatal exposure to insulin alone. The
crude RR was 0.82 (95% CI 0.60–1.12) for
metformin versus insulin, and, after ad-
justment for confounders, the estimated
RR was 0.84 (0.46–1.54) (Table 2). Covariates
d Seventh, to further disentangle the po-
tential teratogenic effect of metformin
from that of diabetes, we selected a dif-
ferent study population of mothers with
a diagnosis of polycystic ovary syndrome
and no pregestational diabetes before
delivery. The exclusion criteria were the
same as for the primary study population
except that we also excluded mothers
with a diagnosis of type 2 diabetes but not
mothers who received assisted reproduc-
tive treatment. We compared the malfor-
mation risk with and without metformin
exposure after adjusting for the same co-
variates as in the primary analyses. Ethics Statement Ethics Statement
The research was approved by applicable
ethics review boards and/or register control-
lers in all study countries (Supplementary
Table 4). CONCLUSIONS Among the 4,023 infants included in this
large Nordic cohort study, we found no
evidence of an increased risk of either
any or major cardiac malformations with
prenatal exposure to metformin com-
pared with insulin alone in pregnancies
affected by type 2 diabetes. These null
findings are in line with other observa-
tional studies (11,20–25). One of the
largest previous studies comparing first
trimester exposure to metformin with in-
sulin was a Taiwanese population-based
cohort study of 1,166 infants born to
mothers with type 2 diabetes. The results Among the 2,852 infants prenatally
exposed to metformin alone, 127 (4.4%)
had a malformation, and among 293 in-
fants exposed to metformin and insulin,
20 (6.8%) had a malformation. The rela-
tive risk for the comparison with insulin
alone did not suggest an increased risk
associated with metformin after adjust-
ing for confounders (Table 2). Data and Resource Availability y
The data that support the findings of this
study are available from the data custo-
dians of the Nordic health registers, but
restrictions apply to the availability of The malformations were most com-
mon in the cardiac organ system. Major Diabetes Care Volume 46, August 2023 1560
Malformation Risk With Metformin in Pregnancy Table 1—Maternal and pregnancy characteristics in the study population of infants with prenatal exposure to metformin
alone or in addition to insulin or insulin alone
Metformin alone or in addition to insulin, n (%)
Insulin alone, n (%)
Total
3,145 (100)
878 (100)
Infant’s country of birth
Finland
1,169 (37.2)
242 (27.6)
Iceland
275 (8.7)
32 (3.6)
Norway
960 (30.5)
168 (19.1)
Sweden
741 (23.6)
436 (49.7)
Infant’s year of birth*
1996–2006
351 (11.2)
226 (25.7)
2007–2009
617 (19.6)
137 (15.6)
2010–2012
578 (18.4)
118 (13.4)
2013–2015
670 (21.3)
184 (21.0)
2016–2020
929 (29.5)
213 (24.3)
Maternal age (years)
<25
218 (6.9)
43 (4.9)
25–29
910 (28.9)
166 (18.9)
30–34
1,106 (35.2)
295 (33.6)
35–39
707 (22.5)
276 (31.4)
$40
204 (6.5)
98 (11.2)
Parity
Nulliparous
1,461 (46.9)
178 (20.6)
Primiparous
965 (30.9)
298 (34.5)
Multiparous
692 (22.2)
387 (44.8)
Missing, n
27
15
Maternal education†
Compulsory
275 (16.7)
96 (18.3)
Preuniversity
714 (43.5)
271 (51.5)
University
653 (39.8)
159 (30.2)
Missing, n
1,503
352
Married/cohabitation with partner‡
2,899 (92.6)
756 (87.0)
Missing, n
16
9
Non-Nordic birth country of mother§
663 (22.1)
345 (40.3)
Missing, n
150
22
BMI in early pregnancy (kg/m2)jj
<18.5
27 (1.1)
<5‡‡
18.5–24
569 (23.2)
83 (13.8)
25–29
620 (25.2)
152 (25.2)
$30
1,240 (50.5)
366 (60.7)
Missing, n
689
<277
Smoking in early pregnancy¶
225 (8.3)
83 (10.5)
Missing, n
448
84
Folate use before/during pregnancy#
797 (40.3)
126 (19.8)
Missing, n
1,169
242
Maternal comorbidities**
Skin/vaginal infection
91 (2.9)
19 (2.2)
Diabetic complication
77 (2.4)
25 (2.8)
Chronic hypertension
248 (7.9)
85 (9.7)
Cardiovascular disease
7 (0.2)
6 (0.7)
Epilepsy
23 (0.7)
8 (0.9)
Severe mental illness
31 (1.0)
14 (1.6)
Maternal comedication††
Suspected teratogenic drugs
190 (6.0)
57 (6.5)
Antihypertensive drugs
144 (4.6)
59 (6.7)
Lipid-modifying agents
57 (1.8)
17 (1.9)
Glucocorticoids
68 (2.2)
26 (3.0)
*Finland contributed births from 1996 to 2016, Iceland from 2004 to 2017, Norway from 2005 to 2020, and Sweden from July 2006 to 2019. Data and Resource Availability †Education was not available for Finland and missing for 21% in Norway and 17% in Sweden in the study population. ‡Married/cohabitation Table 1—Maternal and pregnancy characteristics in the study population of infants with prenatal exposure to metformin
alone or in addition to insulin or insulin alone
Metformin alone or in addition to insulin n (%)
Insulin alone n (%) and pregnancy characteristics in the study population of infants with prenatal exposure to metformin
on to insulin or insulin alone Table 1—Continued However,
there were only seven cases in the insulin
comparison group, and the result was not
statistically significant. The other analyses
on cardiac malformations did not suggest
a significant harmful effect of metformin. An even larger Finnish nationwide co-
hort study by Brand et al. (26) of 10,129
infants also compared metformin with in-
sulin in pregnancy but was not exclusive
to type 2 diabetes and had median expo-
sure after the first trimester. Their point
estimates suggested a somewhat more
protective effect of metformin than ours. We excluded infants born to mothers
with other indications than type 2 diabe-
tes to make the comparison arms more
similar regarding indication and glycemic
control, which may explain why our point
estimates are closer to null. Our study has a cohort design based on
pooled register data from the full popula-
tion of infants and mothers across four
Nordic countries with universal and tax-
funded health care. Personal identity
numbers unique to all residents enable in-
dividual-level linkage of the registers to
provide long and complete follow-up of
both mother and infant (12). Unlike many
other studies, we focused on prenatal ex-
posure during organogenesis in the first
trimester. Our data include better mea-
surement of gestational age than most
large data sets since the information is pri-
marily based on ultrasound routinely of-
fered during pregnancy. To avoid selection
bias, we included stillbirths in addition to
live births, which is rarely done by other
studies. In total, >3,000 infants with pre-
natal exposure to metformin were in-
cluded in the study. To our knowledge,
this study is the largest to date to investi-
gate the risk of major congenital malfor-
mations associated with metformin use in The results of RCTs are similarly reassur-
ing that there is no strong teratogenic ef-
fect of metformin. A 2021 meta-analysis of
nine RCTs comparing metformin alone or
in combination with insulin with insulin
alone in pregnancies affected by gesta-
tional diabetes mellitus or type 2 diabetes
did not find a difference in the risk of con-
genital malformations in infants (9). Table 1—Continued Table 1—Continued 55% in Iceland, 48% in Norway, 6% in Sweden, and 13% in Finland. ¶Smoking status was not available for Iceland and missing for 13% in Nor-
way, 4% in Sweden, and 3% in Finland in the study population. #Folate use before pregnancy was not available for Finland and Sweden. Fo-
late use during pregnancy was not available for Finland and was assumed missing for Iceland since we were not able to adequately capture
use from prescribed drug dispensations. **The look-back period for maternal comorbidities was from 365 days before last menstrual period
to end of first trimester (97 days after last menstrual period) for skin/vaginal infections and from 365 days before last menstrual period to
birth for the other comorbidities. Comorbidities are defined in Supplementary Table 3. ††The look-back period for maternal comedication
was 90 days before last menstrual period to end of first trimester. Comedications are defined in Supplementary Table 3. ‡‡Numbers between
1 and 4 are not shown to protect confidentiality. the first trimester generally and in type 2
diabetes specifically. Because of the large
sample size, we were able to include
analyses on cardiac malformations, which
have not been well captured in the exist-
ing evidence (11). Diabetes in Pregnancy (MiTy) trial compar-
ing metformin with placebo, in addition to
insulin, among pregnant patients with
type 2 diabetes (27). However, on aver-
age, the randomization occurred after
the first trimester in this and the other
trials (9). were comparable to ours regarding any
congenital malformations. However, the
authors did not analyze more specific
malformations, such as the cardiac sub-
group, possibly because of a small sample
size (20). An exception is a European population-
based case-control study that reported a
signal for an increased risk of pulmonary
valve atresia associated with prenatal
metformin exposure (21). The authors
suggested that the finding might be by
chance because of multiple testing of
many congenital malformations. We did
not have a sufficient sample size to test
for single malformations. However, for
prenatal exposure to metformin in addi-
tion to insulin, the point estimate sug-
gested a moderately increased risk of
cardiac malformations compared with in-
sulin alone. Similarly, in the sensitivity
analysis restricted to infants of mothers
with recorded type 2 diabetes, exposure
to any metformin was associated with
twice the risk of cardiac malformations
compared with insulin alone. Downloaded from http://diabetesjournals.org/care/article-pdf/46/8/1556/729552/dc230256.pdf by guest on 29 July 2023 *Finland contributed births from 1996 to 2016, Iceland from 2004 to 2017, Norway from 2005 to 2020, and Sweden from July 2006 to 2019. †Education was not available for Finland and missing for 21% in Norway and 17% in Sweden in the study population. ‡Married/cohabitation
status was missing for 0.6% of the study population. §Non-Nordic birth country of mother was missing for 4.3% of the study population, in-
cluding 12% in Finland (for which only Finnish/non-Finnish maternal citizenship was available in the research material). jjBMI was missing for 1561 Kjerpeseth and Associates diabetesjournals.org/care Table 1—Continued ‡Infants born to mothers with one dispensing of metfor-
min (alone or in addition to insulin) in the first trimester and at least one other dispensing of metformin at any time during pregnancy were
compared with infants born to mothers with one dispensing of insulin alone in the first trimester and at least one other dispensing of insulin
at any time during pregnancy. §Only mother-child pairs with complete information on covariates were included in the analysis. jjMaternal ed-
ucation level at delivery, folate use before pregnancy, folate use during pregnancy, and smoking in early pregnancy were added as covariates
in the model used in the primary analyses. Missing values were imputed 100 times as in the primary analyses. ¶High-dimensional propensity
score with predefined plus top 100 empirically selected covariates. The 200 most prevalent codes were identified in six dimensions of codes
recorded within 1 year of the start of pregnancy: inpatient and outpatient diagnosis codes, inpatient and outpatient procedure codes, primary
care codes, and drug codes. We selected the top 100 binary empirical codes for inclusion in the propensity score model in addition to coun-
try and year of childbirth and maternal age, BMI, cohabitation with a partner, and country of birth (non-Nordic country/non-Finnish citizen). Missing values were imputed 100 times as in the primary analyses. #Infants born to mothers with a diagnosis of polycystic ovary syndrome
and no pregestational diabetes before delivery. We compared the malformation risk with and without metformin exposure after adjusting for
the same covariates as in the primary analyses. **Adjusted for country and year of birth of infant; maternal characteristics including age, coun-
try of birth, cohabitation with a partner, BMI, epilepsy, severe mental illness, chronic hypertension, cardiovascular disease, skin/vaginal infections,
and other diabetic complications; and prenatal exposure to suspected teratogenic drugs, glucocorticoids, lipid-modifying drugs, and antihyperten-
sive drugs. Table 1—Continued ‡Infants born to mothers with one dispensing of metfor-
min (alone or in addition to insulin) in the first trimester and at least one other dispensing of metformin at any time during pregnancy were
compared with infants born to mothers with one dispensing of insulin alone in the first trimester and at least one other dispensing of insulin
at any time during pregnancy. §Only mother-child pairs with complete information on covariates were included in the analysis. jjMaternal ed-
ucation level at delivery, folate use before pregnancy, folate use during pregnancy, and smoking in early pregnancy were added as covariates
in the model used in the primary analyses. Missing values were imputed 100 times as in the primary analyses. ¶High-dimensional propensity
score with predefined plus top 100 empirically selected covariates. The 200 most prevalent codes were identified in six dimensions of codes
recorded within 1 year of the start of pregnancy: inpatient and outpatient diagnosis codes, inpatient and outpatient procedure codes, primary
care codes, and drug codes. We selected the top 100 binary empirical codes for inclusion in the propensity score model in addition to coun-
try and year of childbirth and maternal age, BMI, cohabitation with a partner, and country of birth (non-Nordic country/non-Finnish citizen). Missing values were imputed 100 times as in the primary analyses. #Infants born to mothers with a diagnosis of polycystic ovary syndrome
and no pregestational diabetes before delivery. We compared the malformation risk with and without metformin exposure after adjusting for
the same covariates as in the primary analyses. **Adjusted for country and year of birth of infant; maternal characteristics including age, coun-
try of birth, cohabitation with a partner, BMI, epilepsy, severe mental illness, chronic hypertension, cardiovascular disease, skin/vaginal infections,
and other diabetic complications; and prenatal exposure to suspected teratogenic drugs, glucocorticoids, lipid-modifying drugs, and antihyperten-
sive drugs. 552/dc230256.pdf by guest on 29 July 2023 In our study population, none had pulmo-
nary valve atresia. However, right ventric-
ular malformations were relatively more
common among infants with prenatal ex-
posure to metformin (Supplementary Fig. 1). This group of malformations has been
found to be associated with pregesta-
tional diabetes (28). Since the increase
was only seen in those with both metfor-
min and insulin exposure or those with
type 2 diabetes recorded, but not in other
analyses, we suggest that this result may
be due to residual confounding. Table 1—Continued Table 3—Sensitivity analyses for the risk of any and cardiac major congenital malformations in infants with prenatal
exposure to metformin alone or in addition to insulin compared with insulin alone
Outcome prevalence, n (%)
RR (95% CI)
Exposure to metformin alone
or in addition to insulin
Exposure to insulin alone
(or no metformin#)
Crude
Adjusted**
Any major congenital malformation
New-user design*
24 of 361 (6.7)
35 of 569 (6.2)
1.08 (0.65–1.79)
1.07 (0.53–2.13)
Recorded type 2 diabetes†
53 of 915 (5.8)
27 of 406 (6.7)
0.87 (0.56–1.36)
0.93 (0.55–1.57)
$2 dispensations in pregnancy‡
58 of 1,248 (4.7)
50 of 829 (6.0)
0.77 (0.53–1.11)
0.83 (0.42–1.64)
Complete case analysis§
115 of 2,302 (5.0)
37 of 580 (6.4)
0.78 (0.55–1.12)
0.88 (0.59–1.32)
Education, folate use, and smoking
included as covariatesjj
147 of 3,145 (4.7)
50 of 878 (5.7)
0.82 (0.60–1.12)
0.85 (0.47–1.53)
High-dimensional propensity score¶
147 of 3,145 (4.7)
50 of 878 (5.7)
0.82 (0.60–1.12)
0.88 (0.52–1.49)
Polycystic ovary syndrome
(metformin vs. no metformin)#
104 of 2,554 (4.1)
1,813 of 51,912 (3.5)
1.17 (0.96–1.42)
1.12 (0.92–1.36)
Cardiac major congenital malformation
New-user design*
8 of 361 (2.2)
13 of 569 (2.3)
0.97 (0.41–2.32)
0.94 (0.33–2.73)
Recorded type 2 diabetes†
26 of 915 (2.8)
7 of 406 (1.7)
1.65 (0.72–3.77)
2.03 (0.89–4.62)
$2 dispensations in pregnancy‡
25 of 1,248 (2.0)
18 of 829 (2.2)
0.92 (0.51–1.68)
1.08 (0.54–2.16)
Complete case analysis§
44 of 2,302 (1.9)
12 of 580 (2.1)
0.92 (0.49–1.74)
1.04 (0.51–2.11)
Education, folate use, and smoking
included as covariatesjj
63 of 3,145 (2.0)
18 of 878 (2.0)
0.98 (0.58–1.64)
1.00 (0.55–1.83)
High-dimensional propensity score¶
63 of 3,145 (2.0)
18 of 878 (2.0)
0.98 (0.58–1.64)
1.04 (0.56–1.93)
Polycystic ovary syndrome
(metformin vs. no metformin)#
44 of 2,554 (1.7)
758 of 51,912 (1.5)
1.18 (0.87–1.59)
1.12 (0.82–1.53) Table 3—Sensitivity analyses for the risk of any and cardiac major congenital malformations in infants with prenatal
exposure to metformin alone or in addition to insulin compared with insulin alone *New use was defined as no dispensing of metformin or insulin from 90 days before to 48 days after last menstrual period. †Including infants
born to mothers with at least one diagnosis of type 2 diabetes recorded at any time in the available look-back period before or on delivery
date. In the metformin group, 260 (28%) of the infants were also exposed to insulin. Table 1—Continued The
results were in large part driven by the
Metformin
in
Women
With
Type
2 Table 2—Risk of any and cardiac major congenital malformations in infants with prenatal exposure to metformin alone or
in addition to insulin compared with insulin alone
Outcome prevalence, n (%)
RR (95% CI)
Exposure to metformin
Exposure to insulin alone
Crude
Adjusted*
Any major congenital malformation
Metformin alone or in addition to insulin
147 of 3,145 (4.7)
50 of 878 (5.7)
0.82 (0.60–1.12)
0.84 (0.46–1.54)
Metformin alone
127 of 2,852 (4.4)
50 of 878 (5.7)
0.78 (0.57–1.08)
0.83 (0.44–1.58)
Metformin and insulin
20 of 293 (6.8)
50 of 878 (5.7)
1.20 (0.73–1.98)
0.98 (0.56–1.69)
Cardiac major congenital malformation
Metformin alone or in addition to insulin
63 of 3,145 (2.0)
18 of 878 (2.1)
0.98 (0.58–1.64)
1.01 (0.55–1.84)
Metformin alone
52 of 2,852 (1.8)
18 of 878 (2.1)
0.89 (0.52–1.51)
0.92 (0.47–1.81)
Metformin and insulin
11 of 293 (3.8)
18 of 878 (2.1)
1.83 (0.88–3.83)
1.72 (0.76–3.91)
*Adjusted for country and year of birth of infant; maternal characteristics, including age, country of birth, cohabitation with a partner, BMI,
epilepsy, severe mental illness, chronic hypertension, cardiovascular disease, skin/vaginal infections, and other diabetic complications; and
prenatal exposure to suspected teratogenic drugs, glucocorticoids, lipid-modifying drugs, and antihypertensive drugs. r congenital malformations in infants with prenatal exposure to metformin alone or
li
l rdiac major congenital malformations in infants with prenatal exposure to metformin alone or
ared with insulin alone *Adjusted for country and year of birth of infant; maternal characteristics, including age, country of birth, cohabitation with a partner, BMI,
epilepsy, severe mental illness, chronic hypertension, cardiovascular disease, skin/vaginal infections, and other diabetic complications; and
prenatal exposure to suspected teratogenic drugs, glucocorticoids, lipid-modifying drugs, and antihypertensive drugs. Table 1—Continued Diabetes Care Volume 46, August 2023 1562
Malformation Risk With Metformin in Pregnancy 1562 Table 3—Sensitivity analyses for the risk of any and cardiac major congenital malformations in infants with prenatal
exposure to metformin alone or in addition to insulin compared with insulin alone
Outcome prevalence, n (%)
RR (95% CI)
Exposure to metformin alone
or in addition to insulin
Exposure to insulin alone
(or no metformin#)
Crude
Adjusted**
Any major congenital malformation
New-user design*
24 of 361 (6.7)
35 of 569 (6.2)
1.08 (0.65–1.79)
1.07 (0.53–2.13)
Recorded type 2 diabetes†
53 of 915 (5.8)
27 of 406 (6.7)
0.87 (0.56–1.36)
0.93 (0.55–1.57)
$2 dispensations in pregnancy‡
58 of 1,248 (4.7)
50 of 829 (6.0)
0.77 (0.53–1.11)
0.83 (0.42–1.64)
Complete case analysis§
115 of 2,302 (5.0)
37 of 580 (6.4)
0.78 (0.55–1.12)
0.88 (0.59–1.32)
Education, folate use, and smoking
included as covariatesjj
147 of 3,145 (4.7)
50 of 878 (5.7)
0.82 (0.60–1.12)
0.85 (0.47–1.53)
High-dimensional propensity score¶
147 of 3,145 (4.7)
50 of 878 (5.7)
0.82 (0.60–1.12)
0.88 (0.52–1.49)
Polycystic ovary syndrome
(metformin vs. no metformin)#
104 of 2,554 (4.1)
1,813 of 51,912 (3.5)
1.17 (0.96–1.42)
1.12 (0.92–1.36)
Cardiac major congenital malformation
New-user design*
8 of 361 (2.2)
13 of 569 (2.3)
0.97 (0.41–2.32)
0.94 (0.33–2.73)
Recorded type 2 diabetes†
26 of 915 (2.8)
7 of 406 (1.7)
1.65 (0.72–3.77)
2.03 (0.89–4.62)
$2 dispensations in pregnancy‡
25 of 1,248 (2.0)
18 of 829 (2.2)
0.92 (0.51–1.68)
1.08 (0.54–2.16)
Complete case analysis§
44 of 2,302 (1.9)
12 of 580 (2.1)
0.92 (0.49–1.74)
1.04 (0.51–2.11)
Education, folate use, and smoking
included as covariatesjj
63 of 3,145 (2.0)
18 of 878 (2.0)
0.98 (0.58–1.64)
1.00 (0.55–1.83)
High-dimensional propensity score¶
63 of 3,145 (2.0)
18 of 878 (2.0)
0.98 (0.58–1.64)
1.04 (0.56–1.93)
Polycystic ovary syndrome
(metformin vs. no metformin)#
44 of 2,554 (1.7)
758 of 51,912 (1.5)
1.18 (0.87–1.59)
1.12 (0.82–1.53)
*New use was defined as no dispensing of metformin or insulin from 90 days before to 48 days after last menstrual period. †Including infants
born to mothers with at least one diagnosis of type 2 diabetes recorded at any time in the available look-back period before or on delivery
date. In the metformin group, 260 (28%) of the infants were also exposed to insulin. References is an employee and
L.P. a former employee of the Centre for Pharma-
coepidemiology at Karolinska Institutet, which re-
ceives funding from pharmaceutical companies
and regulatory authorities for drug safety/utiliza-
tion studies, unrelated to the submitted work. K.F. and Ø.K. reported participation in research
projects funded by pharmaceutical companies
(one on diabetes drugs), all regulator-mandated
phase IV studies (postauthorization safety stud-
ies) unrelated to the submitted work, all with
funds paid to the institution (no personal fees). M.G. and M.K.L. reported receiving grants from
the Finnish Medicines Agency and from the Inno-
vative Medicines Initiative (Building an Ecosystem
for Better Monitoring and Communicating the
Safety of Medicines’ Use in Pregnancy and
Breastfeeding: Validated and Regulatory En-
dorsed Workflows for Fast, Optimized Evidence
Generation, IMI ConcePTION grant agreement
821520) during the conduct of the study. No
other potential conflicts of interests relevant to
this article were reported. Duality of Interest. C.E.C. is an employee and
L.P. a former employee of the Centre for Pharma-
coepidemiology at Karolinska Institutet, which re-
ceives funding from pharmaceutical companies
and regulatory authorities for drug safety/utiliza-
tion studies, unrelated to the submitted work. K.F. and Ø.K. reported participation in research
projects funded by pharmaceutical companies
(one on diabetes drugs), all regulator-mandated
phase IV studies (postauthorization safety stud-
ies) unrelated to the submitted work, all with
funds paid to the institution (no personal fees). M.G. and M.K.L. reported receiving grants from
the Finnish Medicines Agency and from the Inno-
vative Medicines Initiative (Building an Ecosystem
for Better Monitoring and Communicating the
Safety of Medicines’ Use in Pregnancy and
Breastfeeding: Validated and Regulatory En-
dorsed Workflows for Fast, Optimized Evidence
Generation, IMI ConcePTION grant agreement
821520) during the conduct of the study. No
other potential conflicts of interests relevant to
this article were reported. 6. Polasek TM, Doogue MP,Thynne TRJ. Metformin
treatment of type 2 diabetes mellitus in pregnancy:
update on safety and efficacy. Ther Adv Drug Saf
2018;9:287–295 7. Cesta CE, Cohen JM, Pazzagli L, et al. Antidiabetic
medication use during pregnancy: an international
utilization study. BMJ Open Diabetes Res Care 2019;
7:e000759 8. ElSayed NA, Aleppo GA, Aroda VR, et al.;
American Diabetes Association. 15. Management
of diabetes in pregnancy: Standards of Care in
Diabetes—2023. Diabetes Care 2023;46(Suppl. 1):
S254–S266 Our findings could support policymakers,
prescribers, and patients weighing the ben-
efits and disadvantages of metformin com-
pared with insulin for glycemic control in
pregnancy. References The null finding for the sensitiv-
ity analysis restricted to mothers with poly-
cystic ovary syndrome suggests that the
results are generalizable to other conditions
as well. Future studies should investigate
the reproductive safety of other noninsulin
glucose-lowering drugs that are increasingly
used in type 2 diabetes. 9. He K, Guo Q, Ge J, Li J, Li C, Jing Z.The efficacy
and safety of metformin alone or as an add-on
therapy to insulin in pregnancy with GDM or
T2DM: a systematic review and meta-analysis of
21 randomized controlled trials. J Clin Pharm
Ther 2022;47:168–177 10. Cassina M, Dona M, Di Gianantonio E, Litta
P,
Clementi
M. First-trimester
exposure
to
metformin and risk of birth defects: a systematic
review and meta-analysis. Hum Reprod Update
2014;20:656–669 Author Contributions. L.J.K. drafted the man-
uscript. L.J.K., C.E.C., M.K.L., and J.M.C. con-
ceived the study design and were essential in
developing the common data model. K.F., M.G.,
H.Z., H.L.G., Ø.K., L.P., and A.E. contributed to
the methodology. L.J.K. and J.M.C. wrote the
syntax to perform the analyses. K.F. obtained
funding for the study. K.F., M.G., H.Z., and M.K.L. acquired data for the study. All authors contrib-
uted to the data interpretation and critical eval-
uation and approved the final submitted version
of the manuscript. L.J.K. is the guarantor of this
work and, as such, had full access to all the data
in the study and takes responsibility for the in-
tegrity of the data and the accuracy of the data
analysis. 11. Abolhassani N, Winterfeld U, Kaplan YC,
et al. Major malformations risk following early
pregnancy exposure to metformin: a systematic
review and meta-analysis. BMJ Open Diabetes
Res Care 2023;11:e002919 In summary, in this cohort study of four
Nordic countries, we found no evidence of
an increased risk of major congenital mal-
formations in the offspring of mothers
with type 2 diabetes treated with metfor-
min compared with insulin during organo-
genesis in early pregnancy. The results
should be interpreted with caution be-
cause of missing information on glycemic
control, which is an important risk factor
for congenital malformations. 12. Laugesen K, Ludvigsson JF, Schmidt M, et al. Nordic health registry-based research: a review
of health care systems and key registries. Clin
Epidemiol 2021;13:533–554 13. Skjaerven R, Gjessing HK, Bakketeig LS. Birthweight by gestational age in Norway. Acta
Obstet Gynecol Scand 2000;79:440–449 Prior Presentation. Table 1—Continued was supported by the
Horizon Europe: Marie Skłodowska-Curie Ac-
tions 2020 research and innovation program un-
der grant agreement 844728. L.P. received and
was supported by a grant from FORTE Swedish
Research Council for Health, Working Life, and
Welfare during the conduct of the study (project
no. 2021-01080). The funders of the study had no role in study referral to specialist care during preg-
nancy, physicians may only use a non-
specific diabetes code. Therefore, we
decided not to limit the study population
to pregnancies of mothers with type 2
diabetes based on recorded diagnosis co-
des. Instead, women with other indications
for metformin or insulin were excluded. Re-
assuringly, the sensitivity analysis limiting
the study population to pregnancies with a
recorded diagnosis of type 2 diabetes gave
a point estimate close to 1. An analysis
with a new-user design was conducted to
further exclude women with other indica-
tions than type 2 diabetes. This analysis
also suggested no clinically relevant in-
crease in the risk of malformations. Nordic
PharmacoEpidemiological
Network,
11 November 2020; and in oral form at the
37th Annual Conference of the International So-
ciety for Pharmacoepidemiology, virtual, 23–25
August 2021. Karolinska Institutet, for contributions to the
data acquisition, design of the study, interpreta-
tion of the data, and feedback on earlier ver-
sions of the manuscript. The authors also thank
Randi Selmer of the Department of Chronic Dis-
eases, Norwegian Institute of Public Health, for
statistical and methodological contributions. Acknowledgments. The authors thank Helle
Kieler of the Centre for Pharmacoepidemiology, References Funding. This study was funded by NordForsk
as part of the Nordic Pregnancy Drug Safety
Studies (NorPreSS) (project no. 83539), by the
Research Council of Norway as part of the Inter-
national Pregnancy Drug Safety Studies (InPreSS)
(project no. 273366), and partly by the Research
Council of Norway through its Centers of Excel-
lence funding scheme (project no. 262700). H.Z. was supported by a University of New South
Wales Scientia Program Award during the con-
duct of the study. C.E.C. was supported by the
Horizon Europe: Marie Skłodowska-Curie Ac-
tions 2020 research and innovation program un-
der grant agreement 844728. L.P. received and
was supported by a grant from FORTE Swedish
Research Council for Health, Working Life, and
Welfare during the conduct of the study (project
no. 2021-01080). 1. Fadl HE, Simmons D. Trends in diabetes in
pregnancy in Sweden 1998-2012. BMJ Open
Diabetes Res Care 2016;4:e000221 2. Arendt LH, Pedersen LH, Pedersen L, et al. Glycemic control in pregnancies complicated by
pre-existing diabetes mellitus and congenital
malformations: a Danish population-based study. Clin Epidemiol 2021;13:615–626 3. Coton SJ, Nazareth I, Petersen I. A cohort
study of trends in the prevalence of pregestational
diabetes in pregnancy recorded in UK general
practice between 1995 and 2012. BMJ Open
2016;6:e009494 4. Bell R, Glinianaia SV, Tennant PWG, Bilous
RW, Rankin J. Peri-conception hyperglycaemia
and nephropathy are associated with risk of
congenital anomaly in women with pre-existing
diabetes: a population-based cohort study. Diabetologia 2012;55:936–947 A further limitation is that information
on potentially important confounders, such
as maternal BMI, education, folate use, and
smoking, were partially or completely miss-
ing for some of the study countries. Still, the
sensitivity analysis with multiple imputation
of maternal education, folate use, and
smoking and the one using complete cases
gave similar results close to null. Although
we know that metformin and insulin were
dispensed at the pharmacies, we cannot
be sure that the mothers actually used the
drugs. The null finding for the sensitivity
analysis requiring at least two dispensa-
tions of metformin or insulin during the
pregnancy was reassuring in this regard. The funders of the study had no role in study
design, data collection, data analysis, data in-
terpretation, or writing of the manuscript. 5. Davidson AJF, Park AL, Berger H, et al. Association of improved periconception hemoglobin
A1c with pregnancy outcomes in women with
diabetes. JAMA Netw Open 2020;3:e2030207 Duality of Interest. C.E.C. Table 1—Continued The find-
ings might also have been caused by
prevalent maternal use of insulin analogs
in the insulin group, which are associated
with a lower risk of cardiac malformations
than human insulin (29). Like other observational studies, a limita-
tion of our study is that information on gly-
cemic control in the mother, an important
risk factor for congenital malformations,
was not available. However, we adjusted
for several comorbidities and comedica-
tions likely related to poor glycemic control,
such as cardiovascular disease and diabe-
tes complications, although these might
not have been sufficient proxies for glyce-
mic control in the pregnancy (30). We
excluded infants of women with other in-
dications for metformin and insulin than
type 2 diabetes and used insulin as an ac-
tive comparator to metformin, thereby
reducing the risk of confounding by indi-
cation and disease severity (18,31). To
test the robustness of this approach, we conducted a sensitivity analysis using the
high-dimensional propensity score method,
which aims to capture proxies for unmeas-
ured confounders (19). This yielded a result
comparable to the primary analyses,
suggesting no increased risk of malforma-
tions overall associated with metformin. Another limitation is the inadequate
recording of type 2 diabetes. Initial diag-
nosis and follow-up of type 2 diabetes
outside of pregnancy usually occurs in
primary care in the study countries. We
had primary care data for Finland and
Norway but not Iceland and Sweden. Furthermore, the International Classifi-
cation of Primary Care codes used in pri-
mary care were not specific to type 2
diabetes before 2014. Even after typical Kjerpeseth and Associates diabetesjournals.org/care 1563 Karolinska Institutet, for contributions to the
data acquisition, design of the study, interpreta-
tion of the data, and feedback on earlier ver-
sions of the manuscript. The authors also thank
Randi Selmer of the Department of Chronic Dis-
eases, Norwegian Institute of Public Health, for
statistical and methodological contributions. Funding. This study was funded by NordForsk
as part of the Nordic Pregnancy Drug Safety
Studies (NorPreSS) (project no. 83539), by the
Research Council of Norway as part of the Inter-
national Pregnancy Drug Safety Studies (InPreSS)
(project no. 273366), and partly by the Research
Council of Norway through its Centers of Excel-
lence funding scheme (project no. 262700). H.Z. was supported by a University of New South
Wales Scientia Program Award during the con-
duct of the study. C.E.C. References Parts of this study were
presented in poster form at the 56th Annual
Meeting of the European Association for the
Study of Diabetes, virtual, 21–25 September
2020; in oral form at a webinar by the Acknowledgments. The authors thank Helle
Kieler of the Centre for Pharmacoepidemiology, 15. Cohen JM, Cesta CE, Kjerpeseth L, et al. A
common data model for harmonization in the Diabetes Care Volume 46, August 2023 Malformation Risk With Metformin in Pregnancy 1564 based cohort study. BMJ Open Diabetes Res Care
2022;10:e002363 Nordic Pregnancy Drug Safety Studies (NorPreSS). Norsk Epidemiol 2021;29(1–2). Accessed 4 October
2021. Available from https://www.ntnu.no/ojs/
index.php/norepid/article/view/4053 Nordic Pregnancy Drug Safety Studies (NorPreSS). Norsk Epidemiol 2021;29(1–2). Accessed 4 October
2021. Available from https://www.ntnu.no/ojs/
index.php/norepid/article/view/4053 21. Given JE, Loane M, Garne E, et al. Metformin
exposure in first trimester of pregnancy and risk of
all or specific congenital anomalies: exploratory
case-control study. BMJ 2018;361:k2477 27. Feig DS, Donovan LE, Zinman B, et al.; MiTy
Collaborative Group. Metformin in women with
type 2 diabetes in pregnancy (MiTy): a multicentre,
international, randomised, placebo-controlled trial. Lancet Diabetes Endocrinol 2020;8:834–844 16. Desai RJ, Rothman KJ, Bateman BT, Hernandez-
Diaz S, Huybrechts KF. A propensity-score-based
fine stratification approach for confounding
adjustment when exposure is infrequent. Epidemiology 2017;28:249–257 22. Panchaud A, Rousson V, Vial T, et al. Pregnancy outcomes in women on metformin for
diabetes or other indications among those seeking
teratology information services. Br J Clin Pharmacol
2018;84:568–578 28. Zhang TN, Huang XM, Zhao XY, Wang W,
Wen R, Gao SY. Risks of specific congenital
anomalies in offspring of women with diabetes: a
systematic review and meta-analysis of population-
based studies including over 80 million births. PLoS
Med 2022;19:e1003900 teratology information services. Br J Clin Pharmacol
2018;84:568–578 17. Sterne JAC,White IR, Carlin JB, et al. Multiple
imputation for missing data in epidemiological
and clinical research: potential and pitfalls. BMJ
2009;338:b2393 23. Dukhovny S, Van Bennekom CM, Gagnon
DR, et al. Metformin in the first trimester and
risks for specific birth defects in the National
Birth Defects Prevention Study. Birth Defects Res
2018;110:579–586 18. Schneeweiss S, Patrick AR, St€urmer T, et al. Increasing levels of restriction in pharma-
coepidemiologic database studies of elderly and
comparison with randomized trial results. Med
Care 2007;45(Suppl. 2):S131–S142 29. Wang H,Wender-Ozegowska E, Garne E, et al. References Insulin analogues use in pregnancy among women
with pregestational diabetes mellitus and risk of
congenital anomaly: a retrospective population-
based cohort study. BMJ Open 2018;8:e014972 29. Wang H,Wender-Ozegowska E, Garne E, et al. Insulin analogues use in pregnancy among women
with pregestational diabetes mellitus and risk of
congenital anomaly: a retrospective population-
based cohort study. BMJ Open 2018;8:e014972
30. Cesta CE, Hernandez-Diaz S, Bateman BT,
Huybrechts KF, Seely EW, Patorno E. The role of
propensity score matching in claims-based studies
for achieving comparability in HbA1c levels
between treatments in pregnant women with
type 2 diabetes [abstract]. Pharmacoepidemiol
Drug Saf 2021;30:62 24. Scherneck S, Schlinke N, Beck E, Grupe K,
Weber-Schoendorfer C, Schaefer C. Pregnancy
outcome after first-trimester exposure to metformin:
a prospective cohort study. Reprod Toxicol 2018;81:
79–83 19. Schneeweiss S, Rassen JA, Glynn RJ, Avorn J,
Mogun
H,
Brookhart
MA. High-dimensional
propensity score adjustment in studies of treatment
effects using health care claims data. Epidemiology
2009;20:512–522 30. Cesta CE, Hernandez-Diaz S, Bateman BT,
Huybrechts KF, Seely EW, Patorno E. The role of
propensity score matching in claims-based studies
for achieving comparability in HbA1c levels
between treatments in pregnant women with
type 2 diabetes [abstract]. Pharmacoepidemiol
Drug Saf 2021;30:62 25. Diav-Citrin O, Steinmetz-Shoob S, Shechtman
S, Ornoy A. In-utero exposure to metformin for
type 2 diabetes or polycystic ovary syndrome: a
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S, Ornoy A. In-utero exposure to metformin for
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Resonant generation of propagating second-harmonic spin waves in nano-waveguides
|
Nature communications
| 2,024
|
cc-by
| 9,735
|
Resonant generation of propagating second-
harmonic spin waves in nano-waveguides K. O. Nikolaev
1,4, S. R. Lake2,4, G. Schmidt
2,3, S. O. Demokritov
1
&
V. E. Demidov
1 Generation of second-harmonic waves is one of the universal nonlinear phe-
nomena that have found numerous technical applications in many modern
technologies, in particular, in photonics. This phenomenon also has great
potential in the field of magnonics, which considers the use of spin waves in
magnetic nanostructures to implement wave-based signal processing and
computing. However, due to the strong frequency dependence of the phase
velocity of spin waves, resonant phase-matched generation of second-
harmonic spin waves has not yet been achieved in practice. Here, we show
experimentally that such a process can be realized using a combination of
different modes of nano-sized spin-wave waveguides based on low-damping
magnetic insulators. We demonstrate that our approach enables efficient
spatially-extended energy transfer between interacting waves, which can be
controlled by the intensity of the initial wave and the static magnetic field. magnetization vector precesses in finite-size magnetic structures. Due
to the dynamic demagnetization effects, the precession trajectory is
typically elliptical under these conditions. This ellipticity initiates a
dynamic magnetization component along to the precession axis at
double the precession frequency5. In other words, magnetic SHG does
not require the use of special media and can be observed in many
magnetic materials and experimental configurations so long as there is
elliptical magnetization precession6–15. This makes magnetic oscilla-
tions excellent candidate for generation of microwave-frequency
harmonics. For example, in ref. 15 it was shown that the nonlinear
response of magnetic domain walls to a driving magnetic field at
megahertz frequencies can lead to the generation of up to 60
harmonics. Second-harmonic generation (SHG) plays an important role in modern
technologies that use waves of different nature for transmission and
processing of information. This phenomenon is particularly important
in the field of photonics as it allows efficient generation of coherent
optical waves that are difficult to generate directly. Because of its great
practical importance, the optical SHG has been intensively studied
over many decades1–4, which has enabled the development of a wide
variety of highly efficient photonic devices.Although SHG is a universal
physical phenomenon, which can potentially be realized for any kind
of waves, its practical realization requires fulfillment of two important
conditions. First, SHG is possible only in media exhibiting nonzero
second-order nonlinear susceptibility. 1Institute of Applied Physics, University of Muenster, 48149 Muenster, Germany. 2Institut für Physik, Martin-Luther-Universität Halle-Wittenberg, 06120
Halle, Germany. 3Interdisziplinäres Zentrum für Materialwissenschaften, Martin-Luther-Universität Halle-Wittenberg, 06120 Halle, Germany. 4These authors
contributed equally: K. O. Nikolaev, S. R. Lake.
e-mail: demokrit@uni-muenster.de Article https://doi.org/10.1038/s41467-024-46108-y Article can be calculated as: makes it difficult to achieve phase matching for waves at frequencies
that differ by a factor of 2. As a result, in previous experiments with
spin waves, only the non-resonant SHG process could be achieved, in
which the generated second-harmonic wave did not belong to the
eigenspectrum of spin waves and represented a forced non-resonant
motion of magnetic moments, i.e., a non-propagating wave6. f p,qðkÞ = γ
2π
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
H + 4πMsð1 PÞ + 2A
Ms
k2
tot
H + 4πMs
k2
k2
ip
P + 2A
Ms
k2
tot
"
#
v
u
u
t
ð1Þ ð1Þ Recently it was shown theoretically11 that phase-matched resonant
SHG can be achieved in a semi-infinite magnetic film for spin waves
propagating in a field-induced potential well near the edge of the
film20,21 (so-called edge modes) and bulk spin-wave modes of the film. However, to satisfy conservation of linear momentum, the second-
harmonic waves must propagate away from the film edge, which
reduces the efficiency of their interaction with the initial wave propa-
gating along the edge. In addition, in real magnetic nano-systems, the
edge modes exhibit enhanced spatial damping due to the scattering
from edge imperfections (see, e.g., ref. 8), which strongly limits the
practical applicability of the approach. Here, k is the component of the wavevector along the axis of the
waveguide, γ is the gyromagnetic ratio, Ms is the saturation magneti-
zation, P =
k2
ip
k2
tot
k4
ip
k4
tot F
1
1 + δ0q, F =
2
kipd 1 ð1Þqekipd
, and A is the
exchange constant. The effective wavevector in the plane of the
waveguide is kip =
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
k2 + k2
z
q
, where kz = πðp + 1Þ=w is the effective
wavevector characterizing the standing wave of the dynamic magne-
tization across the waveguide width36 (see insets in Fig. 1b). The total
effective wavevector is ktot =
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
k2
ip + κ2
q
, where κ = πq=d is the effective
wavevector characterizing the standing wave of the dynamic magne-
tization through the thickness of the waveguide. This model assumes
standard boundary conditions for dynamic magnetization37. The
boundary conditions at the surfaces of the waveguide correspond to
“unpinned” dynamic magnetization, which is typical for thin magnetic
films. Article In contrast, the boundary conditions at the waveguide edges are
determined by dipolar effects and correspond to “pinned” dynamic
magnetization. The calculations are performed at H = 500 Oe using
material parameters described in Methods. p
pp
y
pp
In this work, we propose and experimentally demonstrate a new
approach that enables fully phase-matched, resonant generation of
second-harmonic spin waves in magnetic nano-waveguides made from
a low-damping magnetic insulator. We base this approach on the
nonlinear interaction of spin-wave modes with different distributions
of the dynamic magnetization through the thickness of the waveguide. We show that by choosing a proper thickness, one can engineer the
dispersion spectrum of modes so that the phase velocities of a spin
wave and its second harmonic become equal. Under these conditions,
the initial spin wave continuously transfers its energy to the second-
harmonic wave, resulting in a long-lasting, spatially-extended growth
of the latter. This process can be achieved for different transverse
modes and can be controlled by varying the intensity of the initial wave
and the static magnetic field. Our experimental data show very good
quantitative agreement with the results of theoretical analysis. The
proposed approach provides new opportunities for the field of mag-
nonics. It enables highly-efficient generation of spin waves with short
wavelengths that are difficult to excite directly22–27. Due to the phase
locking of the initial wave and its second harmonic, the approach can
also be used for the implementation of new interference-based devices
that utilize interference effects at the fundamental and doubled fre-
quencies simultaneously. The fundamental mode of the waveguide (q = 0, p = 0) is char-
acterized by a uniform distribution of dynamic magnetization through
its thickness (inset in Fig. 1b). This mode interacts most efficiently with
the dynamic magnetic field of the antenna and, therefore, can be dri-
ven to a large-amplitude strongly-nonlinear regime using moderate
powers of the excitation signal of the order of 10−4 W. As shown in the
inset in Fig. 1a, in this regime, the ellipticity of the precession of
magnetization M leads to the appearance of a sizable component of
the dynamic magnetization along the z-axis. The projection of M onto
the axis z, can be written as: Mz =
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
M2
s m2
q
≈Ms 1 1
2
m2
M2
s
, where m is
the dynamic component of the magnetization in the x-y plane. Studied system and approach Figure 1a shows the schematics of the experiment. We study spin
waves propagating in a waveguide with the width w = 500 nm fabri-
cated from a film of Yttrium Iron Garnet (YIG)28–33 with the thickness
d = 80 nm. The spin waves are excited using a 500-nm wide and 200-
nm thick Au antenna carrying microwave electric current. The wave-
guide is magnetized in plane by a static magnetic field H applied per-
pendicular to its axis. The propagation of spin waves is analyzed with
spatial and spectral resolution using micro-focus Brillouin light scat-
tering (BLS) spectroscopy34 (see Methods for details). This technique
yields a signal, referred to as BLS intensity, which is proportional to the
intensity of spin waves at the position, where the probing light is
focused (Fig. 1a). This allows a direct imaging of spin waves with high
spatial resolution. Thanks to the spectral resolution of the BLS tech-
nique, spin waves at different frequencies can be imaged indepen-
dently. Additionally, we use the ability of BLS to detect the phase of
propagating spin waves, which allows direct determination of their
wavelength and phase velocity. Mz ≈Ms
1
4Ms
m2
x + m2
y
1
4Ms
m2
x m2
y
cosð2π2f t 2kxÞ
ð2Þ ð2Þ The first two terms in Eq. (2) are time independent. The third term
mz oscillates at frequency 2f while its spatial dependence is char-
acterized by a wavevector 2k. The amplitude of this magnetization
component is proportional to m2
x m2
y, i.e. to the ellipticity of pre-
cession. Due to demagnetization effects in the narrow waveguide and
the non-zero wavevector, this component creates a dynamic dipole
magnetic field that is not strictly parallel to the static field H and can
linearly excite magnetization dynamics (see ref. 6). Similarly to mz, this
field oscillates with a frequency 2f and vary in space with a wavevector
2k, where f and k are the frequency and the wavevector of the initially
excited fundamental wave, respectively. Therefore, this dipole field is
expected to couple the initial wave with the wave at 2f and 2k. This process can also be considered as the confluence of two
magnons with energy hf and linear momentum hk into a magnon with
energy 2hf and momentum 2hk according to the energy and the linear
momentum conservation laws. Article Sub-
stituting
into
this
expression
the
dependence
in
the
form
m2 = m2
xcos2 2πf t kx
ð
Þ + m2
ysin2 2πf t kx
ð
Þ, where mx and my are the
amplitudes of the dynamic magnetization in the x and y directions,
respectively, we obtain: Resonant generation of propagating second-
harmonic spin waves in nano-waveguides Second, efficient SHG requires
exact matching of the phase velocities (commonly referred to as phase
matching) of the initial wave and the second-harmonic wave. In optics,
the first requirement is met in a large class of non-centrosymmetric,
nonlinear crystals. Thanks to the weak dispersion of light waves, the
second condition of phase velocity matching can also be easily
achieved by using a number of well-developed approaches2–4. A significantly more complex task is the implementation of a
phase-matched resonant SHG for propagating waves of dynamic
magnetization (spin waves), which are believed to be one of the most
promising candidates for nano-scale wave-based signal processing and
computing16–19. In contrast to electromagnetic waves, spin waves
exhibit strong dispersion, i.e., a strongly nonlinear dependence of the
frequency on the wavevector. Accordingly, the phase velocity of spin
waves changes significantly with the increase of their frequency, which In the case of magnetic systems, the requirement of nonzero
second-order nonlinear dynamic susceptibility can be satisfied rela-
tively
easily. The
second-order
nonlinearity
arises
when
the 1Institute of Applied Physics, University of Muenster, 48149 Muenster, Germany. 2Institut für Physik, Martin-Luther-Universität Halle-Wittenberg, 06120
Halle, Germany. 3Interdisziplinäres Zentrum für Materialwissenschaften, Martin-Luther-Universität Halle-Wittenberg, 06120 Halle, Germany. 4These authors
contributed equally: K. O. Nikolaev, S. R. Lake. e-mail: demokrit@uni-muenster.de Nature Communications| (2024) 15:1827 1 https://doi.org/10.1038/s41467-024-46108-y Article Studied system and approach The ellipticity of the precession of
the magnetization M leads to the appearance of a sizable double-frequency com-
ponent of the dynamic magnetization mz / m2
x m2
y (inset), which results in the
excitation of the second-harmonic spin wave. Both waves are independently
detected by BLS. b Calculated dispersion spectrum of spin-wave modes. The phase-
matching condition between the fundamental mode (q = 0, p = 0) and the first-
order thickness modes (q = 1) is satisfied at the points corresponding to the inter-
section of the dashed curve 2f(2k) with the dispersion curves of the q = 1 modes. This enables a resonant energy transfer, as indicated by the dashed arrows. Sym-
bols correspond to the resonantly interacting waves, as observed in the experi-
ment. Insets schematically show the distributions of dynamic magnetization over
the thickness and the width of the waveguide. The data are obtained at H = 500 Oe. be satisfied for a single spin-wave mode. However, as seen in Fig. 1b, in
nanoscale YIG waveguides, the dashed curve can intersect with dis-
persion curves of first-order thickness modes (q = 1) characterized by a
non-uniform distribution of dynamic magnetization through the
thickness40,41 (inset in Fig. 1b). Note that, by definition, the intersection
points correspond to the condition of equality of the phase velocities
vph = 2πf/k of two spin-wave modes – the fundamental mode and the
mode with doubled frequency. Therefore, it becomes possible to
achieve a completely phase-matched inter-mode resonant energy
exchange, as shown in Fig. 1b by arrows. We emphasize that for a given
thickness of the waveguide, the resonant process is possible in a cer-
tain range of the static field H. By varying the field, one can shift the
intersection points towards shorter or longer wavelengths. have oppositely directed wavevectors, and the resulting magnon has a
nearly zero wavevector38,39. In this case, the energy and momentum
conservation rules can be easily satisfied even for waves, whose fre-
quency vs wavevector dependence is not linear. However, to imple-
ment efficient resonant generation of the second harmonic, the three-
magnon confluence process must involve two initial magnons with
equal and collinear wavevectors and a resulting magnon with a dou-
bled wavevector. Simultaneously, the frequency of the resulting
magnon must be twice the frequency of the initial magnon. Studied system and approach We emphasize, however, that this
process can be efficient only if the phase-space point (2f, 2k) corre-
sponds to an eigenexcitation of the system, which is difficult to
implement in practice using one spin-wave mode. Three-magnon
confluence processes can be easily realized when the initial magnons Figure 1b illustrates the main idea of our work – inter-mode
resonant generation of second-harmonic spin waves. It shows the
spectrum of spin-wave modes in a 500-nm wide YIG waveguide cal-
culated using the analytical theory35 and the approach developed in
ref. 36 According to this approach, the frequency of spin-wave modes Nature Communications| (2024) 15:1827 2 https://doi.org/10.1038/s41467-024-46108-y Article have oppositely directed wavevectors and the resulting magnon has a
be satisfied for a single spin-wave mode. However, as seen in Fig. 1b, in
nanoscale YIG waveguides, the dashed curve can intersect with dis-
persion curves of first-order thickness modes (q = 1) characterized by a
non-uniform distribution of dynamic magnetization through the
thickness40,41 (inset in Fig. 1b). Note that, by definition, the intersection
points correspond to the condition of equality of the phase velocities
vph = 2πf/k of two spin-wave modes – the fundamental mode and the
mode with doubled frequency. Therefore, it becomes possible to
achieve a completely phase-matched inter-mode resonant energy
exchange, as shown in Fig. 1b by arrows. We emphasize that for a given
thickness of the waveguide, the resonant process is possible in a cer-
tain range of the static field H. By varying the field, one can shift the
intersection points towards shorter or longer wavelengths. Fig. 1 | Implementation of resonant generation of second-harmonic spin waves. a Schematics of the experiment. Spin waves in a 500-nm wide and 80-nm thick YIG
waveguide are excited using a Au strip antenna. The ellipticity of the precession of
the magnetization M leads to the appearance of a sizable double-frequency com-
ponent of the dynamic magnetization mz / m2
x m2
y (inset), which results in the
excitation of the second-harmonic spin wave. Both waves are independently
detected by BLS. b Calculated dispersion spectrum of spin-wave modes. The phase-
matching condition between the fundamental mode (q = 0, p = 0) and the first-
order thickness modes (q = 1) is satisfied at the points corresponding to the inter-
section of the dashed curve 2f(2k) with the dispersion curves of the q = 1 modes. Studied system and approach The ellipticity of the precession of
the magnetization M leads to the appearance of a sizable double-frequency com-
ponent of the dynamic magnetization mz / m2
x m2
y (inset), which results in the
excitation of the second-harmonic spin wave. Both waves are independently
detected by BLS. b Calculated dispersion spectrum of spin-wave modes. The phase-
matching condition between the fundamental mode (q = 0, p = 0) and the first-
order thickness modes (q = 1) is satisfied at the points corresponding to the inter-
section of the dashed curve 2f(2k) with the dispersion curves of the q = 1 modes. This enables a resonant energy transfer, as indicated by the dashed arrows. Sym-
bols correspond to the resonantly interacting waves, as observed in the experi-
ment. Insets schematically show the distributions of dynamic magnetization over
the thickness and the width of the waveguide. The data are obtained at H = 500 Oe. Fig. 1 | Implementation of resonant generation of second-harmonic spin waves. Fig. 1 | Implementation of resonant generation of second-harmonic spin waves. a Schematics of the experiment. Spin waves in a 500-nm wide and 80-nm thick YIG
waveguide are excited using a Au strip antenna. The ellipticity of the precession of
the magnetization M leads to the appearance of a sizable double-frequency com-
ponent of the dynamic magnetization mz / m2
x m2
y (inset), which results in the
excitation of the second-harmonic spin wave. Both waves are independently
detected by BLS. b Calculated dispersion spectrum of spin-wave modes. The phase-
matching condition between the fundamental mode (q = 0, p = 0) and the first-
order thickness modes (q = 1) is satisfied at the points corresponding to the inter-
section of the dashed curve 2f(2k) with the dispersion curves of the q = 1 modes. This enables a resonant energy transfer, as indicated by the dashed arrows. Sym-
bols correspond to the resonantly interacting waves, as observed in the experi-
ment. Insets schematically show the distributions of dynamic magnetization over
the thickness and the width of the waveguide. The data are obtained at H = 500 Oe. Fig. 1 | Implementation of resonant generation of second-harmonic spin waves. a Schematics of the experiment. Spin waves in a 500-nm wide and 80-nm thick YIG
waveguide are excited using a Au strip antenna. Studied system and approach The ellipticity of the precession of
the magnetization M leads to the appearance of a sizable double-frequency com-
ponent of the dynamic magnetization mz / m2
x m2
y (inset), which results in the
excitation of the second-harmonic spin wave. Both waves are independently
detected by BLS. b Calculated dispersion spectrum of spin-wave modes. The phase-
matching condition between the fundamental mode (q = 0, p = 0) and the first-
order thickness modes (q = 1) is satisfied at the points corresponding to the inter-
section of the dashed curve 2f(2k) with the dispersion curves of the q = 1 modes. This enables a resonant energy transfer, as indicated by the dashed arrows. Sym-
bols correspond to the resonantly interacting waves, as observed in the experi-
ment. Insets schematically show the distributions of dynamic magnetization over
the thickness and the width of the waveguide. The data are obtained at H = 500 Oe. 1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
0.0
0.5
1.0
(
ytis
n
etni
S
L
B
)
u
. a
stin
b
r Detection
GHz)
frequency ( Fig. 2 | Experimental evidence for resonant wave interaction. a BLS intensity
detected at a frequency twice the frequency of the initial spin wave fexc as a function
of the latter. Note two narrow resonant peaks at fexc = 2.47 and 2.56 GHz. b Complete BLS spectra recorded at two excitation frequencies corresponding to
the observed resonances, as labeled. The data are obtained at H = 500 Oe at a
distance x = 10 μm. Power of the excitation signal P = 0.1 mW. Fig. 2 | Experimental evidence for resonant wave interaction. a BLS intensity
detected at a frequency twice the frequency of the initial spin wave fexc as a function
of the latter. Note two narrow resonant peaks at fexc = 2.47 and 2.56 GHz. b Complete BLS spectra recorded at two excitation frequencies corresponding to
the observed resonances, as labeled. The data are obtained at H = 500 Oe at a
distance x = 10 μm. Power of the excitation signal P = 0.1 mW. Fig. 1 | Implementation of resonant generation of second-harmonic spin waves. a Schematics of the experiment. Spin waves in a 500-nm wide and 80-nm thick YIG
waveguide are excited using a Au strip antenna. Nature Communications| (2024) 15:1827 Studied system and approach In contrast
to the process involving counter-propagating magnons, such a process
is difficult to implement due to the nonlinear dependence of the fre-
quency of spin waves on the wavevector. Studied system and approach This enables a resonant energy transfer, as indicated by the dashed arrows. Sym-
bols correspond to the resonantly interacting waves, as observed in the experi-
ment. Insets schematically show the distributions of dynamic magnetization over
the thickness and the width of the waveguide. The data are obtained at H = 500 Oe. 1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
0.0
0.5
1.0
Excitation frequency
(GHz)
2.0
2.2
2.4
2.6
2.8
3.0
0.0
0.5
1.0
ta
ytis
n
etni
S
L
B
(
)
u
. a
stin
b
r
Detection
GHz)
frequency (
(
ytis
n
etni
S
L
B
)
u
. a
stin
b
r
Fig. 2 | Experimental evidence for resonant wave interaction. a BLS intensity
detected at a frequency twice the frequency of the initial spin wave fexc as a function
of the latter. Note two narrow resonant peaks at fexc = 2.47 and 2.56 GHz. b Complete BLS spectra recorded at two excitation frequencies corresponding to
the observed resonances, as labeled. The data are obtained at H = 500 Oe at a
distance x = 10 μm. Power of the excitation signal P = 0.1 mW. 1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
0.0
0.5
1.0
Excitation frequency
(GHz)
2.0
2.2
2.4
2.6
2.8
3.0
0.0
0.5
1.0
ta
ytis
n
etni
S
L
B
(
)
u
. a
stin
b
r
Detection
GHz)
frequency (
(
ytis
n
etni
S
L
B
)
u
. a
stin
b
r
Fig. 2 | Experimental evidence for resonant wave interaction. a BLS intensity
detected at a frequency twice the frequency of the initial spin wave fexc as a function
of the latter. Note two narrow resonant peaks at fexc = 2.47 and 2.56 GHz. b Complete BLS spectra recorded at two excitation frequencies corresponding to
the observed resonances, as labeled. The data are obtained at H = 500 Oe at a
distance x = 10 μm. Power of the excitation signal P = 0.1 mW. Excitation frequency
(GHz)
2.0
2.2
2.4
2.6
2.8
3.0
0.0
0.5
1.0
ta
ytis
n
etni
S
L
B
(
)
u
. a
stin
b
r Fig. 1 | Implementation of resonant generation of second-harmonic spin waves. a Schematics of the experiment. Spin waves in a 500-nm wide and 80-nm thick YIG
waveguide are excited using a Au strip antenna. Evidence of resonant generation of second-harmonic spin waves Power of the excitation signal P = 0.1 mW. Fig. 3 | Spatial mapping of resonantly interacting waves. Left column corre-
sponds to the resonance at fexc = 2.47 GHz, right column corresponds to the reso-
nance at fexc = 2.56 GHz. a, d Intensity and phase maps of the initially excited wave. b, e Intensity and phase maps of the second-harmonic wave. c, f Spatial for both resonances. This indicates a very efficient energy transfer
from the initially excited wave to the second-harmonic wave. at 2.47 GHz (Fig. 3a). However, the spatial maps of their second har-
monics differ substantially. While the wave at 4.94 GHz (Fig. 3b) is
characterized by an intensity maximum in the center of the waveguide
and a uniform distribution of phase across the waveguide width, the
intensity of the wave at 5.12 GHz exhibits a minimum in the center and
the phase shows a variation by π across the waveguide section. These
differences indicate that the two resonances correspond to two dif-
ferent q = 1 modes that possess symmetric (p = 0) and antisymmetric
(p = 1) transverse profiles. We study this processes in more detail in the space domain using
the space- and phase-resolution of BLS. Figure 3 shows the results of
spatial mapping of the intensity and phase (cos(φ)) of spin waves
corresponding to two observed resonances. Figure 3a–c characterizes
the resonance at fexc = 2.47 GHz, while Fig. 3d–f characterizes the
resonance at fexc = 2.56 GHz. Figure 3a shows spatial maps of the
intensity and phase corresponding to the initial wave at 2.47 GHz,
while Fig. 3b shows the same maps for the second-harmonic wave at
4.94 GHz. The intensity of the initial wave decreases with propagation
distance, while the intensity of the second harmonic, which is negli-
gible near the antenna, gradually increases in space. Figure 3c shows a
direct comparison of the spatial dependences of the intensities of the
initial wave and the second-harmonic wave. The initial wave exhibits a
well-defined exponential decay (note the logarithmic scale of the
vertical axis) characterized by the decay length of 54 μm (for com-
parison, the independently determined decay length of the wave at
4.94 GHz is 11 μm). The intensity of the second harmonic increases in
the range x = 0–10 μm and then saturates. The intensities of the two
waves quickly become equal at x ≈4 μm. Evidence of resonant generation of second-harmonic spin waves p
p
This conclusion is in excellent agreement with the results of cal-
culations (Fig. 1b). From the phase maps in Fig. 3, we obtain the
wavelengths of the initial wave, λ0, and the second harmonic, λSH, for
the first (λ0 = 1.65 μm, λSH = 0.82 μm) and the second (λ0 = 1.38 μm,
λSH = 0.69 μm) resonances. Taking into account the frequencies of
these waves found from the previous analysis, we can plot the
experimental points on the calculated dispersion diagram (symbols in
Fig. 1b). As seen from these data, the point-up triangles, corresponding
to the initial wave, coincide well with the dispersion curve of the fun-
damental mode, and the point-down triangles, corresponding to the
second harmonic, are located at the intersections of the dashed curve
with the dispersion curves of q = 1 modes with transverse quantization
numbers p = 0 and 1, i.e., symmetric and antisymmetric transverse
modes of the waveguide34. Interestingly, at x > 5 μm, the intensity of the second harmonic
becomes larger than the maximum intensity of the initial wave. We
associate this with a difference in the group velocities of the twowaves. In fact, although the waves have equal phase velocities, the second-
harmonic wave possesses the group velocity of 0.2 μm ns−1, which is 4
times smaller than the velocity of the initial wave of 0.8 μm ns−1. The
energy transferred by a wave is proportionalto the product of intensity
and group velocity. Therefore, when a wave with a large group velocity
is converted into a wave with a smaller group velocity, the intensity
must increase to satisfy the law of conservation of energy flux42. In
agreement with this interpretation, the maximum intensity of the
second harmonic does not exceed four times the maximum intensity
of the initial wave. Note, however, that the ratio of these intensities is
close to 4, reinforcing that there is high-efficiency energy transfer. Comparison of the data of Fig. 3c, f allows us to draw one more
important conclusion. The intensity of the second-harmonic wave at
5.12 GHz (Fig. 3f) growswith the propagation distance noticeably faster
than that of the wave at 4.94 GHz (Fig. 3c). These data show that the
second harmonic generation efficiency is higher for the mode p = 1. Evidence of resonant generation of second-harmonic spin waves https://doi.org/10.1038/s41467-024-46108-y 0
5
10
15
0.1
1
10
0
5
10
15
0.1
1
10
4.94 GHz
2.47 GHz
Intensity
Propagation
Intensity
2.47 G z
H
4.9
H
4 G z
)
u
. a
S
L
B
(
ytis
n
etni
stin
b
r
5.12 GHz
2. GHz
56
Intensity
Intensity
2. G z
56
H
5.12
H
G z
1
m
)
u
. a
S
L
B
(
ytis
n
etni
stin
b
r
Fig. 3 | Spatial mapping of resonantly interacting waves. Left column corre-
sponds to the resonance at fexc = 2.47 GHz, right column corresponds to the reso-
nance at fexc = 2.56 GHz. a, d Intensity and phase maps of the initially excited wave. b, e Intensity and phase maps of the second-harmonic wave. c, f Spatial
dependences of the intensity of the initial wave and the second-harmonic wave. Lines show the exponential fit of the data obtained for the initial wave. The data are
obtained at H = 500 Oe. Power of the excitation signal P = 0.1 mW. 0
5
10
15
0.1
1
10
5.12 GHz
2. GHz
56
Intensity
Intensity
2. G z
56
H
5.12
H
G z
)
u
. a
S
L
B
(
ytis
n
etni
stin
b
r 0
5
10
15
0.1
1
10
4.94 GHz
2.47 GHz
Intensity
Propagation
Intensity
2.47 G z
H
4.9
H
4 G z
)
u
. a
S
L
B
(
ytis
n
etni
stin
b
r
1
m 0
5
10
15
0.1
1
10
4.94 GHz
2.47 GHz
Intensity
Propagation
Intensity
2.47 G z
H
4.9
H
4 G z
)
u
. a
S
L
B
(
ytis
n
etni
stin
b
r
1
m
Fig. 3 | Spatial mapping of resonantly interacting waves. Left column corre-
sponds to the resonance at fexc = 2.47 GHz, right column corresponds to the reso-
nance at fexc = 2.56 GHz. a, d Intensity and phase maps of the initially excited wave. b, e Intensity and phase maps of the second-harmonic wave. c, f Spatial )
u
. a
S
L
B
(
ytis
n
etni
stin
b
r dependences of the intensity of the initial wave and the second-harmonic wave. Lines show the exponential fit of the data obtained for the initial wave. The data are
obtained at H = 500 Oe. Evidence of resonant generation of second-harmonic spin waves Evidence of resonant generation of second-harmonic spin waves
To prove the possibility of the resonant inter-mode process in practice,
we first perform measurements at H = 500 Oe. We apply to the antenna
an excitation signal at a frequency fexc varying from 2 to 3 GHz, which
corresponds to the frequency range of the fundamental mode (Fig. 1b),
and record the BLS signal at a frequency 2fexc to observe possible SHG. Figure 2a shows the frequency dependence recorded at a distance
x = 10 μm from the antenna. This curve exhibits two narrow peaks at
fexc = 2.47 and 2.56 GHz, while the intensity found at other frequencies
is below the noise background. This clearly shows that efficient second
harmonic generation is possible only at specific frequencies, which
indicates the resonant character of this process. In order to find the frequencies at which 2f and 2k match magnon
states in the sample, we plot the curve 2f(2k) on the dispersion diagram
(red dashed line in Fig. 1b). Any intersection of the 2f(2k) curve with
other magnon branches indicates a dedicated frequency for which
SHG can become a highly efficient process. As seen from Fig. 1b, the
dashed curve never intersects the curve for the fundamental mode
(q = 0, p = 0). In other words, the required resonant condition cannot Figure 2b shows the complete BLS spectra recorded at two exci-
tation frequencies corresponding to the observed resonances. These
spectra allow one to simultaneously observe signals at the excitation
frequency, as well asthosecorresponding to the second harmonic. The
data show that, at x = 10 μm, the intensity of the second harmonic
exceeds that of the initially excited wave by more than a factor of two Nature Communications| (2024) 15:1827 3 https://doi.org/10.1038/s41467-024-46108-y Article 0
5
10
15
0.1
1
10
5.12 GHz
2. GHz
56
Intensity
Intensity
2. G z
56
H
5.12
H
G z
)
u
. a
S
L
B
(
ytis
n
etni
stin
b
r
dependences of the intensity of the initial wave and the second-harmonic wave. Lines show the exponential fit of the data obtained for the initial wave. The data are
obtained at H = 500 Oe. Power of the excitation signal P = 0.1 mW. Evidence of resonant generation of second-harmonic spin waves This is understandable, since the confluence of two magnons is also
expected to cause a doubling of the transverse component of the
wavevector, which favors generation of the mode p = 1. We also note,
that the spatial decay of the initial wave at 2.56 GHz occurs faster than
at 2.47 GHz. This is also the result of the faster energy transfer from the
initial wave to the second harmonic due to the higher efficiency of the
process. Generally speaking, the efficiency of the inter-mode SHG
process is expected to be nonzero also for modes with p > 1. However,
these modes possess a very short effective wavelength in the direction
across the width of the waveguide and cannot be detected by our
measurement setup. Analysis of the data obtained for the second resonance at
fexc = 2.56 GHz (Fig. 3d–f) demonstrates the main difference between
the two observed resonances. Characteristics of the initial wave at
2.56 GHz (Fig. 3d) are not significantly different from those of the wave Nature Communications| (2024) 15:1827 4 https://doi.org/10.1038/s41467-024-46108-y Article y
c
n
e
u
q
erf
n
oitatic
x
E
(
)z
H
G
ta
ytis
n
etni
S
L
B
(
)
u
. a
stin
b
r
0
5
10
15
2.40
2.45
2.50
2.55
2.60
0.01
0.1
1
= 2.47 GHz
= 2.4 GHz
5
0
1 BLS intensity at 2
(a
. u
)
rb
nits
Fig. 4 | Off-resonance interaction. a Color-coded intensity of the second harmonic
in frequency-space coordinates. b Spatial dependences of the intensity of the
second harmonic recorded at fexc = 2.47 GHz (at resonance) and 2.45 GHz (out of
resonance). The data are obtained at H = 500 Oe. Power of the excitation sig-
nal P = 0.1 mW. 0.01
0.1
1
10
0.00
0.05
0.10
0.15
0.20
0
1
2
3
4
5
ta
ytis
n
etni
S
L
B
(
)
u
. a
stin
b
r
= 2.47 GHz
= 2. GHz
56
ta
ytis
n
etni
S
L
B
(
)
u
. a
stin
b
r
Fig. 5 | Dependence on the excitation power. a Intensity of the second-harmonic
wave for the two resonances, as labeled, as a function of the power of the excitation
signal, P. The data are recorded at a distance x = 10 μm. Symbols show experimental
data. Off-resonance interaction Let us now discuss the generation of the second harmonic at fre-
quencies outside the resonances. We vary the excitation frequency fexc
in the range 2.4–2.6 GHz around the found resonances and record
spatial dependences of the intensity of the second harmonic at 2fexc. The obtained results (Fig. 4a) show that the spectral width of the
resonant peaks strongly decreases with increasing propagation dis-
tance x. This is a natural feature of the resonant interaction, which
requires phase matching between the initial wave and the second
harmonic along the entire interaction path. At large interaction dis-
tances, the result becomes more sensitive to the difference in the
phase velocities of the interacting waves. Correspondingly, efficient
energy exchange can only be achieved over a narrow frequency
interval. On the contrary, at small distances, a significant mismatch of
phase velocities does not lead to a strong mismatch of the phases of
the interacting waves, facilitating energy exchange over a wider
spectral region. We emphasize, however, that the maximum achiev-
able amplitudes of the second harmonic are much smaller in this case. This is demonstrated in Fig. 4b, which shows sections of the map
Fig. 4a for the resonant frequency 2.47 GHz and an off-resonance fre-
quency of 2.45 GHz. As seen from these data, in the region x = 0–2 μm,
the intensity of the second harmonic grows similarly for both fre-
quencies. However, in the non-resonant case (fexc = 2.45 GHz), the
intensity starts to decrease at x > 2 μm until it completely vanishes at
x = 4 μm, only to return periodically for larger distances. This dimin-
ishment arises as the initial wave periodically becomes out of phase
relative to the second-harmonic wave and, thus, suppresses it. The
observed behaviors are similar to those found in optical systems3,
where the frequency dependence of the refractive index tends to lead
to a phase mismatch between the initial wave and the second harmo-
nic, unless special phase-matching approaches are used. As discussed above, generation of the second harmonic relies on the
component of the dynamic magnetization mz / m2
x m2
y (Fig. 1a),
which, in the first approximation, is proportional the square of the
amplitude of dynamic magnetization at the frequency of initially exci-
ted precession5. Therefore, we expect that the intensity of the second
harmonic will be proportional to the square of the intensity of the initial
wave. Evidence of resonant generation of second-harmonic spin waves a Intensity of the second-harmonic
wave for the two resonances, as labeled, as a function of the power of the excitation
signal, P. The data are recorded at a distance x = 10 μm. Symbols show experimental
data. Curves show the fit by a parabolic function. b Spatial dependences of the
intensity of the second harmonic recorded at P = 0.1 mW and 0.2 mW, as labeled. The data are obtained at H = 500 Oe. Fig. 4 | Off-resonance interaction. a Color-coded intensity of the second harmonic
in frequency-space coordinates. b Spatial dependences of the intensity of the
second harmonic recorded at fexc = 2.47 GHz (at resonance) and 2.45 GHz (out of
resonance). The data are obtained at H = 500 Oe. Power of the excitation sig-
nal P = 0.1 mW. Fig. 4 | Off-resonance interaction. a Color-coded intensity of the second harmonic
in frequency-space coordinates. b Spatial dependences of the intensity of the
second harmonic recorded at fexc = 2.47 GHz (at resonance) and 2.45 GHz (out of
resonance). The data are obtained at H = 500 Oe. Power of the excitation sig-
nal P = 0.1 mW. Evidence of resonant generation of second-harmonic spin waves Curves show the fit by a parabolic function. b Spatial dependences of the
intensity of the second harmonic recorded at P = 0.1 mW and 0.2 mW, as labeled. The data are obtained at H = 500 Oe. y
c
n
e
u
q
erf
n
oitatic
x
E
(
)z
H
G
0
5
10
15
2.40
2.45
2.50
2.55
2.60
0
1 BLS intensity at 2
(a
. u
)
rb
nits 0.00
0.05
0.10
0.15
0.20
0
1
2
3
4
5
ta
ytis
n
etni
S
L
B
(
)
u
. a
stin
b
r
= 2.47 GHz
= 2. GHz
56 0.01
0.1
1
10
0.00
0.05
0.10
0.15
0.20
0
1
2
3
4
5
ta
ytis
n
etni
S
L
B
(
)
u
. a
stin
b
r
= 2.47 GHz
= 2. GHz
56
ta
ytis
n
etni
S
L
B
(
)
u
. a
stin
b
r
Fig. 5 | Dependence on the excitation power. a Intensity of the second-harmonic
wave for the two resonances, as labeled, as a function of the power of the excitation
signal, P. The data are recorded at a distance x = 10 μm. Symbols show experimental
data. Curves show the fit by a parabolic function. b Spatial dependences of the
intensity of the second harmonic recorded at P = 0.1 mW and 0.2 mW, as labeled. The data are obtained at H = 500 Oe. 0.01
0.1
1
10
ta
ytis
n
etni
S
L
B
(
)
u
. a
stin
b
r ta
ytis
n
etni
S
L
B
(
)
u
. a
stin
b
r
0.01
0.1
1
= 2.47 GHz
= 2.4 GHz
5 Fig. 5 | Dependence on the excitation power. a Intensity of the second-harmonic
wave for the two resonances, as labeled, as a function of the power of the excitation
signal, P. The data are recorded at a distance x = 10 μm. Symbols show experimental
data. Curves show the fit by a parabolic function. b Spatial dependences of the
intensity of the second harmonic recorded at P = 0.1 mW and 0.2 mW, as labeled. The data are obtained at H = 500 Oe. Fig. 5 | Dependence on the excitation power. Off-resonance interaction This is confirmed by the results presented in Fig. 5a, where we plot
the intensity of the second-harmonic signal for the two resonances as a
function of the power of the excitation signal, P, which is proportional
to the intensity of the initial spin wave. As seen from Fig. 5a, the
experimental data (symbols) are fit well with parabolic functions (solid
curves). We note that the second harmonic generation is a non-
threshold process that can be observed at arbitrarily small intensities of
the initial wave. However, due to the nonlinear dependence of the
intensity of the second harmonic on the intensity of the initial wave, it
only becomes clearly pronounced at large intensities of the initial wave. y
y p
g
Figure 5b shows spatial dependences of the second harmonic
intensity obtained for the first resonance (fexc = 2.45 GHz) at excitation
powers P = 0.1 and 0.2 mW. According to the aforementioned quad-
ratic dependence, at a given distance x ≤10 μm, the intensity of the
second harmonic increases by about four times when the intensity of
the initial wave is doubled. This implies that the spatial rate of the
energy transfer from the initial wave to the second harmonic increases
with the increase in P. This greater efficiency gives rise to faster spatial
attenuation of the initial wave at P = 0.2 mW in comparison with
P = 0.1 mW. As a result, at this power, the energy transfer from the
initial wave beyond x = 10 μm can no longer fully compensate the
attenuation of the second-harmonic wave and the intensity of the
latter starts to decrease, in contrast with P = 0.1 mW, where this
decrease occurs at x > 15 μm. Nature Communications| (2024) 15:1827 5 5 https://doi.org/10.1038/s41467-024-46108-y Article 350
400
450
500
0.3
0.4
0.5
0.6
0.7
0.8
0.9
(
htg
n
ele
v
a
W
)
m
Fig. 6 | Dependence on the static magnetic field. Field dependences of the
wavelengths of the second-harmonic waves for two resonances corresponding to
q = 1 modes with transverse quantization numbers p = 0 and 1, as labeled. Symbols
show experimental data. Curves show theoretical dependences obtained from
calculations of dispersion spectra. Experimental data are obtained at P = 0.1 mW. 350
400
450
500
0.3
0.4
0.5
0.6
0.7
0.8
0.9
(
htg
n
ele
v
a
W
)
m within the field. Calculation of the dispersion spectrum We use the nominal geometrical parameters of the waveguide and
standard
for
YIG
saturation
magnetization
4πMs = 1750 G. The
exchange constant A is used as an adjustable parameter. An excellent
agreement between the experimental and the calculated dispersion is
achieved in a broad range of the static magnetic field for exchange
constant of 3.25 erg/cm, which is very close to the standard for YIG
3.66 erg/cm. Off-resonance interaction Additionally, the demonstrated approach is funda-
mental and is not limited to spin waves. Indeed, engineering the inter-
mode second-harmonic generation in other thin-film nanostructure
systems may just grant the ability to exploit other types of waves (e.g. elastic waves) as well. Micro-focus BLS measurements Micro focus BLS measurements
Measurements are performed at room temperature. For the
magneto-optical detection of propagating spin waves, we focus the
probing laser light into a diffraction-limited spot on the surface of
the YIG waveguide using a high-performance 100× microscope
objective lens with a numerical aperture of 0.9. The probing light
with a wavelength of 437 nm and a power of 0.25 mW is produced by
a single-frequency laser. The spectrum of the light inelastically
scattered from magnetic oscillations is analyzed using six-pass
Fabry–Perot interferometer. The measured intensity of the scat-
tered light is proportional to the intensity of spin waves. To obtain
additional resolution with respect to the phase of spin waves, we use
the interference of the scattered light with the light modulated by
the signal used to excite spin waves. After processing, we obtain a
value proportional to cos(φ), where φ is the difference of the phase
of the spin wave at the measurement position and the phase of the
signal applied to the antenna. In conclusion, our results provide direct experimental evidence of
highly efficient resonant second-harmonic generation by spin waves
enabled by the engineering of the dispersion spectrum of spin waves in
nanoscale YIG waveguides. This engineering allows one to fulfill the
resonant
conditions
for
the
three-magnon
interaction
processes38,39,43–45, in which the non-zero wavevector and the frequency
of magnons can be doubled simultaneously. The demonstrated
approach is flexible and can be customized for different microwave
frequency ranges by simply varying the thickness of the magnetic
waveguide. For example, for YIG waveguides with a thickness of
10–20 nm, the resonant conditions can be fulfilled for sub-THz band
frequencies. The use of films with smaller thicknesses also makes it
possible to tune the dispersion spectrum to achieve the fulfillment of
the resonant conditions for the generation of higher-order harmonics. In addition to clear-cut and bountiful opportunities of generating high-
frequency, ultra-short spin waves, resonant second-harmonic genera-
tion can also be used to implement novel magnonic devices. For
instance, until now, magnonic devices exploiting wave-interference
effects could only operate with signals carried by waves of the same
frequency. The phase-locked second-harmonic generation process
allows such devices to operate simultaneously at the fundamental and
double frequencies. These possibilities will help to extend the func-
tionalities of magnonic circuits and will propel new developments Data availability The data that support the findings of this study are available from the
corresponding author upon reasonable request. Sample fabrication To fabricate the YIG waveguides, first a double-layer of PMMA resist
was spin coated onto GGG < 111 > , then 8 nm of gold was evaporated
to provide a conductive layer, and lastly the structures were pat-
terned using e-beam lithography. Afterwards, the sample was
placed in a potassium iodide solution to etch away the gold layer
and then was developed in pure isopropanol. It was further pro-
cessed with oxygen plasma to remove any remaining resist in the
developed areas. Using the recipe established by Hauser et al.32,
nominally 100 nm of YIG was deposited at room temperature by
pulsed laser deposition and lifted-off in acetone. The sample was
annealed in oxygen for 3 h at 800 degrees, followed by a phosphoric
acid etch to remove about 20 nm of YIG for precise thickness
engineering and smoother edges. In order to do high frequency
measurements on the sample, microstrip antennas had to be over-
layed on top of the YIG waveguides. The same fabrication process
used for the YIG waveguides, up until the PLD step, was used to
pattern gold antennas on top. At this stage, 10 nm of titanium and
200 nm of gold were deposited by e-beam evaporation and then
lifted off in acetone to complete the fabrication of the gold
antennas. Fig. 6 | Dependence on the static magnetic field. Field dependences of the
wavelengths of the second-harmonic waves for two resonances corresponding to
q = 1 modes with transverse quantization numbers p = 0 and 1, as labeled. Symbols
show experimental data. Curves show theoretical dependences obtained from
calculations of dispersion spectra. Experimental data are obtained at P = 0.1 mW. References
1.
Franken, P., Hill, A., Peters, C. & Weinreich, G. Generation of Optical
Harmonics. Phys. Rev. Lett. 7, 118–119 (1961).
2.
Powers, P. E. & Haus, J. W. Fundamentals of Nonlinear Optics (CRC
Press, London, 2017). Dependence on the static magnetic field Dependence on the static magnetic field
Finally, we discuss the effects of the static magnetic field H on the
studied phenomena. In the first approximation, a decrease in H
shifts the dispersion spectrum (Fig. 1b) down without significantly
affecting the frequency gap between the fundamental mode and
q = 1 modes, which is determined by the thickness of the magnetic
film, as well as by its saturation magnetization and the exchange
constant35. Under these conditions, the intersection points corre-
sponding to the resonant interaction shift towards larger wave-
vectors (smaller wavelengths). To demonstrate this, we vary the
static magnetic field, determine the resonant frequencies, and find
the wavelengths of the second-harmonic waves from phase-
resolved measurements. The results of these measurements are
summarized in Fig. 6. The values of the wavelengths obtained from
the experiment (symbols) are in good agreement with the results of
analytical calculations (curves). As seen from Fig. 6, by decreasing H
to 350 Oe, second-harmonic waves with the wavelength below
500 nm can be resonantly generated. Such short waves cannot be
excited directly by the used antenna34. In other words, the resonant
second-harmonic generation process can be used to achieve effi-
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based microwave technology. Nat. Commun. 7, 11255 (2016). K.O.N. performed measurements and data analysis. S.R.L grew and
characterized the films, performed nanofabrication and data analysis. G.S., S.O.D. and V.E.D. formulated the experimental approach and
supervised the project. All authors co-wrote the manuscript. 26. Liu, C. et al. Long-distance propagation of short-wavelength spin
waves. Nat. Commun. 9, 738 (2018). 27. Sluka, V. et al. Emission and propagation of 1D and 2D spin waves
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Nanotech 14, 328 (2019). Funding Open Access funding enabled and organized by Projekt DEAL. Open Access funding enabled and organized by Projekt DEAL. 28. Sun, Y. et al. Growth and ferromagnetic resonance properties of
nanometer-thick yttrium iron garnet films. Appl. Phys. Lett. 101,
152405 (2012). Competing interests
The authors declare no competing interests. Competing interests
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Гончаров И. В. Гончаров И. В. Особенности функционирования механизма государственной власти современной России: традиции и зарубежный опыт Особенности функционирования механизма государственной власти современной России: традиции и зарубежный опыт © Гончаров И. В., 2018
*
Гончаров Игорь Владимирович, доктор юридических наук, профессор, заведующий кафедрой государ-
ственного строительства и права Университета прокуратуры Российской Федерации, профессор кафед-
ры государственно-правовых дисциплин Академии управления МВД Российской Федерации
goncharov-i@mail.ru
107078, Россия, г. Москва, ул. Новая Басманная, д. 10 © Гончаров И. В., 2018 ВВЕДЕНИЕ правления, предполагающей особое место гла-
вы государства в механизме государственной
власти и весьма специфичный механизм реали-
зации конституционного принципа разделения
властей во взаимоотношениях органов государ-
ственной власти. В этом плане Российская Фе-
дерация исключением не является. правления, предполагающей особое место гла-
вы государства в механизме государственной
власти и весьма специфичный механизм реали-
зации конституционного принципа разделения
властей во взаимоотношениях органов государ-
ственной власти. В этом плане Российская Фе-
дерация исключением не является. Принятие 12 декабря 1993 г. Конституции Рос-
сийской Федерации явилось важнейшей вехой
в становлении российской государственности. Ее принятие позволило Российской Федерации
пережить непростой этап кардинального ре-
формирования общественного и политическо-
го строя страны, обусловленного кризисными
явлениями, имевшими место в развитии рос-
сийского государства, в том числе в сфере на-
циональных отношений, усилением различного
рода центробежных тенденций во взаимоотно-
шениях между федеральным центром и субъ-
ектами Федерации, имевших место в начале
1990‑х гг. ОСОБОЕ МЕСТО ПРЕЗИДЕНТА РФ
В СИСТЕМЕ ГОСУДАРСТВЕННОЙ ВЛАСТИ Президент Российской Федерации также за-
нимает особое место в системе органов госу-
дарственной власти. В соответствии с Консти-
туцией РФ Президент — это прежде всего глава
государства, он не входит ни в одну из ветвей
власти и в этом статусе, реализуя свои полно-
мочия, обеспечивает согласованное взаимодей-
ствие всех органов государственной власти как
на федеральном уровне, так и во взаимоотно-
шениях федеральных органов власти и органов
власти субъектов Российской Федерации. Кон-
ституция для этого наделяет его всеми необхо-
димыми полномочиями. Более того, именно
Президент РФ в соответствии с ч. 3 ст. 80 Кон-
ституции РФ определяет основные направления
внутренней и внешней политики государства. Причем подавляющее большинство своих кон-
ституционных полномочий Президент реализует
самостоятельно. И в этом проявляется уже рос-
сийский подход к пониманию роли главы госу-
дарства в управлении страной. Если посмотреть
на конституционный опыт Франции, то надо
признать, что и французский президент — это
«сильный» глава государства. Но в соответствии
со ст. 19 действующей Конституции Франции
большинство из своих полномочий глава госу-
дарства реализует через институт «контрассиг-
нации» «премьер-министром и в случае необ-
ходимости ответственными министрами». В Рос-
сийской Федерации глава государства в этом
плане ничем не связан. И. В. Гончаров*
ОСОБЕННОСТИ ФУНКЦИОНИРОВАНИЯ
МЕХАНИЗМА ГОСУДАРСТВЕННОЙ ВЛАСТИ
СОВРЕМЕННОЙ РОССИИ:
ТРАДИЦИИ И ЗАРУБЕЖНЫЙ ОПЫТ Аннотация. В статье рассматриваются особенности формы правления Российской Феде-
рации, определяющие специфику механизма взаимодействия главы государства и органов
исполнительной и законодательной власти Российской Федерации. Роль Конституции Рос-
сийской Федерации в становлении механизма государственной власти трудно переоце-
нить, поскольку она была призвана как решить вопросы организации власти, так и найти
баланс отношений «Федерация — субъект Федерации», а также соблюдать принципы
федерализма и единства системы государственной власти. Во многом именно принятие
Конституции 1993 г. позволило стабилизировать общественно-политическую ситуацию
в Российской Федерации. Уделено пристальное внимание специфической форме правления
Российской Федерации — так называемой полупрезидентской, или смешанной, республике. Крайне занимателен статус Президента Российской Федерации, учитывая закрепленную
Конституцией форму правления, поскольку личностный фактор, по мнению многих уче-
ных, играет далеко не последнюю роль в государственном строительстве и в истори-
ческой перспективе, особенно учитывая то, насколько Россия преобразилась на рубеже
столетий. Анализ конституционных норм и уже имеющаяся практика взаимоотношений
Президента РФ с законодательными органами государственной власти РФ позволяют
сделать выводы о том, что настоящая Конституция РФ юридически использует при за-
креплении полномочий органов государственной власти Российской Федерации «теорию
рационализированного парламентаризма», предполагающую правовое конституционное
закрепление ограничения роли парламента в управлении государством и господство в го-
сударственном механизме в первую очередь главы государства и исполнительной власти. По мнению разработчиков данной теории, это необходимо для повышения эффективно-
сти государственного управления. Рассматриваются влияние исторических традиций организации власти в российском го-
сударстве и соответствующего зарубежного опыта на функционирование механизма го-
сударственной власти в Российской Федерации. Ключевые слова: Президент Российской Федерации, Федеральное собрание Российской Фе-
дерации, Правительство Российской Федерации, форма правления, теория «рационали-
зированного парламентаризма», принцип разделения властей, контрольные полномочия
парламента. DOI: 10.17803/1729-5920.2018.144.11.145-150 LEX RUSSICA 145 № 11 (144) ноябрь 2018 ЭВОЛЮЦИЯ КОНСТИТУЦИОННОГО РЕГУЛИРОВАНИЯ
СИСТЕМЫ ОРГАНОВ ПУБЛИЧНОЙ ВЛАСТИ РОССИЯ — ПОЛУПРЕЗИДЕНТСКАЯ РЕСПУБЛИКА Действующая Конституция РФ закрепляет весь-
ма специфичную форму правления Российской
Федерации, в теории конституционного права
называемую полупрезидентской республикой
или республикой смешанного типа. Впервые
данная форма правления была закреплена
в действующей Конституции Франции 1958 г. и, в принципе, доказала свою эффективность
в государственной практике Пятой республи-
ки. Во многом благодаря ей удалось справить-
ся с кризисом французской государственности
в 1960‑х гг., когда это государство переживало
далеко не лучшие времена, обусловленные
крахом Франции как колониальной державы,
военным мятежом в Алжире (так называемое
«движение 13 мая»), мятежом на острове Кор-
сике, аналогичным алжирскому, усилением
антиправительственных выступлений внутри
страны. Российская Федерация в 1990‑х гг. также пе-
реживала далеко не простые времена, во мно-
гом схожие с событиями во Франции 1960‑х гг. И разработчики проекта Конституции РФ не
могли не учитывать соответствующий француз-
ский опыт. В настоящее время уже можно кон-
статировать, что во многом именно принятие
Конституции РФ 1993 г. позволило стабилизи-
ровать общественно-политическую ситуацию
в Российской Федерации. Весьма специфичны взаимоотношения Пре-
зидента РФ с органами законодательной власти
Российской Федерации, прежде всего с Государ-
ственной Думой Федерального Собрания РФ. Анализ соответствующих конституционных
норм и уже имеющаяся практика взаимоотно-
шений Президента с законодательными органа-
ми государственной власти РФ позволяет сде-
лать выводы, что действующая Конституция РФ
юридически использует при закреплении
полномочий органов государственной власти «Полупрезидентская республика» как форма
правления не получила большого распростра-
нения в государственном механизме современ-
ных государств. Как представляется, это проис-
ходит именно в силу специфики данной формы 146 № 11 (144) ноябрь 2018 Гончаров И. В. Особенности функционирования механизма государственной власти современной России: традиции и зарубежный опыт р
Особенности функционирования механизма государственной власти современной России: традиции и зарубежный опыт Российской Федерации «теорию рационализи-
рованного парламентаризма», предполагаю-
щую правовое конституционное закрепление
ограничения роли парламента в управлении
государством и доминирование в государствен-
ном механизме прежде всего главы государства
и исполнительной власти. По мнению разработ-
чиков данной теории, это необходимо прежде
всего для повышения эффективности государ-
ственного управления. ция которого в механизме разделения властей
заключается в осуществлении именно законо-
дательной власти. Позиции ученых по поводу сокращения вы-
шеназванных полномочий парламента различ-
ны, вплоть до полярных. Одни считают, что это
«отражает реальный федерализм, построенный
на строгом разделении предметов ве́дения
и полномочий органов государственной вла-
сти Российской Федерации»1. Другие, наобо-
рот, полагают, что это нарушает конституцион-
ный принцип разделения властей в правовом
государстве2. В этом уместно посмотреть аме-
риканскую конституционную модель взаимоот-
ношений между исполнительной властью, воз-
главляемой Президентом США, и Конгрессом
США. РОССИЯ — ПОЛУПРЕЗИДЕНТСКАЯ РЕСПУБЛИКА По-разному можно относиться к этой мо-
дели, но «сильный» американский президент
в необходимых случаях решениями Конгресса
может быть значительно ограничен в реализа-
ции своих полномочий3. Руководствуясь этим и используя уже упо-
минавшийся французский опыт, создатели рос-
сийской Конституции закрепили в ст. 102, 103
исчерпывающий перечень полномочий Совета
Федерации и Государственной Думы, который
не носит расширительного характера. Такое
теоретически возможно в функционировании
государственного механизма, но фактически
в определенных случаях может сложиться си-
туация, когда реализация Государственной Ду-
мой своих конституционных полномочий мо-
жет привести к ее роспуску, что не должно быть
в принципе. Такая ситуация может сложиться,
например, при даче согласия Государственной
Думы Президенту РФ на назначение Предсе-
дателя Правительства РФ. Кроме того, п. «б»
ст. 103 Конституции РФ относит к ве́дению Го-
сударственной Думы «решение вопроса о до-
верии Правительству Российской Федерации»,
но реализация указанных полномочий также
может привести к роспуску нижней палаты
российского парламента решением главы го-
сударства. 1
Баглай М. В. Конституционное право Российской Федерации. М., 1999. С. 427.
2
Баранов П. П. Власть и общество: испытание Конституцией // Независимая газета. № 105 (7234).
29.05.2018.
3
Уже имеющаяся практика взаимоотношений действующего Президента США Д. Трампа и Конгресса
США только служит подтверждением этого. 3
Уже имеющаяся практика взаимоотношений действующего Президента США Д. Трампа и Конгресса
США только служит подтверждением этого. LEX RUSSICA Опалева, «персонификация вла-
сти предполагает восприятие власти не как
политического института, а как конкретной
личности, в которой эта власть воплощает-
ся. Личным качествам представителя власти
придается большее значение, чем законо
творчеству, устройству и функционированию
госаппарата»7; — во-первых, парламентские формы правле-
ния характерны для стран с уже сложив-
шимися демократическими институтами,
устойчивыми парламентскими традициями. К сожалению, к числу таких стран Россий-
скую Федерацию пока отнести еще сложно; Кроме того, в России федеральный парла-
мент осуществляет контрольные полномочия,
в том числе посредством специально создава-
емых органов. Так, например, в соответствии
с ч. 1 ст. 101 Конституции РФ «для осуществле-
ния контроля за исполнением федерального
бюджета Совет Федерации и Государственная
Дума образуют Счетную палату, состав и поря-
док деятельности которой определяется соот-
ветствующим федеральным законом»4. Пункт «е» ст. 103 Конституции РФ наделяет
Государственную Думу полномочиями по на-
значению на должность Уполномоченного по
правам человека в РФ, деятельность которого
регулируется соответствующим Федеральным
конституционным законом «Об Уполномочен-
ном по правам человека в Российской Федера-
ции»5. Данное должностное лицо призвано рас-
сматривать соответствующие жалобы граждан
России и находящихся на территории Россий-
ской Федерации иностранных граждан и лиц
без гражданства на решения или действия орга-
нов государственной власти, органов местного
самоуправления, их должностных лиц. То есть
через призму деятельности Уполномоченного
по правам человека законодательная власть
реализует свои контрольные полномочия за
деятельностью органов исполнительной власти
Российской Федерации. — в-четвертых, данная форма правления
предполагает четкую вертикаль власти, где
отношения в системе органов исполнитель-
ной власти строятся на принципах суборди-
нации и подчинения. И это тоже российские
реалии. К сожалению, приходится констати-
ровать, что на данном этапе исторического
развития без сильной вертикали властных
отношений Россия как единое государство LEX RUSSICA комиссии в целях проведения расследования
тех или иных фактов либо рассмотрения какого-
либо вопроса государственной важности. Без
соответствующего решения парламента невоз-
можно ввести на территории Российской Феде-
рации или в отдельных ее частях специальные
правовые режимы: военный или чрезвычайного
положения. комиссии в целях проведения расследования
тех или иных фактов либо рассмотрения какого-
либо вопроса государственной важности. Без
соответствующего решения парламента невоз-
можно ввести на территории Российской Феде-
рации или в отдельных ее частях специальные
правовые режимы: военный или чрезвычайного
положения. ламента6, но тем не менее на данный период
времени эта форма правления представляет-
ся оптимальной для Российской Федерации. И тому есть свои причины: — во-первых, парламентские формы правле-
ния характерны для стран с уже сложив-
шимися демократическими институтами,
устойчивыми парламентскими традициями. К сожалению, к числу таких стран Россий-
скую Федерацию пока отнести еще сложно;
— во-вторых, парламентские формы правле-
ния объективно имеют определенные не-
достатки, связанные с естественной инер-
ционностью при принятии необходимых
оперативных решений при управлении го-
сударством. Особенно это касается ситуа-
ций, когда необходимо принимать быстрые
управленческие решения при возникно-
вении в стране каких-либо чрезвычайных
и экстраординарных ситуаций. В этом плане
в Российской Федерации также еще не все
так просто в плане стабильного развития
общественно-политических процессов;
— в-третьих, нельзя не учитывать исторические
традиции России. Они во все времена пред-
полагали четкую персонифицированность
власти. А это нехарактерно для парламент-
ских форм правления. Как справедливо от-
мечает А. А. Опалева, «персонификация вла-
сти предполагает восприятие власти не как
политического института, а как конкретной
личности, в которой эта власть воплощает-
ся. Личным качествам представителя власти
придается большее значение, чем законо
творчеству, устройству и функционированию
госаппарата»7; — во-первых, парламентские формы правле-
ния характерны для стран с уже сложив-
шимися демократическими институтами,
устойчивыми парламентскими традициями. К сожалению, к числу таких стран Россий-
скую Федерацию пока отнести еще сложно;
— во-вторых, парламентские формы правле-
ния объективно имеют определенные не-
достатки, связанные с естественной инер-
ционностью при принятии необходимых
оперативных решений при управлении го-
сударством. Особенно это касается ситуа-
ций, когда необходимо принимать быстрые
управленческие решения при возникно-
вении в стране каких-либо чрезвычайных
и экстраординарных ситуаций. В этом плане
в Российской Федерации также еще не все
так просто в плане стабильного развития
общественно-политических процессов;
— в-третьих, нельзя не учитывать исторические
традиции России. Они во все времена пред-
полагали четкую персонифицированность
власти. А это нехарактерно для парламент-
ских форм правления. Как справедливо от-
мечает А. А. 4
Федеральный закон от 5 апреля 2013 г. № 41‑ФЗ «О Счетной палате Российской Федерации» (в ред. от
28.12.2016) // Российская газета. Федеральный выпуск. № 6053 (77). 10.04.2013. 28.12.2016) // Российская газета. Федеральный выпуск. № 6053 (77). 10.04.2013.
5
Федеральный конституционный закон от 27 февраля 1997 г. № 1-ФКЗ «Об Уполномоченном по правам
человека в Российской Федерации» (в ред. от 06.04.2015) // Российская газета. Федеральный выпуск.
№ 6644 (73). 08.04.2015. (
)
6
Есть подсчеты, что в России президентские полномочия в 4 раза превышают парламентские. 7
Опалева А. А. Безопасность личности в системе национальной безопасности // Взаимодействие между-
народного и внутригосударственного права Российской Федерации по обеспечению личности, обще- ФЕДЕРАЛЬНЫЙ ПАРЛАМЕНТ
В СИСТЕМЕ РАЗДЕЛЕНИЯ ВЛАСТЕЙ Конечно, нельзя и неправильно упрощать ме-
сто и роль федерального парламента в государ-
ственно-правовом механизме Российской Феде-
рации. Конституция России наделяет федераль-
ный парламент соответствующими контроль-
ными полномочиями. Перечень контрольных
полномочий парламента весьма значителен. К ним относятся отрешение Президента РФ от
должности, заслушивание Председателя Прави-
тельства РФ о проделанной работе за отчетный
год, контроль за исполнением бюджета (п. «а»
ч. 1 ст. 114 Конституции РФ возлагает на Прави-
тельство РФ обязанность представлять Государ-
ственной Думе отчет о его исполнении), приня-
тие решения по постановке вопроса о доверии
правительству; депутатские запросы, представ-
ляющие собой обращения к конкретным долж-
ностным лицам исполнительной власти в целях
получения соответствующей информации или
необходимых комментариев по конкретным
фактам; право обеих палат федерального пар-
ламента создавать специальные парламентские Действующая Конституция РФ не предус-
матривает конкретных полномочий исполни-
тельно-распорядительного характера в ком-
петенции Федерального Собрания, а объем
имеющихся контрольных полномочий зна-
чительно сокращен. По мнению разработчи-
ков проекта Конституции, это юридически на
конституционном уровне закрепляет пере-
ход от советской парламентской концепции,
согласно которой парламент является един-
ственным высшим органом государственной
власти, к концепции парламента как органа
законодательной власти. Современный рос-
сийский парламент — это представительный
орган государственной власти, основная функ- LEX RUSSICA № 11 (144) ноябрь 2018 147 ЭВОЛЮЦИЯ КОНСТИТУЦИОННОГО РЕГУЛИРОВАНИЯ
СИСТЕМЫ ОРГАНОВ ПУБЛИЧНОЙ ВЛАСТИ РОЛЬ ЛИЧНОСТНОГО ФАКТОРА
В ГОСУДАРСТВЕННОМ СТРОИТЕЛЬСТВЕ Конечно, приходится констатировать, что со-
отношение президентских и парламентских
полномочий явно не в пользу российского пар- 4
Федеральный закон от 5 апреля 2013 г. № 41‑ФЗ «О Счетной палате Российской Федерации» (в ред. от
28.12.2016) // Российская газета. Федеральный выпуск. № 6053 (77). 10.04.2013. 5
Федеральный конституционный закон от 27 февраля 1997 г. № 1-ФКЗ «Об Уполномоченном по правам
человека в Российской Федерации» (в ред. от 06.04.2015) // Российская газета. Федеральный выпуск. № 6644 (73). 08.04.2015. 6
Есть подсчеты, что в России президентские полномочия в 4 раза превышают парламентские. 7
Опалева А. А. Безопасность личности в системе национальной безопасности // Взаимодействие между-
народного и внутригосударственного права Российской Федерации по обеспечению личности, обще- 148 № 11 (144) ноябрь 2018 Гончаров И. В. Гончаров И. В. Особенности функционирования механизма государственной власти современной России: традиции и зарубежный опыт р
Особенности функционирования механизма государственной власти современной России: традиции и зарубежный опыт ет Н. А. Романович, «персонификация власти
имеет глубокие идеологические мировоззрен-
ческие корни и вплетена в политическую рос-
сийскую культуру»10. Позитивная динамика
развития российского государства в ХХI в., из-
менение места и роли Российской Федерации
в мире, приведшие к тому, что однополярный
мир в его американской версии утрачивает
способность к самоупорядочиванию, как раз
служит лучшим подтверждением правильно-
сти выбора правовых форм развития нашего го-
сударства. А именно: Конституция закрепляет
основы существующего государственного строя,
являясь каркасом, на котором держится обще-
ственное здание нашего государства. И в этой
связи с удовлетворением можно констатиро-
вать, что за прошедшие 25 лет (что, в общем-то,
совсем немного в историческом периоде разви-
тия государства) действующая Конституция РФ
сумела выполнить возложенные на нее функ-
ции и обеспечила стабильность политического
и экономического развития российского госу-
дарства, уникального по своим национально-
территориальным, религиозным, социокультур-
ным и геополитическим особенностям. существовать вообще не сможет либо ее
будут сотрясать перманентные кризисы,
сопровождающиеся снижением уровня
управляемости общественными, полити-
ческим и социально-экономическими про-
цессами. Как утверждает Ю. С. Пивоваров, «русская
власть предполагает режим персонификации»8. И это отмечают даже в «западном» мире. По
мнению многих западных политологов, «лич-
ностный фактор» сыграл далеко не последнюю,
а скорее всего, главную роль в том, что Россия
к XXI в. стала реально сильнее во многих отно-
шениях, чем была раньше9. Насколько это эффективно с точки зрения
функционирования государственного механиз-
ма? С точки зрения западных подходов — это
скорее вопрос риторический, так как «инсти-
туциональное устройство», традиции поли-
тической системы должны, как они считают,
преобладать над личностью, нивелировать
ее персональные качества. 9
Джонас Е А. «Путин изменил ландшафт России»: почему западные СМИ боятся рассказывать прав-
ду о президенте РФ и его стране // URL: https://rueconomics.ru/303776-putin-izmenil-landshaft-rossii-
pochemu-zapadnye-smi-boyatsya-rasskazyvat-pravdu-o-prezidente-rf-i-ego-strane (дата обращения: 14
июня 2018 г.). РОЛЬ ЛИЧНОСТНОГО ФАКТОРА
В ГОСУДАРСТВЕННОМ СТРОИТЕЛЬСТВЕ Но с точки зрения
наших реалий такое положение дел представ-
ляется неприемлемым для российского обще-
ственного сознания. Как справедливо отмеча- ства и государства : материалы межвузовской науч.-практ. конференции. М. : Академия управления
МВД РФ, 2012. С. 120. 10 Романович Н. А. К вопросу о персонализации власти в России // Власть. № 9. 2009. С. 13—16 Д
,
8
Пивоваров Ю. С. Русская власть и публичная политика // Полис. 2006. № 1. С. 26. БИБЛИОГРАФИЯ 1. Алексис Д. Е. «Путин изменил ландшафт России»: почему западные СМИ боятся рассказывать прав-
ду о президенте РФ и его стране // URL: https://rueconomics.ru/303776-putin-izmenil-landshaft-rossii-
pochemu-zapadnye-smi-boyatsya-rasskazyvat-pravdu-o-prezidente-rf-i-ego-strane (дата обращения: 14
июня 2018 г.). 1. Алексис Д. Е. «Путин изменил ландшафт России»: почему западные СМИ боятся рассказывать прав-
ду о президенте РФ и его стране // URL: https://rueconomics.ru/303776-putin-izmenil-landshaft-rossii-
pochemu-zapadnye-smi-boyatsya-rasskazyvat-pravdu-o-prezidente-rf-i-ego-strane (дата обращения: 14
июня 2018 г.). 2. Баглай М. В. Конституционное право Российской Федерации. — М., 1999. 2. Баглай М. В. Конституционное право Российской Федерации. — М., 1999. 3. Баранов П. П. Власть и общество: испытание Конституцией // Независимая газета. — № 105 (7234). —
29.05.2018. 3. Баранов П. П. Власть и общество: испытание Конституцией // Независимая газета. — № 105 (7234). —
29.05.2018. 4. Опалева А. А. Безопасность личности в системе национальной безопасности // Взаимодействие между-
народного и внутригосударственного права Российской Федерации по обеспечению личности, обще-
ства и государства : материалы межвузовской научно-практической конференции. — М. : Академия
управления МВД РФ, 2012. — С. 118—124. 4. Опалева А. А. Безопасность личности в системе национальной безопасности // Взаимодействие между-
народного и внутригосударственного права Российской Федерации по обеспечению личности, обще-
ства и государства : материалы межвузовской научно-практической конференции. — М. : Академия
управления МВД РФ, 2012. — С. 118—124. у р
Д
5. Пивоваров Ю. С. Русская власть и публичная политика // Полис. — 2006. — № 1. — С. 12—32. // 5. Пивоваров Ю. С. Русская власть и публичная политика // Полис. — 2006. — № 1. — С. 12—32. 6. Романович Н. А. К вопросу о персонализации власти в России // Власть. — 2009. — № 9. — С. 13—16. 5. Пивоваров Ю. С. Русская власть и публичная политика // Полис. — 2006. — № 1. — С. 12—32. 6. Романович Н. А. К вопросу о персонализации власти в России // Власть. — 2009. — № 9. — С PECULIARITIES OF MECHANISM OF STATE POWER OPERATION IN MODERN RUSSIA:
TRADITIONS AND FOREIGN EXPERIENCE The influence of historical traditions of the organization of powers in the Russian State and the corresponding
foreign experience on the mechanism of state power operation in the Russian Federation is considered. Keywords: President of the Russian Federation, Federal Assembly of the Russian Federation, government of the
Russian Federation, form of government, theory of “rationalized parliamentarism”, principle of separation of
powers, control powers of Parliament. PECULIARITIES OF MECHANISM OF STATE POWER OPERATION IN MODERN RUSSIA:
TRADITIONS AND FOREIGN EXPERIENCE GONCHAROV Igor Vladimirovich, Doctor of Law, Professor, Head of the Department of State
Construction and Law of the University of the Prosecutor’s Office of the Russian Federation, Professor
of the Department of State and Legal Disciplines of the Academy of Management of the Ministry of
Internal Affairs of the Russian Federation
goncharov-i@mail.ru
107078, Russia, Moscow, ul. Novaya Basmannaya, d. 10 Abstract. The article discusses the features of the form of government of the Russian Federation, which determine
the specifics of the mechanism of interaction between the head of the State and the executive and legislative
authorities of the Russian Federation. The role of the Constitution of the Russian Federation in the formation of
the mechanism of state power is difficult to overestimate. It was designed to solve the issues of organization of
power, and to find a balance of relations “Federation — constituent entity of the Federation”, as well as to comply
with the principles of federalism and unity of the system of state power. In many ways, it was the adoption of the
1993 Constitution that made it possible to stabilize the social and political situation in the Russian Federation. Close attention is given to the specific form of government of the Russian Federation — the so-called semi-
presidential or mixed Republic. The status of the President of the Russian Federation is extremely interesting,
given the form of government enshrined in the Constitution. The personal factor, according to many scientists,
plays a significant role in state-building and in historical perspective, especially given how Russia has changed at
the turn of the century. The analysis of the constitutional norms and the existing practice of relations between
the President of the Russian Federation and the legislative bodies of state power of the Russian Federation
makes it possible to draw conclusions that this Constitution of the Russian Federation legally uses the “theory of
rationalized parliamentarism” in the consolidation of the powers of the state authorities. It presupposes the legal
constitutional consolidation of the limitation of the role of Parliament in the state administration and domination
in the state mechanism, first of all of the head of state and the executive power. According to the developers of
this theory, it is necessary to improve the efficiency of public administration. Материал поступил в редакцию 18 июля 2018 г. Материал поступил в редакцию 18 июля 2018 г. Материал поступил в редакцию 18 июля 2018 г. ства и государства : материалы межвузовской науч.-практ. конференции. М. : Академия управления
МВД РФ, 2012. С. 120. Д
,
воваров Ю. С. Русская власть и публичная политика // Полис. 2006. № 1. С. 26. 9
Джонас Е А. «Путин изменил ландшафт России»: почему западные СМИ боятся рассказывать прав-
ду о президенте РФ и его стране // URL: https://rueconomics.ru/303776-putin-izmenil-landshaft-rossii-
pochemu-zapadnye-smi-boyatsya-rasskazyvat-pravdu-o-prezidente-rf-i-ego-strane (дата обращения: 14
июня 2018 г.). 10 Романович Н. А. К вопросу о персонализации власти в России // Власть. № 9. 2009. С. 13—16 LEX RUSSICA 149 № 11 (144) ноябрь 2018 ЭВОЛЮЦИЯ КОНСТИТУЦИОННОГО РЕГУЛИРОВАНИЯ
СИСТЕМЫ ОРГАНОВ ПУБЛИЧНОЙ ВЛАСТИ LEX RUSSICA LEX RUSSICA REFERENCES 1. Aleksis D.E. «Putin izmenil landshaft Rossii»: pochemu zapadnye smi boyatsya rasskazyvat pravdu o prezidente
RF i ego strane [“Putin has changed the landscape of Russia»: why Western media are afraid to tell the truth
about the Russian President and his country]. URL: https://rueconomics.ru/303776-putin-izmenil-landshaft-
rossii-pochemu-zapadnye-smi-boyatsya-rasskazyvat-pravdu-o-prezidente-rf-i-ego-strane (accessed: 14 June
2018). 1. Aleksis D.E. «Putin izmenil landshaft Rossii»: pochemu zapadnye smi boyatsya rasskazyvat pravdu o prezidente
RF i ego strane [“Putin has changed the landscape of Russia»: why Western media are afraid to tell the truth
about the Russian President and his country]. URL: https://rueconomics.ru/303776-putin-izmenil-landshaft-
rossii-pochemu-zapadnye-smi-boyatsya-rasskazyvat-pravdu-o-prezidente-rf-i-ego-strane (accessed: 14 June
2018).iiii 1. Aleksis D.E. «Putin izmenil landshaft Rossii»: pochemu zapadnye smi boyatsya rasskazyvat pravdu o prezidente
RF i ego strane [“Putin has changed the landscape of Russia»: why Western media are afraid to tell the truth
about the Russian President and his country]. URL: https://rueconomics.ru/303776-putin-izmenil-landshaft-
rossii-pochemu-zapadnye-smi-boyatsya-rasskazyvat-pravdu-o-prezidente-rf-i-ego-strane (accessed: 14 June
2018).iiii 3. Baranov P.P. Vlast i obshchestvo: ispytanie konstitutsiey [Authorities and society: test of the Constitution]. Nezavisimaya Gazeta. No. 105 (7234). 29.05.2018.ii 3. Baranov P.P. Vlast i obshchestvo: ispytanie konstitutsiey [Authorities and society: test of the Constitution]. Nezavisimaya Gazeta. No. 105 (7234). 29.05.2018. l
l hi
l
bi
[
l
h
f 4. Opaleva A. A. Bezopasnost lichnosti v sisteme natsionalnoy bezopasnosti [Personal security in the system of
national security]. Vzaimodeystvie mezhdunarodnogo i vnutrigosudarstvennogo prava Rossiyskoy Federatsii
po obespecheniyu lichnosti, obshchestva i gosudarstva: materialy mezhvuzovskoy nauchno-prakticheskoy
konferentsii [Interaction of international and domestic law of the Russian Federation to ensure the individ-
ual, society and state: materials of the interuniversity scientific-practical conference]. Moscow: Akademiya
upravleniya MVD RF, 2012. Pp. 118—124.i p
y
p
5. Pivovarov Y.S. Russkaya vlast i publichnaya politika [Russian government and public policy]. Polis. 2006. No. 1. Pp. 12—32.ii 6. Romanovich N.A. K voprosu o personalizatsii vlasti v Rossii [On the issue of personalization of power in Russia]. Vlast [Power]. 2009. No. 9. Pp. 13-16. № 11 (144) ноябрь 2018 150
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Review of: "Growing Confidence and Remaining Uncertainty About Animal Consciousness"
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Qeios, CC-BY 4.0 · Review, February 4, 2024 Review of: "Growing Confidence and Remaining Uncertainty
About Animal Consciousness" Christopher Rourk Potential competing interests: No potential competing interests to declare. The only thing that might be identified as a "competing interest" is the noted
disagreement between the reviewer's research and the author's conclusions, but that seems to be the purpose of peer review. Review of Growing Confidence and Remaining Uncertainty About Animal Consciousness The paper presents a review of the neuroscience literature that addresses phenomenological consciousness and
identifies five areas of broad consensus. It also concludes that there are three “major mysteries” that remain about it: (1)
the neurological correlates of consciousness, (2) the apparent gap between phenomenology and mechanism, and (3) the
agent that monitors the brain activity admitted into consciousness. The paper provides a thorough review of the neuroscience literature and reaches what appears to be a valid conclusion
that there are a number of areas of consensus. It is also correct that classical neuroscience cannot explain
phenomenological consciousness. It is very well written and informative, and if the major problem discussed below is
corrected, it will receive a “5-star” review. Paper weaknesses Qeios ID: QXUNGB · https://doi.org/10.32388/QXUNGB Paper weaknesses The paper states that “Most neuroscientists agree with the argument that consciousness arises from a physical substrate
in a manner that will be uncovered in time, when newer data and insights conveyed through a more suitable vocabulary
will clarify the link between mechanism and experience (P.S. Churchland, 1982; P.S. Churchland and Sejnowski, 1988;
P.M. Churchland, 2013). We are not there yet though.” However, the work I have been doing has provided evidence and
insight into the physics of that physical substrate that appears to be an important part of the link between mechanism and
experience. Because the paper completely ignores that evidence, I have to give it a “1-star” rating. The author might not
agree with some or all of my work, but at a minimum, any objective observations about that physical mechanism should
be discussed if the paper is to meaningfully address and support the conclusion that there are “major mysteries.” I will provide a brief overview of that evidence for the benefit of the author. In 2015, I read “Consciousness and the Brain:
Deciphering How the Brain Codes Our Thoughts” by Stanislas Dehaene and reached the same conclusion as the author
does in this paper - classical neuroscience cannot answer the question of why there is phenomenological consciousness. I also read Life on the Edge: The Coming of Age of Quantum Biology by Johnjoe McFadden and Jim Al-Khalili, which
provides an excellent introduction to the new field of quantum biology, a field of research that many neuroscientists I have
spoken with are unfamiliar with. Dehaene discusses some of the “quantum consciousness” ideas in his book, and I Qeios ID: QXUNGB · https://doi.org/10.32388/QXUNGB 1/5 Qeios, CC-BY 4.0 · Review, February 4, 2024 generally agree with his assessments, including that they fail to provide an explanation for phenomenological
consciousness. However, I also noted that none of them was discussed as quantum biological mechanisms by McFadden
and Al-Khalili, and given the inability of classical neuroscience to explain phenomenological consciousness, it seemed
that the new field of quantum biology might be worth investigating as it could relate to neuroscience. generally agree with his assessments, including that they fail to provide an explanation for phenomenological
consciousness. Paper weaknesses However, I also noted that none of them was discussed as quantum biological mechanisms by McFadden
and Al-Khalili, and given the inability of classical neuroscience to explain phenomenological consciousness, it seemed
that the new field of quantum biology might be worth investigating as it could relate to neuroscience. I decided to investigate whether there are pigment-protein structures in the brain that are similar to the pigment-protein
structures that support excitonic electron transport in photosynthesis. While I am not a neuroscientist, I do have a solid
background in mesoscopic devices and electron physics (expertise that many neuroscientists probably do not have), and I
was able to identify that ferritin and neuromelanin in catecholaminergic neurons appeared to have the right distribution
and concentration to form structures that could transfer energy between the soma of those neurons. The function
associated with this hypothesized neural signaling mechanism in those neurons (the substantia nigra pars compacta
(SNc), the locus coeruleus (LC), and the ventral tegmental area, among others) is action selection, which the author notes
at a number of points in his paper is integral with many of the aspects of consciousness for which there is agreement,
such as: - “consciousness is acknowledged as and often taken to be synonymous with current experience.” - “consciousness is acknowledged as and often taken to be synonymous with current experience - “Humans report and other animals behave as though they are attending to or aware of one thing at a time. This does not
preclude the fact that awareness of peripheral or unrelated sensory perceptions is present, but that the organism’s
attention is focused on one particular subject or event at a given instant.” - “Humans report and other animals behave as though they are attending to or aware of one thing at a time. Paper weaknesses Sara Ostrowski at EAG Labs in Silicon Valley to perform conductive atomic force microscopy
tests on fixed human SNc tissue, which provided compelling evidence of predicted ferritin and neuromelanin structures
that are capable of supporting widespread electron tunneling in that tissue. The results of those tests were also published
(2). This is unusual evidence that has never before been observed, and nobody has refuted it. A number of researchers
have cited it. The 2018 hypothesis was shown to have predictive power. I then commissioned Prof. Cai Shen to test for the predicted long-distance electron transport through disordered ferritin
arrays and for the predicted switching function that could be performed by those disordered ferritin arrays. Those tests
provided compelling evidence of those predicted physical phenomena that was also published (3). This is also unusual
evidence that has never before been observed, and nobody has refuted it. At least one researcher has independently
confirmed it. The predictive power of the 2018 hypothesis was further demonstrated. I subsequently published explanations of how the hypothesized catecholaminergic neuron electron transport (CNET)
hypothesis relates to Integrated Information Theory (4, 5). I am in the process of publishing an updated explanation of the
CNET hypothesis (6) as it relates to action selection as part of the cortico-striatal processing loop and other neural
processing loops. I have also published a paper with a number of the leading researchers in the field of electron tunneling
in proteins that describes how electron tunneling associated with ferritin appears to be involved in biological processes in
mitochondria, the retina, the cochlea, macrophages, as part of ROS homeostasis, as part of magnetosensation, and other
biological processes (7), and is not just limited to the brain. It appears to be present in nearly every living cell. As such, a
large number of unusual and unexpected phenomena that were predicted by the CNET hypothesis have been observed,
and it should be given serious consideration by neuroscientists and should be further investigated if such investigation is
deemed worthwhile after serious consideration. This information is provided in hopes that the author will consider this evidence of a mechanism that directly relates to and
in some instances refutes the three “major mysteries” conclusion that is made by the author: (1) the neurological correlates of consciousness – independent tests by Prof. Paper weaknesses This does not
preclude the fact that awareness of peripheral or unrelated sensory perceptions is present, but that the organism’s
attention is focused on one particular subject or event at a given instant.” - “All the individual sensations or fragments of memory appear as an integrated and meaningful whole rather than a
fragmented target of attention.” - “All motile organisms show goal-directed behavior, and the role of consciousness in carrying it out has frequently been
noted.” - “Intentional movement is the way an organism interacts volitionally with its environment, and a consciousness of spatial
orientation and extent is central to this ability.” - “It also produced animals who handle their dealings with the world in a way that includes a tacit sense of self.” - “Global accessibility and broadcast: the capacity to link and integrate information from perception, memory, and
evaluative systems and broadcast the output back to input and executive systems that lead to relevant actions.” - “Value attribution: the capacity to alter and update the rewarding or punishing values of actions and sensory stimuli.” When the SNc and LC are destroyed by Parkinson’s Disease, death results, and as they are destroyed, there is
progressive loss of motive and cognitive function. In addition, the neural mechanism associated with the selection of
specific actions is still unknown. As such, there appeared to be a very clear connection between a signaling mechanism
in those nuclei associated with action selection and consciousness. I published my first paper on the hypothesis in 2018,
after receiving encouragement from a neuroscientist who performs research on catecholaminergic neurons (1). Qeios ID: QXUNGB · https://doi.org/10.32388/QXUNGB 2/5 Qeios, CC-BY 4.0 · Review, February 4, 2024 I then commissioned Dr. Sara Ostrowski at EAG Labs in Silicon Valley to perform conductive atomic force microscopy
tests on fixed human SNc tissue, which provided compelling evidence of predicted ferritin and neuromelanin structures
that are capable of supporting widespread electron tunneling in that tissue. The results of those tests were also published
(2). This is unusual evidence that has never before been observed, and nobody has refuted it. A number of researchers
have cited it. The 2018 hypothesis was shown to have predictive power. I then commissioned Dr. Qeios ID: QXUNGB · https://doi.org/10.32388/QXUNGB Paper weaknesses Pascal Kaeser at Harvard Medical School
have confirmed another unusual and unexpected prediction made by the CNET hypothesis, namely, that afferent cortical
signals provided to striatal dendrites mediate action selection by stimulating the axons of SNc dopamine neurons to
generate action potentials and cause the release of dopamine (see, e.g. (8, 9)). Stimulation of axons to create action
potentials is very unusual – it’s essentially the opposite of how most neurons function, but it was predicted in 2018. Those
afferent signals can be integrated by the CNET mechanism, which would explain how neural signals from widespread
sensory and cognitive processing neural systems are tied together, namely, as part of the action selection mechanism
(that the author acknowledges is an important part of consciousness). The CNET mechanism can thus integrate the
observed NCCs as part of phenomenological consciousness. (2) The apparent gap between phenomenology and mechanism – the CNET mechanism can help to explain that gap, but
a better understanding of the CNET mechanism will be needed to fully explain it. It involves strongly correlated electrons,
which are largely understood as a solid-state mechanism. I am the first person to provide evidence that it is also a Qeios ID: QXUNGB · https://doi.org/10.32388/QXUNGB 3/5 Qeios, CC-BY 4.0 · Review, February 4, 2024 biological mechanism. biological mechanism. (3) The agent that monitors the brain activity admitted into consciousness – CNET indicates that this “agent” is a physical
component of the unusual neural signaling mechanism in catecholaminergic neurons, and that brain activity is “admitted
into consciousness” when associated afferent signals to the CNET mechanism reach a level that influences the physical
behavior of that mechanism for the purposes of selecting whether to act or not to act. Not acting is an important part of
that mechanism, as the wrong action can have fatal consequences, which is why consciousness persists continuously and
not just when acting. Choosing the right food to eat, whether it is safe to go to the water to drink, and other choices
required for deciding whether and when to act involve many aspects of qualia and would need to be practiced by any
evolutionarily successful animal. See also (10) and related works, which are consistent with CNET and Kaeser’s work. In
that regard, the basal ganglia has been conserved over 500 million years of evolution, and catecholaminergic neurons
predate the basal ganglia, such as in C. elegans. Paper weaknesses While many neuroscientists have been dismissive of my work and have failed to give it any substantive consideration (in
fact, most do not even respond to polite enquiries, and when I have taken the time to follow up, I have been abused and
treated rudely and unprofessionally by some of them), a number of neuroscientists and other scientists have reviewed it in
detail and are supportive of it. However, as I am the only person that I am aware of who is working on this subject, I have
the moral and ethical obligation to try to raise awareness of it with people who might be able to appreciate it, even if it
means being ignored or dismissed out of hand most of the time. As shown by the IEEE paper, there is compelling
evidence that electron tunneling associated with ferritin could be a factor in many biological processes and that
understanding it may be necessary to treat diseases and disorders. As far as the paper goes, it would be of much greater
import if it addressed these recent discoveries; otherwise, it is just rehashing information that has been mostly known
since Dehaene published his book in 2015, a point which is generally acknowledged by the author (“Rather, it focuses on
those aspects of consciousness about which there is a growing body of agreement in principle, if not in detail.”) (1) Rourk, Christopher John. "Ferritin and neuromelanin “quantum dot” array structures in dopamine neurons of the
substantia nigra pars compacta and norepinephrine neurons of the locus coeruleus." Biosystems 171 (2018): 48-58. (2) Rourk, Christopher J. "Indication of quantum mechanical electron transport in human substantia nigra tissue from
conductive atomic force microscopy analysis." Biosystems 179 (2019): 30-38. (2) Rourk, Christopher J. "Indication of quantum mechanical electron transport in human substantia nigra tissue from
conductive atomic force microscopy analysis." Biosystems 179 (2019): 30-38. (3) Rourk, Christopher, et al. "Indication of Strongly Correlated Electron Transport and Mott Insulator in Disordered
Multilayer Ferritin Structures (DMFS)." Materials 14.16 (2021): 4527. (3) Rourk, Christopher, et al. "Indication of Strongly Correlated Electron Transport and Mott Insulator in Disordered
Multilayer Ferritin Structures (DMFS)." Materials 14.16 (2021): 4527. (3) Rourk, Christopher, et al. "Indication of Strongly Correlated Electron Transport and Mott Insulator in Disordered
Multilayer Ferritin Structures (DMFS)." Materials 14.16 (2021): 4527. (4) Rourk, Chris. Qeios ID: QXUNGB · https://doi.org/10.32388/QXUNGB Paper weaknesses "Application of the Catecholaminergic Neuron Electron Transport (CNET) Physical Substrate for
Consciousness and Action Selection to Integrated Information Theory." Entropy 24.1 (2022): 91. (4) Rourk, Chris. "Application of the Catecholaminergic Neuron Electron Transport (CNET) Physical Substrate for
Consciousness and Action Selection to Integrated Information Theory." Entropy 24.1 (2022): 91. (5) https://www.mdpi.com/1099-4300/25/10/1436 (6) Rourk, Christopher. "Catecholaminergic Neuron Electron Transport (CNET): A Neural Signaling Mechanism." Qeios
(2023). 4/5 Qeios ID: QXUNGB · https://doi.org/10.32388/QXUNGB Qeios, CC-BY 4.0 · Review, February 4, 2024 (7) Perez, Ismael Diez, et al. "Electron tunneling in ferritin and associated biosystems." IEEE Transactions on Molecular,
Biological and Multi-Scale Communications (2023). (7) Perez, Ismael Diez, et al. "Electron tunneling in ferritin and associated biosystems." IEEE Transactions on Molecular,
Biological and Multi-Scale Communications (2023). (8) Liu, Changliang, Pragya Goel, and Pascal S. Kaeser. "Spatial and temporal scales of dopamine transmission." Nature
Reviews Neuroscience 22.6 (2021): 345-358. (8) Liu, Changliang, Pragya Goel, and Pascal S. Kaeser. "Spatial and temporal scales of dopamine transmission." Nature
Reviews Neuroscience 22.6 (2021): 345-358. (9) Liu, Changliang, et al. "An action potential initiation mechanism in distal axons for the control of dopamine release."
Science 375.6587 (2022): 1378-1385. (9) Liu, Changliang, et al. "An action potential initiation mechanism in distal axons for the control of dopamine release."
Science 375.6587 (2022): 1378-1385. (10) Gurney, Kevin, Tony J. Prescott, and Peter Redgrave. "A computational model of action selection in the basal
ganglia. I. A new functional anatomy." Biological cybernetics 84 (2001): 401-410) (10) Gurney, Kevin, Tony J. Prescott, and Peter Redgrave. "A computational model of action selection in the basal
ganglia. I. A new functional anatomy." Biological cybernetics 84 (2001): 401-410) 5/5
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Auditory salience using natural scenes: An online study
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OCTOBER 19 2021
Auditory salience using natural scenes: An online study Sandeep Reddy Kothinti; Nicholas Huang; Mounya Elhilali J. Acoust. Soc. Am. 150, 2952–2966 (2021)
https://doi.org/10.1121/10.0006750 I. INTRODUCTION that can more efficiently process information in real-life
scenarios. In everyday life, a multitude of information-bearing
sources impinges on our senses that it is almost impossible
to process all the information at once with the same resolu-
tion. Attention plays a vital role in focusing perceptual and
cognitive resources in navigating the real-world (Driver,
2001). Attention can be employed voluntarily to perform a
task efficiently, such as paying attention to a particular indi-
vidual at a cocktail party (Cherry, 1953). This is an example
of top-down attention, where a listener’s goal orients her
perceptual and cognitive resources to facilitate listening to
the desired signal amidst other distractors (Baluch and Itti,
2011). In contrast, a source or stimulus can also capture our
attention because of its inherent properties, known as
salience; for instance, the sound of glass shattering, if some-
one drops a glass at the same party, would be a salient sound
that attracts our attention regardless of our state of cognitive
control or attentional focus. Salience presents itself in vari-
ous perceptual modalities; a flashing traffic sign on the road
(visual), a brewing smell of coffee (olfaction), and the ear-
lier example of glass breaking (auditory) are all salient but
in different ways. Understanding salience mechanisms pro-
vide insights not only into perceptual and cognitive systems
in the brain but also guides the development of technologies The literature on sensory salience has varied greatly,
particularly in terms of appropriate experimental paradigms
best suited to shed light on underlying physical, neural, and
perceptual underpinnings of salience encoding in the brain. In visual salience studies, gaze-tracking paradigms have
become a standard approach to track the eye movements of
a subject when presented with a static image or video in free
viewing tasks. In the absence of any task demands, the ten-
dency to fixate gaze on specific locations is guided by image
features which inform of salience attributes of both low-
level and high-level visual information in the image (Bruce
and Tsotsos, 2009; Judd et al., 2009; Zhao and Koch, 2013). In contrast, in the auditory domain, there is no established
framework to study salience (Kaya and Elhilali, 2017). a)ORCID: 0000-0002-1574-3339.
b)ORCID: 0000-0001-5993-8325.
c)Electronic mail: mounya@jhu.edu, ORCID: 0000-0003-2597-738X. ABSTRACT: Salience is the quality of a sensory signal that attracts involuntary attention in humans. While it primarily reflects
conspicuous physical attributes of a scene, our understanding of processes underlying what makes a certain object or
event salient remains limited. In the vision literature, experimental results, theoretical accounts, and large amounts
of eye-tracking data using rich stimuli have shed light on some of the underpinnings of visual salience in the brain. In contrast, studies of auditory salience have lagged behind due to limitations in both experimental designs and stim-
ulus datasets used to probe the question of salience in complex everyday soundscapes. In this work, we deploy an
online platform to study salience using a dichotic listening paradigm with natural auditory stimuli. The study vali-
dates crowd-sourcing as a reliable platform to collect behavioral responses to auditory salience by comparing experi-
mental outcomes to findings acquired in a controlled laboratory setting. A model-based analysis demonstrates the
benefits of extending behavioral measures of salience to broader selection of auditory scenes and larger pools of sub-
jects. Overall, this effort extends our current knowledge of auditory salience in everyday soundscapes and highlights
the limitations of low-level acoustic attributes in capturing the richness of natural soundscapes. p
g
p
V
C 2021 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons
Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1121/10.0006750 (Received 15 April 2021; revised 13 August 2021; accepted 29 September 2021; published online 19 October 2021)
[Editor: Christian Lorenzi]
Pages: 2952–2966 (Received 15 April 2021; revised 13 August 2021; accepted 29 September 2021; published online 19 October 2021)
[Editor: Christian Lorenzi]
Pages: 2952–2966 24 October 2024 04:05:07 24 October 2024 04:05:07 Auditory salience using natural scenes: An online study
Sandeep Reddy Kothinti,1,a) Nicholas Huang,2,b) and Mounya Elhilali1,c)
1Department of Electrical and Computer Engineering, Center for Language and Speech Processing, The Johns Hopkins University,
Baltimore, Maryland 21218, USA Auditory salience using natural scenes: An online study
Sandeep Reddy Kothinti,1,a) Nicholas Huang,2,b) and Mounya Elhilali1,c)
1Department of Electrical and Computer Engineering, Center for Language and Speech Processing, The Johns Hopkins University,
Baltimore, Maryland 21218, USA Auditory salience using natural scenes: An online study
Sandeep Reddy Kothinti,1,a) Nicholas Huang,2,b) and Mounya Elhilali1,c)
1Department of Electrical and Computer Engineering, Center for Language and Speech Processing, The Johns Hopkins University,
Baltimore, Maryland 21218, USA 2Department of Biomedical Engineering, The Johns Hopkins University, Baltimore, Maryland 21218, USA ARTICLE
................................... ARTICLE
................................... Articles You May Be Interested In Articles You May Be Interested In
Auditory salience using natural soundscapes
J. Acoust. Soc. Am. (March 2017)
A computational model for auditory saliency of environmental sound. J Acoust Soc Am (April 2009)
Using auditory saliency to interpret complex auditory scenes
J Acoust Soc Am (May 2007) Auditory salience using natural soundscapes 24 October 2024 04:05:07 https://doi.org/10.1121/10.0006750 Ultimately, these tasks yield different outcomes in terms
of discrete versus continuous measures of salience over the
course of an entire acoustic signal. They also probe various
aspects related to auditory salience as it pertains to either the
encoding of specific events or entire scenes or its effect on
attentional deployment and perceptual or cognitive load neces-
sary to perform the specific task at hand. Given that sounds
unfold over time, it is advantageous to obtain a continuous tem-
poral measure of salience as humans interact with complex
soundscapes with dynamic attentional guidance based on the
attributes of the sound events in the scene. stimulus is presented (Kaya and Elhilali, 2014; Kayser et al.,
2005). Ultimately, these tasks yield different outcomes in terms
of discrete versus continuous measures of salience over the
course of an entire acoustic signal. They also probe various
aspects related to auditory salience as it pertains to either the
encoding of specific events or entire scenes or its effect on
attentional deployment and perceptual or cognitive load neces-
sary to perform the specific task at hand. Given that sounds
unfold over time, it is advantageous to obtain a continuous tem-
poral measure of salience as humans interact with complex
soundscapes with dynamic attentional guidance based on the
attributes of the sound events in the scene. In addition, measures of salience are heavily informed
and sometimes biased towards the choice of stimuli used in
the experiments. Early visual salience studies used abstract
shapes such as lines and polygons to identify effects of simi-
larity and differences in simple attributes like colors and
shapes (Treisman and Gelade, 1980). Salience models
developed based on these simplified stimuli often failed to
generalize to complex natural scenes (Itti and Koch, 2000;
Judd et al., 2009). In recent years, visual salience has been
more commonly explored using complex natural scenes
such as faces (Ramanathan et al., 2010), natural images
(Judd et al., 2009), and images with rich contextual informa-
tion (Jiang et al., 2015); hence resulting in richer and more
generalizable models and theories of visual salience. In the
auditory domain, synthetic or simulated data such as sequen-
ces of tones (Duangudom and Anderson, 2013) or odd-ball
musical sequences (Kaya and Elhilali, 2014) were used to
identify salience effects along known auditory attributes,
such as loudness, pitch, and timbre. https://doi.org/10.1121/10.0006750 such tests in the laboratory, but also to broaden access to a
larger pool of volunteers and cover more diversity in age,
race, and gender (Buhrmester et al., 2011; Buhrmester et al.,
2018). Nonetheless, relinquishing control and rigor of an
experimental setup in the lab under the scrutiny of the
researcher comes at the cost of questionable quality and
interpretability of the crowd-sourced results. Despite the
skepticism regarding online data collection, visual salience
studies have embraced crowd-sourcing as the defacto plat-
form for large-scale data collection over the years. In visual
salience studies, eye-tracking and visual search paradigms
are prevalent paradigms. While visual search paradigms
measure salience as an attribute of a target object in the
image, eye-tracking-based paradigms employ free-viewing
(Borji et al., 2013; Zhao and Koch, 2013) and track eye-
movement while the subject processes the image. Eye-
tracking paradigms provide a salience map of each image
and can be used to study natural images. Crowd-sourcing
has been extensively used to collect eye-tracking data by
using the web camera (webcam) at the side of the subject
(Huang et al., 2019; Kim et al., 2015; Xu et al., 2015) or
mouse-contingent
paradigms
(Gomez
et
al.,
2017;
Lyudvichenko and Vatolin, 2019; Newman et al., 2020),
which emulate human visual exploration by tracking
the mouse movement. Several studies have compared
eye-tracking data on datasets collected in laboratory envi-
ronments with crowd-sourced data and reported that crowd-
sourced data match closely with traditional data collection
paradigms (Jiang et al., 2015; Rudoy et al., 2012). This con-
sistency has led to the deployment of crowd-sourcing for
large-scale salience data collection. SALICON (Jiang et al.,
2015) is an eye-tracking dataset collected using a mouse-
contingent paradigm for 10 000 images and is presently the
largest salience dataset. Several deep learning models have
been successfully trained on this dataset (Borji, 2021;
Cornia et al., 2018; Kruthiventi et al., 2017). By establish-
ing crowd-sourcing as a reliable framework for data collec-
tion, visual salience datasets have not only exponentially
grown in size, but also opened new avenues to developing
improved models for various downstream tasks including
objective video quality assessment (Le Meur et al., 2007)
and region of interest (ROI) identification in video (Rai
et al., 2016). stimulus is presented (Kaya and Elhilali, 2014; Kayser et al.,
2005). I. INTRODUCTION Some experimental schemes examine auditory salience as
the ability of the stimulus to pop-out while the subject is
actively engaged in the task (Kaya et al., 2020; Southwell
et al., 2017), whereas others use attention tracking mecha-
nisms to measure salience continuously while subjects are
attending to the stimuli (Huang and Elhilali, 2017; Zhao
et al., 2019b). In addition, distractor paradigms are consid-
ered in some studies to probe attentional deployment in the
presence of distracting salient events (Petsas et al., 2016;
Southwell et al., 2017; Vachon et al., 2017); alternatively,
detection tasks collect feedback from subjects about their
judgments of event salience or relative salience after a V
C Author(s) 2021. 2952
J. Acoust. Soc. Am. 150 (4), October 2021 0001-4966/2021/150(4)/2952/15 https://doi.org/10.1121/10.0006750 scenes from the JHU-DNSS database previously used to col-
lect booth data. An additional 40 scenes (DNSS-Ext) were
selected to extend the DNSS dataset, both to a larger size
and greater variety of scenes. Scenes were chosen specifi-
cally to include more events of underrepresented classes in
the original database (e.g., animal sounds and non-speech
human vocalizations). In addition, scenes were chosen to
include mostly acoustically dense scenes, i.e., scenes with
continuous presence of one or more overlapping auditory
objects and fewer quieter moments. Those scenes were
found to have events that were difficult to predict from
acoustic models (Huang and Elhilali, 2017). All the 40 new
scenes were exactly two minutes in length and were taken
from the Freesound audio library (Freesound, 2021). All
auditory stimuli were sampled or resampled at 22 kHz with
32 bits per sample. Any stereo recordings were converted to
mono by taking the average of the two channels. The present study aims to provide insight into adopting
crowd-sourcing as the primary experimental platform for
auditory salience experiments. For this purpose, we adopt
the dichotic listening task used in an earlier study (Huang
and Elhilali, 2017) for a web-based crowd-sourcing plat-
form. The study sets out three main goals. First, it evaluates
the use of a crowd-sourcing platform to yield high-quality
salience measures using a dichotic listening paradigm. Salience data collected in the laboratory in the previous study
[JHU-DNSS (Dichotic Natural Salience Soundscapes)] is
compared with data collected using the popular crowd-
sourcing platform Amazon Mechanical Turk (MTurk). To
establish consistency, a cross-platform comparison is per-
formed on responses from the two settings, as well as
salience models derived from the two platforms. Second, it
extends the selection of stimuli used previously (JHU-
DNSS) to encompass a wider collection of event types and
environments; particularly focusing on acoustically dense
scenes that cause more challenges of interpretability and
predictability for salience models. Third, it evaluates salience
responses derived from a larger and diverse pool of subjects
using data-driven salience models. The effect of larger
subject size on training and evaluating salience models
is assessed. Ultimately, the study aims to expand the
frontiers of auditory salience using larger datasets of com-
plex sounds. II. METHODS Behavioral paradigm: Following the same procedure
adopted to collect booth data [see Huang and Elhilali
(2017)], online participants were presented with two audi-
tory scenes dichotically. Subjects listened concurrently to
both scenes, one delivered to each ear, and indicated their
attentional focus continuously by positioning their cursor on
the screen. They were instructed to move their cursor to the
right side of the screen when the scene played in the right
ear grabbed their attention, and vice versa when attending to
the scene in their left ear. When attending to both scenes or
neither of them, subjects were instructed to keep their cursor
in the center of the screen. Participants were instructed to
move the cursor as soon as they noticed that their attention
had shifted, and vertical lines divided the screens into sec-
tions to delineate the three response areas (left, right, and
middle). https://doi.org/10.1121/10.0006750 For data collection purposes, auditory stimuli were
divided into three blocks with 16 scenes from the original
study as one block (DNSS) and two subsets of 20 scenes
from the DNSS-Ext stimuli as two separate blocks. Table I
summarizes the scene breakup for DNSS and DNSS-Ext
blocks. More detailed description of the scenes is provided
in the Appendix. Scenes were subjectively labeled by the
experimenter as dense if there were one or more object pre-
sent throughout the scene and as sparse if there are multiple
segments of the audio without any acoustic energy. 24 October 2024 04:05:07 https://doi.org/10.1121/10.0006750 Similarly, natural audi-
tory stimuli present more complexity when compared to
simulated data and require a higher level of control on the
biases introduced by familiarity, semantic information, as
well as sound context. Moreover, natural stimuli present an
additional challenge in terms of large variations or configu-
rations in which certain aspects of the scene can be pre-
sented. To eliminate some of these confounding factors
when probing general effects of auditory salience, it is
important to explore a large variety of natural scenes. 24 October 2024 04:05:07 The experimental design and complexity of incorporat-
ing richer stimuli also raise challenges with regard to the
number of subjects and variability arising from pool selec-
tion (de Haas et al., 2019). Conventionally, salience experi-
ments are conducted in laboratory settings, which offer
control over the testing conditions and the quality of the
subject responses. However, laboratory experiments are
time-consuming and often lack subject diversity due to limi-
tations on both subject pool and size, which can bias the
study findings. In recent years, online platforms have
emerged as a medium for large-scale data collection, allow-
ing data acquisition and curation to grow in orders of magni-
tude [e.g., 14 106 annotated images in ImageNet (Deng
et al., 2009), 5.8 thousand hours of audio in AudioSet
(Gemmeke et al., 2017)]. Crowd-sourcing has also been lev-
eraged more recently in several behavioral and psychophysi-
cal studies to not only overcome challenges with performing In comparison, scaling up auditory salience datasets
remains in its infancy. A recent salience study adopted
crowd-sourcing for salience judgment tasks (Zhao et al.,
2019a) and showed a strong agreement between the labora-
tory data and the crowd-sourced data. Still, several hurdles
remain in terms of scaling up the study of auditory salience,
both in terms of scope, size, and diversity of stimuli, as well
as the choice of paradigms that—at the very least—yield
temporal salience maps that can be leveraged to tie in multi-
scale representations of natural sounds (from low-level
acoustics to high-level semantics). It is therefore important
to scale studies of auditory salience to not only broaden the
scope and diversity of stimuli but also examine effects
across larger pools of subjects. Kothinti et al. 2953 J. Acoust. Soc. Am. 150 (4), October 2021 A. Behavioral procedure Three example trials are shown in the figure with the same scene (scene 4) being paired with different opposing scenes. The middle panel
shows responses for the example trials. Responses were averaged across subjects to achieve a salience measure called average behavioral salience as shown
in the bottom-most panel. 24 October 2024 04:05:07 24 October 2024 04:05:07 FIG. 1. Salience measurement paradigm. Two scenes were dichotically presented, and subjects indicated continuously which scene they were attending to at
any given time. Three example trials are shown in the figure with the same scene (scene 4) being paired with different opposing scenes. The middle panel
shows responses for the example trials. Responses were averaged across subjects to achieve a salience measure called average behavioral salience as shown
in the bottom-most panel. listens to each scene only once. Experiments were designed
such that each scene is paired on average with 18 different
scenes, and a specific pair of scenes repeated once in every
18 subjects on average. This procedure aligns with the pro-
cedure used for booth data. Subjects spent 30 min of experi-
mental time on average excluding the breaks in both
laboratory and crowd-sourced settings. movement or switching rate across the three positions in the
screen (left, right, middle). The average rate of attentional
switches (a shift of attention from one scene to the other)
was used as a criterion to flag trials with outlier behavior. Trials with disproportionately large (above 1/s) or small
(0.025/s) switches were considered outliers and removed
from further analysis. Subjects with more than half of the
trials flagged as anomalous were identified as outlier sub-
jects and were excluded from further data analysis. Participants: In this study, a total of 275 subjects (154
male, 110 female, 11 non-binary/unspecified) with average
age of 36.4 years (standard deviation ¼ 12.8 years) were
recruited as part of crowd data. Subjects were asked to report
the languages they speak, their dominant hand, and whether
they have normal hearing. Subjects were compensated for the
task after data collection following a study protocol approved
by the Johns Hopkins Institutional Review Board (IRB). Defining salient events: A continuous salience measure
was defined as the fraction of subjects listening to a given
scene at any given time, averaged across many subjects and
competing scenes, as shown in Fig. 1, bottom middle panel. A. Behavioral procedure Experimental setup: Behavioral data from an online
platform were collected using Amazon Mechanical Turk
(MTurk). The experiment was conducted using a web server
hosted on Amazon Web Services (AWS), and the interface
with
AWS
was
enabled
by
the
psiTurk
framework
(Gureckis et al., 2016), which uploaded the experiment as
an MTurk Human Intelligence Task (HIT). Once the task
was posted, subjects were able to find the HIT, complete the
task, and receive payment without further interaction with
the experimenter. The experiment presentation was imple-
mented using the jsPsych library (de Leeuw, 2015). Crowd-
sourced participants were instructed to use headphones for
the task. Subjects were instructed to adjust the volume to a
comfortable level before beginning the experiment. A train-
ing phase was conducted to familiarize subjects with the
interface, check if the orientation of the headphones was
correct, and confirm that dichotic listening was achieved. An optional break of 30 s was provided between trials. This
crowd-sourced data (referred to as crowd) was compared
against data previously collected in a booth in a laboratory
setting (referred to as booth), with subjects seated in a
soundproof booth and audio presented over Sennheiser
HD595 headphones, under close supervision by an experi-
menter (Huang and Elhilali, 2017). Each scene was paired with different opposing scenes
across subjects and trials, and the responses from all the tri-
als were averaged to get the mean salience measure of the
scene over time across subjects and opposing scenes
(Fig. 1). Each experiment consisted of 10 trials, during
which two scenes from a block of scenes were chosen ran-
domly without replacement which ensured that a subject TABLE I. Description and grouping of the scenes used in the study. TABLE I. Description and grouping of the scenes used in the study. Set
# Blocks
# Scenes
Duration
# Dense
DNSS
1
16
1.13–2.22
10
DNSS-Ext
2
40
2.00
40
Kothinti et al. Auditory stimuli: In total, 56 audio recordings of natural
scenes with a total duration of 112 min were used as stimuli
for behavioral data collection. These scenes included 16 Kothinti et al. 2954
J. Acoust. Soc. Am. 150 (4), October 2021 FIG. 1. Salience measurement paradigm. Two scenes were dichotically presented, and subjects indicated continuously which scene they were attending to at
any given time. A. Behavioral procedure For each scene in a trial, subject responses were mapped to
1 for subjects listening to the scene and 0 for subjects listen-
ing to the opposite scene or neither scenes nor both scenes
(quantitatively
and
qualitatively
similar
results
were
obtained if responses to the center of the screen and opposite
scene were differentiated). An average response curve called
average behavioral salience was obtained by taking the aver-
age of subject responses for each scene. Subjects for data collection in the sound-booth were
recruited on the university campus. Booth data consisted of
responses from 50 subjects (16 male, 34 female) with average
age of 21.8 years (std dev ¼ 3.8 years). Subject populations in
booth were significantly younger and comprised of higher
percentage of female participants when compared to crowd. Onsets of salient events were defined as peaks in the
slope of the average behavioral salience curve. These events
were moments in the scenes when a large number of sub-
jects began attending to that particular scene, regardless of
the contents of any opposing scene. To identify peak https://doi.org/10.1121/10.0006750 Interobserver agreement in terms of AUROC
was computed for the shuffled dataset by finding salient
events from the average behavioral salience computed from
the shuffled dataset and performing the detection analysis
from the individual responses as described above. This pro-
cedure was repeated 50 times each for various subsample
sizes. The AUROC values produced in this way indicated a
chance level interobserver agreement. The strength of each event was computed as a sum of
two factors: (1) height of the local maximum of the 1st order
derivative of salience, which measures temporal agreement
among subjects, and (2) maximum salience within 4 s after
the event scaled by 75th percentile of slopes, which reflects
absolute consensus irrespective of time. Events identified
using this procedure were sorted based on salience strength,
and the top 50% events were chosen as the final events. This
additional pruning was performed to remove small peaks
that can arise because of noise in behavioral responses. Reaction times: Reaction times were computed for each
subject by comparing individual subject responses to the
onset of salient events (event onsets are defined as the time
when the “average subject” responded with 1 s subtracted
for nominal reaction time). A subject was considered to
have responded to a salient event if they moved towards the
particular scene between 0.5 and 1.5 s after the event onset. The difference between the event timestamp and the time
when the individual subject moved the mouse towards the
scene was considered as the event reaction time. 24 October 2024 04:05:07 Behavioral response consistency: A subset of the crowd
data (only matching DNSS stimuli) was compared to booth
data, to check for consistency across platforms on the same
stimuli using two metrics: correlation and subject-wise F-
scores. (1) Correlations were defined for individual scenes. These
were
computed
as
sample
Pearson
correlation
between the average behavioral salience of matched scenes
from crowd and booth by assuming average behavioral
salience at different time-points as samples; then correlation
coefficients were averaged across scenes. (2) F-scores were
computed for individual subjects. Instances, when the sub-
ject moved towards a given scene, were identified as events
detected by that subject; reference salient events were
derived from the average behavioral salience of the remain-
ing subjects for that scene (following the procedure to derive
salient events described earlier). A detection analysis was
performed to evaluate the subject’s detected events against
reference events. https://doi.org/10.1121/10.0006750 locations, the salience curve for each scene was first
smoothed with three equally weighted moving average oper-
ations of duration 1.5 s. Then a peak-detection method was
applied to find timestamps of local maxima in the first-order
difference of the smoothed salience curve. A fixed duration
of 1 s was subtracted from the peak timestamps to obtain
salient event onsets. This adjustment was done to account
for subject reaction times. 1 s was found to be a typical time
difference between a peak in loudness change and the next
salient onset in both booth and crowd datasets [see Huang
and Elhilali (2017) for details]. average behavioral salience profiles from booth and crowd
match; while subject-wise F-scores indicated how well a
subject agreed with the average behavioral salience around
salient moments in a scene and hence reflected a level of
uniformity in subjects’ responses in both platforms. y
j
p
p
Effect of subject size: The number of online subjects
recruited for the study can affect variability in the data. The
interobserver agreement on events from crowd was evalu-
ated as a function of number of subjects. It quantified how
well subjects agreed with salient events derived from the
average behavioral salience of all the subjects in crowd
data. This analysis was performed on the DNSS scenes only
since these scenes were tested in both booth and crowd set-
tings. To evaluate interobserver agreement, scenes were
divided into segments of 1 s with 75% overlap, and each
segment was labeled 1 or 0 based on whether a salient event
was present in the segment or not, respectively. The percent-
age of subjects who switched their attention within each seg-
ment was used as the detection signal. Hits and false alarms
were computed with different threshold levels. The area
under the ROC curve (AUROC) (Fawcett, 2006) was com-
puted as a concise metric of detection performance. A simi-
lar analysis was performed on the booth data using the
entire booth subject pool and used as benchmark for the
crowd data. Along with the booth interobserver AUROC, a
chance-level benchmark was computed by shuffling the
dataset to form randomized datasets. For each scene,
responses from the crowd data were sampled without
replacement from the pool of responses from all subjects
and scenes. https://doi.org/10.1121/10.0006750 Matched detections (within 1 s) were con-
sidered as hits. Reference events without a close-by detected
event (within 1 s) were considered false negatives, and
detected events without any close-by reference event were
considered false positives. F-score was computed as a har-
monic mean of precision and recall with appropriate flooring
(Mesaros et al., 2010). These two metrics were chosen
because they reflect different aspects of consistency across
platforms: The correlation metric indicated how well the B. Behavioral data analysis Data quality control: Subject responses in both crowd
and booth data were analyzed based on the speed of cursor Kothinti et al. 2955 J. Acoust. Soc. Am. 150 (4), October 2021 https://doi.org/10.1121/10.0006750 (6) Irregularity (IR) measures jaggedness in the spectrum
and was computed as a sum of squares of the differ-
ence between consecutive spectral magnitudes divided
by the sum of squares of spectral magnitudes. (7) Flatness (FL) was computed as a ratio of geometric
mean to the arithmetic mean of spectral magnitudes. (7) Flatness (FL) was computed as a ratio of geometric
mean to the arithmetic mean of spectral magnitudes. (8) Average slow temporal modulations (or low rates -LR)
were computed using the NSL toolbox (Chi et al.,
2005) by decomposing spectrogram y(t, f) into tempo-
ral modulations z(t, r) for r 2 ½1; 20 Hz, then averaging
the energy across modulation filter outputs. (9) Average fast temporal modulations [or high rates
(HR)] were computed similarly as above, by averaging
energy in temporal filters r 2 ½20; 100 Hz. This aver-
age energy is commensurate with a measure of rough-
ness previously found to correlate with aversion to
auditory events (Arnal et al., 2019). 24 October 2024 04:05:07 (10) Rate centroid (RC) was computed as the centroid of
temporal modulations over the range r 2 ½1; 32 Hz. (11) Scale centroid (SC) was computed similarly to rate by
first decomposing the spectrogram y(t, f) into spectral
modulations w(t, s) for s 2 ½0:25; 8 cyc/oct and then
taking the centroid. Data augmentation: Two different sets of LDA models
were trained using booth and crowd data for DNSS scenes
and were evaluated with held-out sets from both the plat-
forms to examine the compatibility of LDA models from
one platform to the other. To understand the benefits of
larger data, additional models were trained using varying
amounts of added training data and again evaluated on held-
out sets. For this analysis, booth and crowd subject data for
matched DNSS scenes were combined. The total data were
randomly sampled 100 times for each training data size to
capture variations in the data. These features were chosen to span a wide range of
potentially relevant acoustic attributes, including many that
have been found to be predictors of salience in prior studies. Temporal and spectral modulations were part of the auditory
salience map model by Kayser et al. (2005). Contrasts in loud-
ness and pitch were demonstrated to be salient by Kaya and
Elhilali (2014), and Tordini et al. (2015) found that less bright
sounds were more likely to be in the foreground. https://doi.org/10.1121/10.0006750 was first split into segments of 1 s with an overlap of 0.75 s,
and each segment was assigned a label 1 if an event was pre-
sent in the segment or 0 if no event was present in the seg-
ment. A change detection based approach was used on the
acoustic features for event prediction. For each feature,
peaks in the slope of the feature were assumed to be pre-
dicted events for the feature-derived salient events. Events
predicted across features were converted to segment-level
binary predictions and combined using the linear discrimi-
nant analysis (LDA) method (Fisher, 1935). The LDA
weights signified how individual acoustic features contrib-
uted to predictions of salience of the scene. Segment-level
salient event labels were used as class assignments, and
feature-based predictions were used as inputs to the LDA
procedure. LDA model parameters were trained and tested
with non-overlapping subsets of the data using 10-fold
cross-validation. During an evaluation with the held-out set,
LDA output was binarized with different thresholds and
compared with the labels to compute hits and false alarms at
each threshold level. Area under the receiver operating char-
acteristic curve (AUROC) was used as a summary statistic to
compare different LDA model performances. While the
detection methodology has similarities to sound event detec-
tion [e.g., DCASE 2017 (Mesaros et al., 2019)], the formula-
tion in this work predicts the onsets derived from behavioral
salience as opposed to just acoustics. This methodology was
adopted to highlight how the acoustic dimensions were driv-
ing the salience by using a data-driven approach. (3) Harmonicity (H) was derived as a measure of the
degree of match between each spectral slice and the
matched pitch template. (4) Brightness (BR), or spectral centroid, was computed as
the weighted average of frequencies with the power
spectrum at each time [yðt; fÞ2] as weights. (4) Brightness (BR), or spectral centroid, was computed as
the weighted average of frequencies with the power
spectrum at each time [yðt; fÞ2] as weights. (5) Bandwidth (BW) was computed as an average of the
absolute difference between the spectral centroid and
frequencies, weighted by the magnitude spectrum. (5) Bandwidth (BW) was computed as an average of the
absolute difference between the spectral centroid and
frequencies, weighted by the magnitude spectrum. https://doi.org/10.1121/10.0006750 While the
remaining features were not studied in the context of auditory
salience prior to the original study, they were recognized to be
useful for various audio applications (Alıas et al., 2016). C. Acoustic analysis Feature analysis: A set of eleven acoustic features that
capture a wide range of spectral, temporal, and spectro-
temporal attributes of auditory scenes were used to analyze
acoustic markers of auditory salience. All spectral features
were computed by first converting the audio waveform x(t) to
an auditory spectrogram y(t, f) using the biomimetic model of
Wang and Shamma (1994) using a frame rate of 125 Hz. (1) Loudness (LD) was derived by taking the average of
envelopes computed on 28 bark frequency bands
(Zwicker et al., 1991) in the range [250 Hz, 12 kHz] with
critical bands matching mammalian auditory periphery. (2) Pitch (P) was computed using the optimum processor
method (Goldstein, 1973), where each spectral slice
was matched with a set of pre-computed pitch tem-
plates and the fundamental frequency was determined
from the best template using maximum likelihood
estimation. 2956
J. Acoust. Soc. Am. 150 (4), October 2021 A. Behavioral results (E) Reaction times from combined booth and crowd data divided by subject age. Error bars represent 61 standard error and significant dif-
ces are indicated by stars. https://doi.org/10.1121/10.0006750 24 October 2024 04:05:07 24 October 2024 04:05:07 FIG. 2. (Color online) (A) Distribution of the number of switches across trials in the sound booth data. 4% of trials had a low switch rate, while 0% of trials
had a high switch rate. (B) Distribution of the number of switches across trials in the Mechanical Turk data. 4% of trials had a low switch rate while 2% of
trials had a high switch rate. (C) Example responses that exhibited low, regular, and high numbers of attentional switches between scenes. Trials with low or
high switches were excluded from the analysis. (D) Box plot of subject reaction times with median, 25th and 75th percentiles for DNSS scenes from booth
and crowd data. (E) Reaction times from combined booth and crowd data divided by subject age. Error bars represent 61 standard error and significant dif-
ferences are indicated by stars. interaction between age and platform) were primarily caused
by imbalances in subject ages in the two paradigms with most
subjects from booth being younger (92% of subjects with age
<25) than crowd (12% of subjects with age <25). To under-
stand the effect of age further, subjects from booth and crowd
were grouped based on subject age to balance the age distribu-
tions more evenly [Fig. 2(D)]. There were no differences
between the young-age (<25) and middle-age (25 and 55)
groups [two-sample t-test, tð128Þ ¼ 1:51, p ¼ 0.09], and the
middle-age and old-age (>55) groups [two-sample t-test, 0.19 switches per second. For booth data, no subjects were
considered as outliers as their switching patterns remained
largely within the nominal switching rates. We also analyzed average reaction times under both
experimental paradigms. Figure 2(D) shows reaction time
distributions for booth and crowd with median, 25th and 75th
percentiles for the DNSS scenes. Reaction times from both
paradigms followed a normal distribution [Kolmogorov-
Smirnov test (Massey, 1951), p(booth) ¼ 0.64, p(crowd)
¼ 0.47] and were not significantly different from each other
[two sample t-test, t(138) ¼ –1.69, p ¼ 0.09]. TABLE II. ANOVA results for subject-wise F-scores and reaction times. Three factors considered were Age (>25, 25), Gender (male, female), and
Platform (booth, crowd). A. Behavioral results Analysis of behavioral responses across subjects compar-
ing booth and crowd data reveals slightly different patterns of
switching rate before any outlier analysis [Fig. 2(A) and 2(B)]. The median switching rate of booth (0.32 switches per second)
was found to be higher than crowd (0.17 switches per second)
[Wilcoxon rank-sum test (Wilcoxon, 1945), p ¼ 2e 61]. Trials with disproportionately large [Fig. 2(C), top] or small
[Fig. 2(C), middle] number of attentional switches were
excluded from further analysis. Within the crowd data,
roughly 6% of trials (upper 2 percentile, lower 4 percentile)
had either a high or a low rate of attentional switches. Overall,
12 of 275 subjects were identified as outlier subjects and all
the trials from these subjects were removed. Removing outlier
trials increased the median switching rate in crowd to All features were resampled to have a uniform sampling
time of 64 ms. They were normalized to have zero mean and
unit variance. To analyze the dependencies across these fea-
tures, Pearson correlations were computed for DNSS and
DNSS-Ext scenes for each pair of features. Changes in fea-
tures around events were analyzed to appraise the contribu-
tion of specific acoustic features in the event salience. The
change in an acoustic feature was computed as the differ-
ence between the average value of the feature in two 0.5 s
intervals: one starting at 0.5 s after event onset and one start-
ing 1 s before event onset. Acoustic prediction of events: A model to predict salient
events was developed based on the acoustic analysis. The
model operates in a segment-wise manner where each scene Kothinti et al. 2957 Kothinti et al. J. Acoust. Soc. Am. 150 (4), October 2021 2957 2. (Color online) (A) Distribution of the number of switches across trials in the sound booth data. 4% of trials had a low switch rate, while 0% of trials
a high switch rate. (B) Distribution of the number of switches across trials in the Mechanical Turk data. 4% of trials had a low switch rate while 2% of
had a high switch rate. (C) Example responses that exhibited low, regular, and high numbers of attentional switches between scenes. Trials with low or
switches were excluded from the analysis. (D) Box plot of subject reaction times with median, 25th and 75th percentiles for DNSS scenes from booth
crowd data. Kothinti et al. 2958
J. Acoust. Soc. Am. 150 (4), October 2021 https://doi.org/10.1121/10.0006750 tð88Þ ¼ 1:56, p ¼ 0.12]; however, the old-age group had
slower
reaction
times
[one-sided
t-test,
tð58Þ ¼ 2:62,
p ¼ 0.015] compared to the younger group. mean of recall and precision (Massey, 1951). Subject-wise F-
scores for booth (p ¼ 0.99) and crowd (p ¼ 0.93) using the
DNSS scenes were found to follow a normal distribution. A
two-sample t-test between subject-wise F-scores for booth
and crowd showed no statistically significant difference
between the two groups [t(136) ¼ 1.30, p ¼ 0.2]. Differences
in variance in the F-scores for the two groups were also not
found to be statistically significant [F(49, 87) ¼ 0.6, p ¼ 0.06]
with higher variance observed in crowd (std¼ 0.07) when
compared to booth (std ¼ 0.05) [Fig. 4(A)]. Next, salience responses across platforms from the
same DNSS auditory scenes were compared. Correlations
between average behavioral salience from booth and crowd
revealed a strong agreement between the behavioral data
under both testing paradigms (average Pearson correlation
q ¼ 0.69 across all scenes; p 1e3 for each DNSS scene). Figure 3(A) depicts a spectrogram of a sample scene over-
laid on average behavioral salience from booth and crowd
data in response to the same scene. While responses were
rather noisy given the subjective and continuous behavioral
feedback by subjects throughout the trial, there was a
remarkable accord in general patterns between the two
responses, revealing increased engagement of subjects with
the scene at specific moments in time (regardless of the
opposite scene or platform). Given the perceptual difference
between scenes based on their acoustic transience, we fur-
ther explored the correlation between booth and crowd aver-
age behavioral salience separately for dense and sparse
DNSS scenes. Sparse scenes tended to give rise to more dis-
tinct objects and resulted in higher correlations (average cor-
relation across scenes, q ¼ 0.79), while denser scenes were
more perceptually continual resulting in noisier (less dis-
tinct) reactions from subjects, though there was still statisti-
cal agreement in responses between booth and crowd data
(average correlation across scenes, q ¼ 0.64). To explore factors
contributing to
differences
in
salience responses between the booth and crowd paradigms,
a three-way ANOVA was performed using age, platform,
and gender as the factors where all factors were defined in a
similar way as for reaction times. https://doi.org/10.1121/10.0006750 Although none of the fac-
tors or interactions were significant (Table II), gender and
age seemed to have a stronger influence on the F-scores than
platform, which suggests that the observed differences in
F-scores were due to differences in populations and not due
to differences in data collection platform. Effects of age on
F-scores in the combined booth and crowd data are shown
in Fig. 4(B). Subjects from booth and crowd were combined
together and then grouped into three groups: young-age
(<25 years), middle-age (25–55) and old-age (>55). The
analysis showed no significant differences among age
groups. Marginally
significant
differences
were
found
between younger participants and middle age groups [two-
sample t-test, t(128) ¼ 1.94, p ¼ 0.05] [Fig. 4(B)]; while no
significant difference were observed between young and old
groups [t(58) ¼ 1.51, p ¼ 0.13] and middle and older age
groups [t(88) ¼ 0.50, p ¼ 0.62]. 24 October 2024 04:05:07 In addition to agreement between booth and crowd data
in average behavioral salience, inter-subject agreement
based on salient event onsets also revealed a consistent con-
currence across behavioral paradigms on average, albeit
with higher variance for the crowd-sourced data. Subject-
wise F-scores were computed as a measure of agreement
between each subject’s switching pattern and salient event
onsets computed from the average behavioral salience for
the same behavioral paradigm (crowd or booth, see Sec. II). A Kolmogorov-Smirnov test was used to confirm that the
distributions were indeed normal since F-score is a harmonic Next, we leveraged the crowd-sourced paradigm to
examine the effect of subject quantity and used a measure of
interobserver AUROC to quantify consistency across sub-
jects as a function of the number of subjects (see Sec. II). Focusing on the DNSS scenes (for which there is a baseline
in booth data), Fig. 5 shows that the interobserver AUROC
increases systematically with the inclusion of more subjects,
confirming increased consistency between subjects in terms FIG. 3. (Color online) Correlation between booth and crowd responses for DNSS scenes with an example. (A) Spectrogram of an example scene with booth,
crowd average behavioral salience overlaid on the spectrogram. (B) Correlation between booth and crowd responses for DNSS scenes with sparse and dense
breakup. Error bars depict 61 SEM. FIG. 3. (Color online) Correlation between booth and crowd responses for DNSS scenes with an example. A. Behavioral results To further explore similarities and differences between
booth and crowd data, a three-way ANOVA on reaction time
was performed using age (<25 and 25), platform (booth
and crowd), and gender (male and female). Subjects without
gender information (four subjects who listened to the DNSS
scenes only) were removed for this analysis. As shown in
Table II, none of the factors or interactions were found to be
statistically significant. No effect of platform indicated that
both paradigms had subject responses with similar latencies. The effect of age and interaction between age and platform
were found to be contributing the most to the differences in
reaction times than other factors. These age effects (and Reaction times
F-scores
Factor
df
Mean. Sq. F
p
Mean. Sq. F
p
Age
1
0.0076
2.28
0.13
0.0009
0.23
0.63
Platform
1
0.0006
0.19
0.66
0.0000
0.01
0.93
Gender
1
0.0000
0
0.99
0.0079
1.94
0.17
Age Platform
1
0.0079
2.35
0.13
0.0000
0.02
0.90
Age Gender
1
0.0001
0.05
0.83
0.0008
0.19
0.66
Platform Gender
1
0.0008
0.24
0.63
0.003
0.76
0.38 2958
J. Acoust. Soc. Am. 150 (4), October 2021 https://doi.org/10.1121/10.0006750 (A) Spectrogram of an example scene with booth,
crowd average behavioral salience overlaid on the spectrogram. (B) Correlation between booth and crowd responses for DNSS scenes with sparse and dense
breakup. Error bars depict 61 SEM. J. Acoust. Soc. Am. 150 (4), October 2021 Kothinti et al. 2959 FIG. 4. (Color online) Subjectwise F-scores indicating agreement of subjects with extracted events. Stars indicate significant differences between groups. Error bars depict 61 SEM. (A) F-scores for booth and crowd subjects for matched scenes. (B) Combined F-scores from booth and crowd divided by age
group. https://doi.org/10.1121/10.0006750 https://doi.org/10.1121/10.0006750 FIG. 4. (Color online) Subjectwise F-scores indicating agreement of subjects with extracted events. Stars indicate significant differences between groups. Error bars depict 61 SEM. (A) F-scores for booth and crowd subjects for matched scenes. (B) Combined F-scores from booth and crowd divided by age
group. of behavioral salience judgments. Furthermore, the analysis
of the crowd data showed that reaching the level of consis-
tency observed in booth data required N ¼ 60 subjects,
which is slightly more than N ¼ 50 subjects used in the
booth (Fig. 5, red line). This increasing trend in interob-
server AUROC was also observed with DNSS-Ext scenes in
crowd data, although the absolute values of AUROC were
lower than for DNSS scenes. Chance level interobserver
AUROC (Fig. 5, green line) was found to be lower than the
booth and crowd interobserver AUROC and remained con-
stant with increasing number of subjects. This difference
supported our claim that a high interobserver AUROC indi-
cated a high agreement across the subjects and the increase
in AUROC was a systematic effect. structure of the scenes themselves. Figure 6(A) quantifies
changes in eleven spectral and temporal acoustic dimensions
(see Sec. II) around the salient events. In the first analysis,
we compared acoustic changes around salient events in
booth versus events in crowd data in the same DNSS scenes. https://doi.org/10.1121/10.0006750 Pair-wise two-sample t-tests for each acoustic dimension
showed no significant differences between booth and crowd
feature changes for DNSS across all features [bandwidth:
t(418) ¼ –0.4, p ¼ 0.70, loudness: t(418) ¼ 0.3, p ¼ 0.76,
pitch: t(418) ¼ –0.5, p ¼ 0.64, brightness: t(418) ¼ –0.5,
p ¼ 0.62,
harmonicity:
t(418) ¼ 0.0,
p ¼ 0.99,
flatness:
t(418) ¼ –0.0, p ¼ 0.99, irregularity: t(418) ¼ –0.5, p ¼ 0.61,
rate: t(418) ¼ –0.0, p ¼ 0.99, scale: t(418) ¼ 1.74, p ¼ 0.09,
high-rate: t(418) ¼ 0.85, p ¼ 0.40, low-rate: t(418) ¼ 0.54,
p ¼ 0.59]. For each dataset, we noted that loudness, bright-
ness, harmonicity, high-rate, and low-rate had significant
positive changes while scale had significant negative
changes
around
events
[one-sample
t-test,
for
booth,
loudness: t(192) ¼ 11.0, p ¼ 6e22, pitch: t(192) ¼ 2.8, 24 October 2024 04:05:07 24 October 2024 04:05:07 p ¼ 0.59]. For each dataset, we noted that loudness, bright-
ness, harmonicity, high-rate, and low-rate had significant
positive changes while scale had significant negative
changes
around
events
[one-sample
t-test,
for
booth,
loudness: t(192) ¼ 11.0, p ¼ 6e22, pitch: t(192) ¼ 2.8, B. Acoustic features p ¼ 0.01, brightness: t(192) ¼ 4.8, p ¼ 1e3, harmonicity:
t(192) ¼ 6.4, p ¼ 1e9, high-rate: t(192) ¼ 11.0, p ¼ 4e22,
low-rate: t(192) ¼ 10.5, p ¼ 1e20, scale: t(192) ¼ –2.5,
p ¼ 0.01, for crowd, loudness: t(226) ¼ 11.8, p ¼ 3e25,
pitch:
t(226) ¼ 4.2,
p ¼ 4e5,
brightness:
t(226) ¼ 6.2,
p ¼ 3e9, harmonicity: t(226) ¼ 7.7, p ¼ 4e13, high-rate:
t(226)¼ 11.1, p¼ 3e22, low-rate: t(226)¼ 10.9, p ¼ 1e22,
scale: t(226) ¼ –3.9, p ¼ 8e4]. Bandwidth was the only
feature which was found to have significant positive changes
for the crowd events [t(226) ¼ 2.7, p ¼ 0.01], whereas for
the booth events the changes in bandwidth were not statisti-
cally significant [t(192) ¼ 1.9, p ¼ 0.06]. These results sug-
gested that subjects under both experimental paradigms
(booth and crowd) were reacting to prominent variations
along the same acoustic dimensions of the scenes, driven by
loudness and mostly spectral attributes. p ¼ 1e9] and scale [t(554) ¼ –6.5, p ¼ 2e10] had signifi-
cant negative changes around DNSS-Ext events. A two-
sample t-test showed changes in bandwidth [t(780) ¼ 2.03,
p ¼ 0.04] and flatness [t(780) ¼ 2.9, p ¼ 3e3] were signifi-
cantly different across DNSS and DNSS-Ext scenes. It is important to note that the acoustic dimensions
explored in this study are not necessarily independent of
each other, and there exist strong correlations between sev-
eral features as shown in Fig. 6(B). For example, loudness is
strongly correlated to harmonicity (q ¼ 0.44, p ¼ 6e3),
high-rate
(q ¼ 0.73,
p ¼ 5e-48),
and
low-rate
features
(q ¼ 0.69, p ¼ 5e70) while flatness is correlated to bright-
ness (q ¼ 0.49, p ¼ 0.01). The differences between DNSS and DNSS-Ext were
largely driven by the variety of scene composition in each
dataset. As noted earlier, the DNSS-Ext scenes consisted of
a higher percentage of human vocalizations and device
sounds. An example DNSS-Ext scene with a large change in
flatness is shown in Fig. 6(D). This particular scene had a
telephone ring event with some speech events in the back-
ground. A sharp dip in flatness can be observed when the
ring happens due to the tone-like nature of the ring. This dip
also coincided with an increase in loudness near t ¼ 25 s, as
well as an increase in spectral irregularity [Fig. B. Acoustic features Next, we examined the relationship between behavioral
responses to salient events and changes in the acoustic FIG. 5. (Color online) Variability as function of number of subjects for the crowd data captured by interobserver ROC for booth (DNSS and DNSS-dense)
and crowd (DNSS and DNSS-Ext scenes). Benchmark variability was calculated using data from all subjects in the booth condition. A chance-level interob-
server AUROC computed from shuffled responses is shown for comparison purposes. Error bars represent standard errors across 50 trials of subsampling. 2960
J. Acoust. Soc. Am. 150 (4), October 2021
Kothinti et al. FIG. 5. (Color online) Variability as function of number of subjects for the crowd data captured by interobserver ROC for booth (DNSS and DNSS-dense)
and crowd (DNSS and DNSS-Ext scenes). Benchmark variability was calculated using data from all subjects in the booth condition. A chance-level interob-
server AUROC computed from shuffled responses is shown for comparison purposes. Error bars represent standard errors across 50 trials of subsampling. 2960
J. Acoust. Soc. Am. 150 (4), October 2021
Kothinti et al. 2960
J. Acoust. Soc. Am. 150 (4), October 2021 Kothinti et al. https://doi.org/10.1121/10.0006750 FIG. 6. (Color online) (A) Acoustic feature change averaged across events, compared between sound booth and crowd for DNSS and DNSS-Ext scenes. All
features are z-score normalized. Error bars represent 61 standard error. (B) Correlations across features computed as Pearson correlation. (C) Example seg-
ment from one of the DNSS stimuli. (D) Example segment from one of the DNSS-Ext stimuli. (Top) Time-frequency spectrum of the signal, with a descrip-
tion of the scene in text and salient events marked by the vertical lines in red. Flatness, loudness, irregularity are shown in the bottom panels for the
corresponding scenes. 24 October 2024 04:05:07 24 October 2024 04:05:07 FIG. 6. (Color online) (A) Acoustic feature change averaged across events, compared between sound booth and crowd for DNSS and DNSS-Ext scenes. All
features are z-score normalized. Error bars represent 61 standard error. (B) Correlations across features computed as Pearson correlation. (C) Example seg-
ment from one of the DNSS stimuli. (D) Example segment from one of the DNSS-Ext stimuli. (Top) Time-frequency spectrum of the signal, with a descrip-
tion of the scene in text and salient events marked by the vertical lines in red. Flatness, loudness, irregularity are shown in the bottom panels for the
corresponding scenes. https://doi.org/10.1121/10.0006750 TABLE III. Cross-modal AUROC averaged across tenfold cross-validation. Models were trained using DNSS scenes in booth and crowd data and tested
on the held out sets from booth and crowd data from the same scenes. Standard error across 10 folds is indicated with 6 sign. performance measure, Fig. 7(A) shows the effect of data
augmentation with different amounts of training data on the
detection performance. The evaluation is performed with
10-folds of test sets (11 min each) and for each test fold,
training data is sampled from the remaining folds in steps of
10 min. Figure 7(A) (right y axis) shows a normalized
AUROC for each fold in cross-validation, where the
AUROC with 10 min of training data were used as the
denominator. For each increment in the training data, a
paired t-test was performed on the AUROC for the 10-folds
to test if the performance improvement is significant. The
analysis
revealed
a
statistically
significant
increase
(p < 0.05) in performance with the inclusion of additional
data up to 70 min [Fig. 7(A), left y axis] and the improve-
ments were not statistically significant after 70 min. The
model with 100 min improves AUROC by 1% relative to the
10 min model. Figure 7(B) shows the impact of the DNSS-
Ext data on performance improvements with receiver oper-
ating characteristic (ROC) curves compared to a model
trained on DNSS data only. Interobserver ROC serves as an
upper limit on the achievable detection performance. As the
figure indicates, higher amounts of data led to better detec-
tion performance. Test data
Train data
Booth
Crowd
Booth
0.759 6 0.027
0.753 6 0.037
Crowd
0.753 6 0.027
0.757 6 0.038 is of a different nature with a different frequency profile
which caused a smaller dip in spectral flatness and not a pro-
nounced change in irregularity [Fig. 6(C), bottom panels]. C. Event prediction With the consistency between booth and crowd data
established with various characterizations based on behav-
ioral data and acoustic changes, salience models derived
from both platforms are expected to be compatible with
each other. To confirm this, a salience computational model
of event prediction based on acoustic changes was evaluated
in a cross-platform manner (see Sec. II for model details). Models were trained separately on booth and crowd data for
DNSS scenes and tested on held-out sets from both plat-
forms using tenfold cross-validation. Table III shows the
performance of the models, quantified using the AUROC. No significant differences were observed on paired t-tests on
cross-validation folds for both models tested on crowd data
[t(9) ¼ –1.3, p ¼ 0.33] and booth [t(9) ¼ 1.0, p ¼ 0.22]. IV. DISCUSSION 24 October 2024 04:05:07 In this study, we presented a detailed analysis of
salience data for natural scenes, collected on a crowd-
sourcing platform, using a dichotic listening paradigm. This
work focused on three main goals: establishing the validity
of dichotic listening in a crowd-sourcing platform as a reli-
able marker of auditory salience in complex natural scenes;
expanding the existing salience dataset to more diverse
scenes; and validating the benefits of large scale salience
datasets to develop accurate salience models. As previously
examined in several earlier studies, there is a close synergy
between acoustic attributes of a sound stimulus and its With access to a larger scene dataset with a bigger pool
of behavioral responses, we quantified the effect of addi-
tional data on acoustic predictions in an incremental man-
ner. This
analysis
examined
the
hypothesis
that
computational salience models would benefit from larger
databases (with more diverse scenes) that become possible
with
crowd-sourced
methods. Using
AUROC
as
a FIG. 7. (Color online) (A) Event detection performance in terms of AUROC from incremental data augmentation. On the left y axis, the average AUROC of
the 10-folds is plotted with each increment step and statistically significant improvements indicated with an asterisk. On the right y axis, each line represents
a cross-validation fold and AUROC for each augmentation step is divided by the AUROC with 10 min of training data to obtain a normalized AUROC mea-
sure. All 56 scenes were used for this evaluation. (B) ROC with models trained on DNSS and DNSS-total when compared with the interobserver perfor-
mance which can be considered an upper-bound to the detection performance. 2962
J Acoust Soc Am 150 (4) October 2021
Kothinti et al FIG. 7. (Color online) (A) Event detection performance in terms of AUROC from incremental data augmentation. On the left y axis, the average AUROC of
the 10-folds is plotted with each increment step and statistically significant improvements indicated with an asterisk. On the right y axis, each line represents
a cross-validation fold and AUROC for each augmentation step is divided by the AUROC with 10 min of training data to obtain a normalized AUROC mea-
sure. All 56 scenes were used for this evaluation. (B) ROC with models trained on DNSS and DNSS-total when compared with the interobserver perfor-
mance which can be considered an upper-bound to the detection performance. 2962
J. Acoust. Soc. Am. B. Acoustic features 6(D), bottom
panels]. An example scene with a telephone ring from a
DNSS scene is shown in Fig. 6(C) for comparison. The ring Taking advantage of the expanded variety of scenes
tested in the crowd paradigm, we compared the effect of
changes in acoustic attributes near salient onsets for DNSS-
Ext scenes in the crowd data [Fig. 6(A)]. A one-sample t-
test showed that loudness [t(554) ¼ 16.9, p ¼ 3e52], pitch
[t(554) ¼ 6.2,
p ¼ 6e10],
brightness
[t(554) ¼ 5.9,
p ¼ 4e9], harmonicity [t(554) ¼ 14.0, p ¼ 1e38], irregu-
larity [t(554) ¼ 3.2, p ¼ 1e3], high-rate [t(554) ¼ 20.4,
p ¼ 1e-69], low-rate [t(554) ¼ 19.9, p ¼ 2e67] had signifi-
cant
positive
changes
while
flatness
[t(554) ¼ –6.2, Kothinti et al. 2961 2961 Kothinti et al. Kothinti et al. J. Acoust. Soc. Am. 150 (4), October 2021 2962
J. Acoust. Soc. Am. 150 (4), October 2021 https://doi.org/10.1121/10.0006750 This observation is consistent with previ-
ous research in salience that hypothesized that violation of
expectation or variations in time along key attributes of the
signal give rise to salient events (R€oer et al., 2014; Vachon
et al., 2017). In the current work, these effects are confirmed
in a larger and more diverse pool of subjects with the crowd
data and provide further support for using dichotic listening
for salience data collection. Moreover, the analysis pre-
sented here extends the investigation of acoustic attributes
to include rate-specific energies (high rates HR and low
rates LR) which are found to have significant changes
around the salient events. The higher modulation energies
(>20 Hz) represent roughness in the audio signal, and the
contribution from high-rate towards salient events coincides
with the known effects of roughness on salience (Arnal
et al., 2019). On the other end of the modulation spectrum,
lower modulation energies (<20 Hz) are commensurate with
dynamics in the audio signal (e.g., syllabic rate in speech)
and play an important role not only in intelligibility and per-
ception of natural sounds (Ding et al., 2017; Elhilali, 2019;
Elliott and Theunissen, 2009) but are found here to be
important markers of auditory salience. 24 October 2024 04:05:07 While changes in key acoustic attributes tend to draw
the attentional focus of listeners, we note that variability and
richness of everyday soundscapes do also result in differ-
ences of effects across different scene subsets, as noted with
features such as flatness or irregularity. These variations
could only be addressed with an expansion of scene datasets
to an even larger size. Furthermore, the exploration of
acoustic effects has to be carefully appraised as changes in
acoustic attributes are heavily interdependent. As shown in
Fig. 6(B), there are strong correlations across the feature
dimensions, and the contribution of features to events is
affected by these inter-dependencies. Earlier behavioral and
neural recordings have shown a great deal of nonlinear inter-
actions between features such as pitch and timbre (Allen
and Oxenham, 2014; Melara and Marks, 1990; Walker
et al., 2011) that underlie the perception of integrated audi-
tory objects. Such interdependence has also been reported in
perceptual and neural measurements of auditory salience
(Kaya et al., 2020) where nonlinear interactions between
acoustic attributes like pitch, intensity, and timbre cause
interdependent responses to changes in individual dimen-
sions. https://doi.org/10.1121/10.0006750 some of the crowd-sourced visual salience studies (Jiang
et al., 2015; Rudoy et al., 2012), a comprehensive compari-
son of the data from booth and crowd sets using various
metrics is performed to justify the adoption of the crowd-
sourcing platform. Correlations of the average behavioral
salience between booth and crowd data indicate consider-
able agreement across platforms [Fig. 3(B)]. Under the
assumptions of normal distribution, this approach is similar
to the divergence measures used in Rudoy et al. (2012). We
also establish similarities across the booth and crowd plat-
forms in terms of inter-subject variability. This variability is
first quantified using subject-wise F-scores which measured
variability within the platform around the salient events. We
observe no statistically significant differences in average
F-scores and variance in F-scores. Further breakdown of the
differences in F-scores across reveals that age and gender
are the main contributors to the small differences observed
in crowd data (Table II). These are subject-specific factors
that point to the diversity in the crowd data. The diversity of
subjects guarantees an unbiased measure of salience, which
is of utmost importance when developing datasets that can
be used for salience models. In addition, interobserver
agreement is used as a secondary measure of variability. As
shown in Fig. 5, crowd data achieves the same interobserver
AUROC as booth data with a slightly higher number of sub-
jects and there is an increasing trend in AUROC with the
number of subjects, which shows the necessity for a larger
number of subjects to reduce variance in salience data. The
analysis with interobserver agreement serves as validation
of using a larger pool of subjects and can also be used as a
guiding principle to choose the number of subjects for a
required level of variance in data. perceptual salience. The acoustic analysis presented in Fig. 6(A) shows that key acoustic attributes like loudness, pitch,
and harmonicity, which were previously shown to drive
salience (Kaya and Elhilali, 2014; Tordini et al., 2015;
Tordini et al., 2013), have statistically significant changes
around event onsets from both the booth and crowd data. This result indicates that any large changes in these features
induce a consistent attention shift in subjects even under
dichotic listening. IV. DISCUSSION 150 (4), October 2021 https://doi.org/10.1121/10.0006750 TABLE IV. Continued
#
Block
Duration
Description
Sparse
Source
16
DNSS
2:14
Casino
No
Freesound
17 DNSS-Ext
2:00
Carnival, crowds
No
Freesound
18 DNSS-Ext
2:00
Arabic Concert
No
Freesound
19 DNSS-Ext
2:00
Cafeteria, Vehicles
No
Freesound
20 DNSS-Ext
2:00
Guns, Heavy Artillery
No
Freesound
21 DNSS-Ext
2:00
Baby Laughing
No
Freesound
22 DNSS-Ext
2:00
Beach, Waves
No
Freesound
23 DNSS-Ext
2:00
Dogs Growling, Birds
No
Freesound
24 DNSS-Ext
2:00
Crowd Chanting, Clapping
No
Freesound
25 DNSS-Ext
2:00
Volleyball Game, Cheering
No
Freesound
26 DNSS-Ext
2:00
Social Gathering, Dishes
No
Freesound
27 DNSS-Ext
2:00
Birds, Insects Chirping
No
Freesound
28 DNSS-Ext
2:00
Rain, Thunders
No
Freesound
29 DNSS-Ext
2:00
Piano Concert
No
Freesound
30 DNSS-Ext
2:00
Goats, Dogs, Horses
No
Freesound
31 DNSS-Ext
2:00
Water Stream, Birds
No
Freesound
32 DNSS-Ext
2:00
Party, Laughing, Music
No
Freesound
33 DNSS-Ext
2:00
Arcade, Songs
No
Freesound
34 DNSS-Ext
2:00
Reception Area, Telephone
No
Freesound
35 DNSS-Ext
2:00
Parade, Cheering
No
Freesound
36 DNSS-Ext
2:00
Motorcycle, Pump
No
Freesound
37 DNSS-Ext
2:00
Crowd Fight, Sword Sounds
No
Freesound
38 DNSS-Ext
2:00
Carpentry Machinery
No
Freesound
39 DNSS-Ext
2:00
Motorbikes, Rain
No
Freesound
40 DNSS-Ext
2:00
Sports Arena, Music
No
Freesound
41 DNSS-Ext
2:00
Ocean Waves
No
Freesound
42 DNSS-Ext
2:00
String Instrument
No
Freesound
43 DNSS-Ext
2:00
Arcade, Gaming
No
Freesound
44 DNSS-Ext
2:00
Street, Drums
No
Freesound
45 DNSS-Ext
2:00
Sports Arena, Cheering
No
Freesound
46 DNSS-Ext
2:00
Market, Crowd
No
Freesound
47 DNSS-Ext
2:00
Restaurant, Dishes
No
Freesound
48 DNSS-Ext
2:00
Office Space, Telephone
No
Freesound
49 DNSS-Ext
2:00
Seagulls, Motorboat,
No
Freesound
50 DNSS-Ext
2:00
Piano Concerto
No
Freesound
51 DNSS-Ext
2:00
Birds
No
Freesound
52 DNSS-Ext
2:00
Birds, Rooster
No
Freesound
53 DNSS-Ext
2:00
Fireworks
No
Freesound
54 DNSS-Ext
2:00
Bar, Music
No
Freesound
55 DNSS-Ext
2:00
Birds, Crickets
No
Freesound
56 DNSS-Ext
2:00
Soccer Game
No
Freesound
https://doi.org/10.1121/10.0006750 2017). These models were trained on crowd-sourced data
and were found to generalize well to eye-fixation bench-
marks. In recent years, there have been numerous studies
that leveraged principles of auditory salience for audio event
detection application (Kothinti et al., 2019; Podwinska
et al., 2019). But none of these studies employed any behav-
ioral data as part of the salience models, which could be
partly attributed to the lack of large-scale salience data. In
this work, we demonstrate the advantage of an extended
auditory salience database; and consistent improvements
from data augmentation support the rationale to scale the
data collection over crowd-sourcing platforms. The perfor-
mance of acoustic prediction is significantly improved by
adding more data to the training set [Fig. 7(A)]. While these
models are by no means comparable in size to deep learning
models of visual salience, the improvement shown with
added data is an encouraging outcome. There is a significant
gap between the performance of the salience model and the
inter-observer agreement which serves as an upper-bound
on detection performance [Fig. 7(B)]. This difference could
be because of the higher-level stimulus attributes such as
scene semantics that could directly impact salience or modu-
late the importance given to different acoustic dimensions. We believe this gap could be bridged by studying a larger
variety of scenes with complex semantics such as speech
and music. 24 October 2024 04:05:07 ACKNOWLEDGMENTS This
work
was
supported
by
Grant
Nos. NIH
U01AG058532, ONR N00014-19-1-2014, and N00014-19-
1-2689. S.R.K. and N.H. contributed equally to this work. https://doi.org/10.1121/10.0006750 Given these multiplexed relationships, it is important
to nuance judgments of which acoustic features are critical
for salience perception and infer that no one feature can
operate on its own as the main driver of sound conspicuity. While it is relatively easier to collect crowd-sourced
data from a large number of subjects, quality control is a
concern that prevents the deployment for behavioral experi-
ments (Br€uhlmann et al., 2020; Kan and Drummey, 2018)
on crowd-sourcing platforms. In this work, we use switching
rates of subject responses as a quality measure of the subject
data. A similar quality control strategy was used in
BubbleView (Kim et al., 2017), a visual exploration para-
digm used for visual salience. Since the dichotic salience
experiment inherently does not have a ground truth, it is dif-
ficult to inject control trials within the experiment, as is the
case with some of the crowd-sourcing paradigms in visual
salience (Othman et al., 2017). While the switching rate
analysis is done post-experiment in this study, it is possible
to inform subjects when their switching rates fall outside the
acceptable range. Another aspect related to the timing of
subject response is the reaction times of the subjects. Although there are no significant differences in average
reaction times across platforms [Fig. 2(D)], ANOVA on
reaction times indicates that the age of the subjects can con-
tribute (though not significant) more than the platform
(Table II). Advantages of having large amounts of data were dem-
onstrated by several visual salience models that leverage
deep learning methods (Jiang et al., 2015; Kruthiventi et al., In line with our initial goal, the study establishes strong
parallels between laboratory and online data. Similar to Kothinti et al. 2963 Kothinti et al. 2963 J. Acoust. Soc. Am. 150 (4), October 2021 2964
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169–178. Fisher, R. A. S. APPENDIX The details of the soundscapes used as part of the study
are listed in Table IV. TABLE IV. Details of the soundscapes used in the study. #
Block
Duration
Description
Sparse
Source
1
DNSS
2:00
Nature, Birds
No
Youtube
2
DNSS
2:15
Sporting Event
Yes
BBC Sounds
3
DNSS
2:00
Cafeteria
No
BBC Sounds
4
DNSS
2:02
Battle, Guns
Yes
Youtube
5
DNSS
1:21
Airplane, Baby
Yes
Soundsnap
6
DNSS
1:13
Fair
No
Freesound
7
DNSS
1:59
Classical Music
No
Youtube
8
DNSS
2:01
Bowling Alley
Yes
BBC Sounds
9
DNSS
2:00
Egypt Protests
No
Youtube
10
DNSS
2:08
Store Counter
No
BBC Sounds
11
DNSS
1:57
Drum Line
No
Youtube
12
DNSS
1:59
Blacksmith
No
Youtube
13
DNSS
2:13
Orchestra Tuning
No
Youtube
14
DNSS
2:07
Japanese Game Show
No
Youtube
15
DNSS
2:22
Dog Park
No
Freesound
2964
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Identification of proteins associated with clinical and pathological features of proliferative diabetic retinopathy in vitreous and fibrovascular membranes
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RESEARCH ARTICLE Editor: Alfred S. Lewin, University of Florida,
UNITED STATES Received: March 25, 2017
Accepted: October 16, 2017
Published: November 2, 2017 Received: March 25, 2017
Accepted: October 16, 2017
Published: November 2, 2017 Ingeborg Klaassen1*, Ewout W. de Vries1, Ilse M. C. Vogels1, Antoine H. C. van Kampen2,
Machteld I. Bosscha3, David H. W. Steel4, Cornelis J. F. Van Noorden1, Sarit Y. Lesnik-
Oberstein5, Reinier O. Schlingemann1 Ingeborg Klaassen1*, Ewout W. de Vries1, Ilse M. C. Vogels1, Antoine H. C. van Kampen2,
Machteld I. Bosscha3, David H. W. Steel4, Cornelis J. F. Van Noorden1, Sarit Y. Lesnik-
Oberstein5, Reinier O. Schlingemann1 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
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a1111111111 1 Ocular Angiogenesis Group, Departments of Ophthalmology and Medical Biology, Academic Medical
Center, University of Amsterdam, Amsterdam, The Netherlands, 2 Bioinformatics Laboratory, Clinical
Epidemiology, Biostatistics and Bioinformatics (KEBB), Academic Medical Center, University of Amsterdam,
Amsterdam, The Netherlands, 3 Department of Ophthalmology, VU University Medical Center, Amsterdam,
The Netherlands, 4 Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne and
Sunderland Eye Infirmary, Sunderland, United Kingdom, 5 The Harley Street Eye Clinic, London, United
Kingdom Abstract Citation: Klaassen I, de Vries EW, Vogels IMC, van
Kampen AHC, Bosscha MI, Steel DHW, et al. (2017) Identification of proteins associated with
clinical and pathological features of proliferative
diabetic retinopathy in vitreous and fibrovascular
membranes. PLoS ONE 12(11): e0187304. https://
doi.org/10.1371/journal.pone.0187304 Purpose To identify the protein profiles in vitreous associated with retinal fibrosis, angiogenesis, and
neurite formation in epiretinal fibrovascular membranes (FVMs) in patients with proliferative
diabetic retinopathy (PDR). Editor: Alfred S. Lewin, University of Florida,
UNITED STATES Methods Vitreous samples of 5 non-diabetic control patients with vitreous debris and 7 patients with
PDR membranes were screened for 507 preselected proteins using the semi-quantitative
RayBio® L-series 507 antibody array. From this array, 60 proteins were selected for a cus-
tom quantitative antibody array (Raybiotech, Human Quantibody® array), analyzing 7 con-
trol patients, 8 PDR patients with FVMs, and 5 PDR patients without FVMs. Additionally,
mRNA levels of proteins of interest were measured in 10 PDR membranes and 11
idiopathic membranes and in retinal tissues and cells to identify possible sources of protein
production. Copyright: © 2017 Klaassen 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 and its Supporting Information
files. * i.klaassen@amc.uva.nl Introduction Proliferative diabetic retinopathy (PDR) is a serious ocular complication of diabetes and is
characterized by retinal neovascularization and microvascular leakage in response to chronic
ischemia. Although anti-VEGF therapy alongside pan-retinal photocoagulation has been
shown to reduce neovascularization and macular edema [1], response to anti-VEGF treatment
is heterogeneous [2]. Additionally, there have been concerns that anti-VEGF treatment may
temporarily increase fibrovascular membrane (FVM) formation and retinal traction [3–7]. Ultimately both retinal detachment and hemorrhages in PDR are the leading causes of perma-
nent vision loss or blindness in adults of working age [8,9]. Therefore, it is imperative that the
molecular pathways leading to FVM formation are better understood in order to identify
novel therapeutic targets. Recently, it has also become clear that PDR is characterized not only by fibrovascular but
also by neuroglial pathology [10,11]. It was previously assumed that in PDR, the neurons of
the retina are incapable of proliferation, and that the total neural cell volume remains either
static, or is reduced due to apoptosis following diabetic damage [10]. Recently however, it has
been shown that PDR-associated FVMs contain neurite extensions growing alongside Mu¨ller
cells, a specialized type of retinal glia cell [12]. These neurites originate from rod photorecep-
tors and various populations of retinal ganglion cells [12–14]. New vessel growth is orches-
trated by chemoattractant and trophic factors derived from neurons [15] and Mu¨ller cells [16],
and Mu¨ller cells also serve as a scaffold for new vasculature [17]. Under these conditions, reti-
nal glial cells, macrophages, monocytes, hyalocytes (resident cells in the vitreous), fibroblasts,
pericytes and vascular endothelial cells will migrate and proliferate into the vitreous body,
hereby forming FVMs [18,19]. The cause and pathological implications of early neurite
recruitment in FVMs and their contribution to retinal angiogenesis and fibrosis are still
unknown. FVM formation occurs secondary to secretion of cytokines and growth factors by the retina
in response to ischemia [4,20]. A number of cytokines and growth factors have been identified
in measurable quantities in the vitreous of PDR patients, and their levels correlated strongly
with PDR disease activity [21,22]. Among them, vascular endothelial growth factor (VEGF)
remains the growth factor that is most-frequently studied [23], although various other growth
factors such as transforming growth factor-ß (TGF- ß), hepatocyte growth factor (HGF),
plasma kallikrein and platelet-derived growth factor (PDGF) have also been implicated in
PDR [24–26]. Conclusions This exploratory study reveals protein networks that potentially contribute to neurite out-
growth, angiogenesis and fibrosis in the formation of fibrovascular membranes in PDR. We
identified a possible role of Ang2 in fibrosis and the formation of FVMs, and of the neuro-
trophic factors NRG1, PDGF and GDNF in neurite growth that occurs in all FVMs in PDR. Competing interests: The authors have declared
that no competing interests exist. Two of the
authors are employed by a commercial company
[DS, SYLO]. This does not alter our adherence to
PLOS ONE policies on sharing data and materials. Identification of proteins involved in PDR membrane formation PDGF to be extensively co-regulated with other proteins, including thrombospondin-1 and
Ang2. mRNA levels of glial-derived and brain/derived neurotrophic factor (GDNF and
BDNF) were elevated in PDR membranes. These results were validated in a second study
of 52 vitreous samples of 32 PDR patients and 20 control patients. (Grant 2011-03). This study was published with the
help of Edward en Marianne Blaauw Fonds voor
Oogheelkunde (Edward and Marianne Blaauw Fund
for Ophthalmology). The funder provided support
in the form of salaries for authors [IK, IMCV], but
did not have any additional role in the study design,
data collection and analysis, decision to publish, or
preparation of the manuscript. The specific roles of
these authors are articulated in the ‘author
contributions’ section. (Grant 2011-03). This study was published with the
help of Edward en Marianne Blaauw Fonds voor
Oogheelkunde (Edward and Marianne Blaauw Fund
for Ophthalmology). The funder provided support
in the form of salaries for authors [IK, IMCV], but
did not have any additional role in the study design,
data collection and analysis, decision to publish, or
preparation of the manuscript. The specific roles of
these authors are articulated in the ‘author
contributions’ section. Results Funding: The study was supported by Landelijke
Stichting voor Blinden en Slechtzienden (LSBS),
Stichting Blinden-Penning and MaculaFonds that
contributed through UitZicht (Grant UitZicht 2011-
15), Stichting Ooglijders, Nederlandse Vereniging
ter Verbetering van het Lot der Blinden and
Stichting Nederlands Oogheelkundig Onderzoek Of the 507 proteins screened, 21 were found to be significantly elevated in PDR patients,
including neurogenic and angiogenic factors such as neuregulin 1 (NRG1), nerve growth
factor receptor (NGFR), placental growth factor (PlGF) and platelet derived growth factor
(PDGF). Angiopoietin-2 (Ang2) concentrations were strongly correlated to the degree of
fibrosis and the presence of FVMs in patients with PDR. Protein correlation analysis showed PLOS ONE | https://doi.org/10.1371/journal.pone.0187304
November 2, 2017 1 / 21 PLOS ONE | https://doi.org/10.1371/journal.pone.0187304
November 2, 2017 Study population For this study, 23 vitreous samples were used, including vitreous samples from PDR patients
(n = 16) and from patients with vitreous floaters (n = 7). In addition, 21 FVMs were collected
from patients with either PDR (n = 10) or non-diabetic patients with a macular pucker
(n = 11) who were operated by pars plana vitrectomy. Clinical variables were assessed by
trained ophthalmologists. The study was approved by the Medical Ethics Committees of the
Academic Medical Center and the VU University Medical Center, Amsterdam, The Nether-
lands. The study was conducted according to the tenets of the Declaration of Helsinki and
written consent was obtained from all patients. Table 1 highlights clinical features of the 23 patients that were included in the study of pro-
teins in vitreous. There was no statistically significant difference in the average age of control
(58 years) and diabetic (52 years) patients (T-test, P = 0.27). Additionally, there was no differ-
ence in the gender ratio between both groups (Fischer’s exact test, P = 0.41). In total, 43% of
participants in the study were male and 57% were female. The majority of the PDR patients
received preoperative pan-retinal laser treatment (n = 14) and 9 out of 16 PDR patients
received preoperative anti-VEGF therapy with bevacizumab (Avastin; Genentech, San Fran-
cisco, California, USA). Table 2 presents the clinical features of the 21 patients from whom membranes were har-
vested during pars plana vitrectomy. The patients where either non-diabetic and operated for
the removal of a macula pucker (n = 11), or suffering from PDR and operated for the removal
of FVMs (n = 10). PDR patients were on average younger (52 years) than control (71 year)
patients (T-test, P = 0.002). No difference in gender ratio between both groups was found
(Fischer’s exact test, P = 0.08). In total, 57% of the participants in the study were male and 43%
were female. Eight out of 10 PDR patients had received pan-retinal laser treatment and another
8 out of 10 patients were treated with bevacizumab at 3 days prior to surgery. We repeated our analysis in an independent set of vitreous samples obtained by the Univer-
sity of Newcastle upon Tyne. PLOS ONE | https://doi.org/10.1371/journal.pone.0187304
November 2, 2017 Identification of proteins involved in PDR membrane formation large number of growth factors and cytokines that may be involved in PDR pathogenesis
necessitates the use of high throughput techniques to identify relevant proteins. In the present study, we used a combination of semi-quantitative and quantitative antibody
arrays to screen over 500 proteins in the vitreous of PDR patients and control patients oper-
ated for vitreous floaters. We included a large series of angiogenic and neurogenic growth fac-
tors to identify those that are involved in PDR pathogenesis and thus are potential therapeutic
targets. In addition, we compared mRNA levels of the corresponding genes in FVMs of PDR
patients and idiopathic epiretinal membranes (ERMs, macular puckers of non-diabetic
patients) and various other retinal tissues to identify the possible sources of these proteins. Introduction Moreover, the balance between connective tissue growth factor (CTGF) and
VEGF is correlated with the degree of retinal fibrosis [4] and the angio-fibrotic switch. The PLOS ONE | https://doi.org/10.1371/journal.pone.0187304
November 2, 2017 2 / 21 3 Degree of fibrosis: 0, no evidence of fibrosis; 1, few pre-retinal membranes; 2, membranes with limited extension into the vitreous; 3, abundant white
membranes reaching into the vitreous body. 4 Degree of neovascularisation: 0, absent; 1, quiescent; 2, active. 5 Array used: L, L507 Array; Q, Quantibody Array. 6 Patient was operated for vitreomacular traction 2 Hemorrhage: 0, no hemorrhage; 1, mild hemorrhage; 2, moderate hemorrhage or heavy hemorrhage more than 2 months ago; 3, heavy hemorrhage
within 2 months. 3 Degree of fibrosis: 0, no evidence of fibrosis; 1, few pre-retinal membranes; 2, membranes with limited extension into the vitreous; 3, abundant white
membranes reaching into the vitreous body. 4 Degree of neovascularisation: 0 absent; 1 quiescent; 2 active 6 Patient was operated for vitreomacular traction groups (Fischer’s exact test, P = 0.13). None of the PDR patients had received pan-retinal laser
treatment or cataract surgery within the last 2 months, none had previous vitrectomy surgery
and none had undergone previous treatments with steroid therapy in the last year. Patients with
confounding retinovascular or other eye disease (e.g. retinal vein occlusion or glaucoma) and
patients who had received anti-VEGF therapy within the last 3 months were excluded. Study population A favorable ethical opinion for the collection of the samples was
obtained from the National Health Service (NHS) research ethics committee (South East Coast
—Surrey research ethics committee reference 12/LO/0130) and the collection carried out at
Sunderland Eye Infirmary under the sponsorship of City Hospitals Sunderland NHS founda-
tion trust. The study was conducted according to the tenets of the Declaration of Helsinki and
written consent was obtained from all patients. Fifty two vitreous samples of PDR patients
(n = 32) and of control patients without diabetes who had undergone vitrectomy for macular
hole or non-inflammatory vitreous opacities (n = 20) were included. Eighteen of the PDR
patients had fibrovascular membranes and 14 had no signs of fibrosis. PDR patients were on
average younger (56 years) than control patients (66 years) (t-test, P = 0.01). In total, 27% of the
52 patients were male and 73% were female with no difference in gender ratio between both PLOS ONE | https://doi.org/10.1371/journal.pone.0187304
November 2, 2017 3 / 21 1 Diabetic status: CON, no diabetes; DM2, diabetes type 2; DM2 + INS, diabetes type 2 with insulin dependency. 2 Hemorrhage: 0, no hemorrhage; 1, mild hemorrhage; 2, moderate hemorrhage or heavy hemorrhage more than 2 months ago; 3, heavy hemorrhage
within 2 months.
3 Degree of fibrosis: 0, no evidence of fibrosis; 1, few pre-retinal membranes; 2, membranes with limited extension into the vitreous; 3, abundant white
membranes reaching into the vitreous body 2 Hemorrhage: 0, no hemorrhage; 1, mild hemorrhage; 2, moderate hemorrhage or heavy hemorrhage more than 2 months ago; 3, heavy hemorrhage
within 2 months.
3 Degree of fibrosis: 0, no evidence of fibrosis; 1, few pre-retinal membranes; 2, membranes with limited extension into the vitreous; 3, abundant white
membranes reaching into the vitreous body.
4 Degree of neovascularisation: 0, absent; 1, quiescent; 2, active.
5 Array used: L, L507 Array; Q, Quantibody Array.
6 P
i
d f
i
l
i Identification of proteins involved in PDR membrane formation Table 1. Clinical characteristics of patients whose vitreous samples were used for protein analysis with antibody arrays. Case
Status1
Ophthalmological
Status
Age
Sex
Hemorrhage2
Degree of
Fibrosis3
Degree of
Neovascularisation4
Laser
Avastin
Array5
1
CON
Vitreal floaters
61
F
0
0
0
no
no
L+Q
2
CON
Vitreal floaters
59
F
0
0
0
no
no
L+Q
3
CON
Vitreal floaters
42
M
0
0
0
no
no
L+Q
4
CON
Vitreal floaters
51
F
0
0
0
no
no
Q
5
CON
Vitreal floaters
49
F
0
0
0
no
no
Q
6
CON
Vitreal floaters
68
M
0
0
0
no
no
L+Q
7
CON
Vitreal floaters
73
F
0
0
0
no
no
L+Q
8
DM2
PDR withoutFVM
54
F
1
0
0
yes
no
Q
9
DM2
PDR with FVM
44
M
3
3
2
yes
no
L+Q
10
DM2
PDR without FVM6
62
F
0
0
0
yes
yes
Q
11
DM2
PDR with FVM
56
M
2
2
2
yes
yes
L+Q
12
DM2
PDR with FVM
54
M
2
2
0
yes
no
Q
13
DM2
PDR with FVM
59
F
0
3
1
yes
yes
Q
14
DM2
PDR without FVM
63
M
1
0
2
yes
yes
Q
15
DM2
PDR without FVM
67
M
3
0
1
yes
no
Q
16
DM2
PDR with FVM
27
M
1
3
2
yes
yes
L
17
DM2
PDR with FVM
35
F
1
3
2
yes
yes
L
18
DM2
+ INS
PDR with FVM
55
F
2
2
1
yes
no
Q
19
DM2
+ INS
PDR with FVM
48
F
2
3
2
no
yes
L+Q
20
DM2
+ INS
PDR with FVM
53
F
0
3
2
yes
no
L+Q
21
DM2
+ INS
PDR without FVM
51
M
3
0
2
yes
no
Q
22
DM2
+ INS
PDR with FVM
51
F
1
2
2
yes
yes
Q
23
DM2
+ INS
PDR with FVM
57
M
0
3
2
no
yes
L characteristics of patients whose vitreous samples were used for protein analysis with antibody arrays. Table 1. Clinical characteristics of patients whose vitr 2 Hemorrhage: 0, no hemorrhage; 1, mild hemorrhage; 2, moderate hemorrhage or heavy hemorrhage more than 2 months ago; 3, heavy hemorrhage
within 2 months. 1 Diabetic status: CON, no diabetes; DM2, diabetes type 2; DM2 + INS, diabetes type 2 with insulin dependency. https://doi.org/10.1371/journal.pone.0187304.t001 2 Hemorrhage: 0, no hemorrhage; 1, mild hemorrhage; 2, moderate hemorrhage or heavy hemorrhage more than 2 months ago; 3, heavy hemorrhage
within 2 months. DM2, diabetes type 2; DM2 + INS, diabetes type 2 with insulin dependency. Identification of proteins involved in PDR membrane formation Table 2. Clinical characteristics of patients whose fibrovascular membranes where used for mRNA analysis. Case
Status1
Ophthalmological Status
Age
Sex
Hemorrhage2
Degree of Fibrosis3
Degree of Neovascularisation4
Laser
Avastin
1
CON
Pucker
84
F
0
1
0
no
no
2
CON
Pucker
66
F
0
1
0
no
no
3
CON
Pucker
72
M
0
1
0
no
no
4
CON
Pucker
79
M
0
1
0
no
no
5
CON
Pucker
62
F
0
1
0
no
no
6
CON
Pucker
73
F
0
1
0
no
no
7
CON
Pucker
70
M
0
1
0
no
no
8
CON
Pucker
66
F
0
1
0
no
no
9
CON
Pucker
80
F
0
1
0
no
no
10
CON
Pucker
63
F
0
1
0
no
no
11
CON
Pucker
69
M
0
1
0
no
no
12
DM1
PDR
41
M
3
2
2
yes
yes
13
DM1
PDR
59
M
2
1
2
yes
no
14
DM1
PDR
43
M
3
1
2
yes
yes
15
DM1
PDR
48
F
0
1
2
yes
yes
16
DM2
PDR
25
M
3
3
2
no
no
17
DM2 + INS
PDR
48
F
2
3
2
no
yes
18
DM2 + INS
PDR
79
M
3
1
2
yes
yes
19
DM2 + INS
PDR
39
M
1
1
2
yes
yes
20
DM2 + INS
PDR
72
M
3
1
2
yes
yes
21
DM2 + INS
PDR
61
M
1
2
2
yes
yes Table 2. Clinical characteristics of patients whose fibrovascular membranes where used for mRNA analysis Table 2. Clinical characteristics of patients whose fibrovascular membrane 3 Degree of fibrosis: 0, no evidence of fibrosis; 1, few pre-retinal membranes; 2, membranes with limited extension into the vitreous; 3, abundant white
membranes reaching into the vitreous body. 4 Degree of neovascularisation: 0, absent; 1, quiescent; 2, active. Additionally, the patient files and per-operative observations, which were recorded using a
standardized form, were used. Fibrosis was graded as 0 when there was no fibrosis, as 1 when
there were a few pre-retinal membranes (limited as in macular pucker), as 2 when pre-retinal
membranes were present with limited extension into the vitreous, and as 3 when abundant
membranes reaching into the vitreous body were observed. Neovascularization was graded as
0 when absent, as 1 (quiescent) when only non-perfused vessels were present, and as 2 (active)
when perfused pre-retinal capillaries were present [27]. Degree of hemorrhage was graded as 0
when all media were clear and all fundus details were visible, as 1 when media were slightly
clouded but the fundus could still be examined, as 2 when the hemorrhage was moderate, or
heavy and more than 2 months ago, and as 3 when the hemorrhage was heavy and less than
two months ago. Clinical measurements of PDR The degree of fibrosis, activity of neovascularization, degree of hemorrhage, and presence and
type of diabetes, were obtained from pre-operative ophthalmic and ultrasound examinations. PLOS ONE | https://doi.org/10.1371/journal.pone.0187304
November 2, 2017 4 / 21 https://doi.org/10.1371/journal.pone.0187304.t002 1 Diabetic status: CON, no diabetes; DM2, diabetes type 2; DM2 + INS, diabetes type 2 with insulin dependency. 1 Diabetic status: CON, no diabetes; DM2, diabetes type 2; DM2 + INS, diabetes type 2 with insulin dependency.
2 Vitreous protein quantitation Vitreous samples of 7 PDR patients with FVMs and of 5 control patients were measured using
a biotin label-based human antibody array (Human Antibody L-series 507 Array; RayBiotech,
Norcross, GA, USA) to screen for potential proteins of interest. The array was performed in
accordance with the manufacturer’s instructions. In brief, vitreous samples were centrifuged
for 15 min at 14,000 g at 4˚C. Supernatant was collected and dialyzed with PBS overnight at
4˚C. After determination of protein concentrations, the appropriate amount of biotin was
added and samples were again dialyzed with PBS overnight at 4˚C. Samples were then hybrid-
ized to the arrays overnight at 4˚C with gentle shaking. Biotinylated proteins captured by the
membrane-bound antibodies were detected by incubation with HRP-streptavidin and analysis
was performed by an Agilent laser scanner (Agilent Technologies, Palo Alto, CA, USA). Spot
intensities were quantified with ScanAlyze software (Michael Eisen, http://rana.lbl.gov/
EisenSoftware.htm) and mean signal and median background values were applied in subse-
quent calculations. Sixty proteins of interest (based on results of the L507 array and relevant literature) were
selected for further quantitative analysis. Quantitation was achieved using a customizable
array-based multiplex immunoassay (RayBiotech, Human Quantibody1 array) in accordance
with the manufacturer’s instructions. Standard curves for each protein were generated and the
lower limit of detection (LOD) was calculated based on the average and standard deviation of
four negative controls (average + 2x standard deviation). Proteins that were undetectable in
more than half of the samples were deemed to be below the LOD. Correlation network construction The correlation network was constructed in the statistical package ‘R’ (version 3.2.3) (https://
www.R-project.org) using the igraph package (version 1.0.1) (igraph.org). First, Pearson corre-
lations between Quantibody protein profiles over all samples were calculated for all pairs of
proteins. Subsequently, the proteins were presented in a correlation network [27] in which the
nodes represent proteins and the interconnecting lines represent the correlation between the
proteins. We discarded all correlations that were lower than 0.7 and removed all unconnected
proteins. Control tissue and cells Control retinas from donor eyes were provided anonymously by the Corneabank Beverwijk
(http://www.eurotissuebank.nl/comeabank/), The Netherlands. In The Netherlands, the use of
donor material is provided for by a law named "Wet op Orgaan Donatie (WOD)". Following
this law, donors provide written informed consent for donation with an opt out for the use of
left-over material for related scientific research. Specific requirements for the use of left-over PLOS ONE | https://doi.org/10.1371/journal.pone.0187304
November 2, 2017 5 / 21 Identification of proteins involved in PDR membrane formation material originating from corneal grafting for scientific research have been described in an
additional document formulated by the Ministry of Health, Welfare, and Sport and the BIS
foundation (Eurotransplant; Leiden, July 21, 1995; 6714.ht). The present study was performed
in accordance with all requirements stated in the WOD and the relevant document and in
accordance with these regulations approval of the local medical ethics committee was not
required as the data were analyzed anonymously. Human primary retinal pigment epithelial cells were also obtained from donor eyes, with a
post mortem time of less than 15 hours, as described previously [28]. Human primary retinal
endothelial cells and pericytes were obtained from donor eyes (up to 24 hours post mortem) as
described for bovine retinas [29]. Endothelial cells were cultured in complete EBM-2 medium
(Lonza, Breda, The Netherlands) with 5% human serum. For glial cells a human astrocytoma
cell line, U-373 MG (Uppsala) (Sigma-Aldrich, Zwijndrecht, The Netherlands) was used. RNA
from blood was obtained as described previously [30]. Detection of vitreous proteins The L507 array allowed us to screen the vitreous for presence of 507 proteins in PDR patients
with FVMs relative to control patients with floaters. 453 proteins were shown to have a higher
and 54 proteins had a lower concentrations in vitreous of PDR eyes. Only 55 of the proteins
were significantly higher in concentration in PDR patients, whereas none of the proteins were
significantly lower in concentration in PDR patients (two-tailed Welch’s t-test). Fifty three
proteins had concentrations in the vitreous of PDR patients more than 2-fold higher than con-
trols, while 25 proteins had a 10-fold higher concentration (S1 Table). Statistical analysis Values of vitreous proteins are reported as a mean (pg/ml) ± standard deviation. Univariable
analysis with two-tailed T-tests assuming unequal variance were performed to identify individ-
ual proteins significantly associated with PDR. For all statistical analyses a P value < 0.05 was
considered to indicate statistically significant differences. RNA isolation and mRNA quantification Fibrous tissue was removed during surgery, placed in 250 μL TRIzol reagent (Life Technolo-
gies, Carlsbad, CA, USA) and stored at -80˚C until further processing. Membranes were then PLOS ONE | https://doi.org/10.1371/journal.pone.0187304
November 2, 2017 6 / 21 Identification of proteins involved in PDR membrane formation homogenized using a pestle and vortexed. RNA from ERMs was extracted and quantified using
techniques described previously [28]. Total RNA yields were measured using a NanoDrop
(ND1000 Spectrophotometer; NanoDrop Technologies, Wilmington, DE, USA) and RNA qual-
ity was assessed using an Experion™Automated Electrophoresis System (Bio-Rad, Hercules,
CA, USA). For all samples, RNA quality indicator (RQI) values ranged between 6.5 and 8.5. A 1-μg aliquot of total RNA was treated with DNAse I (Amplification Grade; Invitrogen)
and reverse transcribed into first strand cDNA using the Maxima1 First Strand cDNA Syn-
thesis Kit (Thermo Scientific, Roskilde, Denmark). Real-time quantitative PCR was performed
on 20x diluted cDNA samples using a CFX96 system (Bio-Rad, Hercules, CA) as described
previously [31]. homogenized using a pestle and vortexed. RNA from ERMs was extracted and quantified using
techniques described previously [28]. Total RNA yields were measured using a NanoDrop
(ND1000 Spectrophotometer; NanoDrop Technologies, Wilmington, DE, USA) and RNA qual-
ity was assessed using an Experion™Automated Electrophoresis System (Bio-Rad, Hercules,
CA, USA). For all samples, RNA quality indicator (RQI) values ranged between 6.5 and 8.5. A 1-μg aliquot of total RNA was treated with DNAse I (Amplification Grade; Invitrogen)
and reverse transcribed into first strand cDNA using the Maxima1 First Strand cDNA Syn-
thesis Kit (Thermo Scientific, Roskilde, Denmark). Real-time quantitative PCR was performed
on 20x diluted cDNA samples using a CFX96 system (Bio-Rad, Hercules, CA) as described
previously [31]. Primer details are listed in S2 Table. The specificity of the primers was confirmed by NCBI
BLAST. The presence of a single PCR product was verified by both the presence of a single
melting temperature peak and detection of a single band of the expected size on 3% agarose
gel. Non-template controls were included to verify the method and the specificity of the prim-
ers. Normalization of data was performed with global mean normalization [32]. PLOS ONE | https://doi.org/10.1371/journal.pone.0187304
November 2, 2017 Identification of proteins involved in PDR membrane formation Table 3. Protein concentrations in vitreous of control and PDR patients. Protein concentrations were determined by Quantibody arrays in patients with
vitreal floaters (CON) and patients with proliferative diabetic retinopathy (PDR) that underwent vitreoretinal surgery. Quantitation of vitreous proteins On the basis of the results of the L507 array and relevant literature, we designed a custom
quantibody array containing antibodies for the detection of 60 proteins in 20 samples (13 PDR
and 7 controls). Although more expensive, the advantage of the quantibody array is that actual
protein concentrations rather than relative yields of protein can be measured. Of the 60 pro-
teins screened, 20 had concentrations below the LOD (defined as >50% of samples below
detection limit) and were excluded from further analysis (AR, b-NGF, BDNF, COCO, E-Selec-
tin, GDNF, ICAM3, IGF1R, IGF2, Insulin, Insulin R, NT3, NT4, PDGF-Ra, PDGF-Rb, Prolac-
tin, TARC, TGFβ1, TPO and XEDAR). For the other 40 proteins, occasional samples that were
below the LOD were approximated as the LOD/p2 [33]. Four proteins showed concentrations
above the highest standards (Adiponectin, IGFBP6, NrCAM and TIMP-1). The results of the quantibody array and the lower limits of detection are presented in
Table 3. Twenty-one proteins were significantly upregulated in PDR patients (Welch’s T-test)
and are highlighted in bold. In addition, 14 of the proteins exhibited a larger than 3-fold rise in
concentration and are highlighted in bold. In a second independent study group of PDR PLOS ONE | https://doi.org/10.1371/journal.pone.0187304
November 2, 2017 7 / 21 Identification of proteins involved in PDR membrane formation Table 3. (Continued)
CON
PDR
LOD
mean
SD
min
max
mean
SD
min
max
Fold Change
P-value
PIGF*
2.5
1.8
0.0
1.8
1.8
102.8
134.5
1.8
467.4
57.30
0.019
Prolactin
151.7
BDL
BDL
TARC
1.7
BDL
BDL
TGFb1
151.4
BDL
BDL
TGF-b2
7.1
30.3
14.1
13.0
43.9
48.9
56.7
5.0
184.7
1.61
0.281
Thrombospondin-1
215.0
980.3
1650.2
152.0
4645.7
10116.4
10082.6
499.9
34455.3
10.32
0.007
TIMP-1*
13.4
60145.6
19198.7
41110.6
88938.0
61745.9
9704.1
47592.0
82709.5
1.03
0.842
TIMP-4
6.5
395.0
333.7
142.0
1054.3
611.8
282.4
196.5
1069.2
1.55
0.173
TPO
139.9
BDL
BDL
Ubiquitin+1
136.8
261.9
197.4
96.7
578.9
897.7
390.9
271.1
1465.3
3.43
<0.001
VCAM-1
794.5
7383.1
7964.7
561.8
23677.8
8143.9
5254.2
2198.5
22644.2
1.10
0.825
VEGF
10.7
23.5
42.2
7.6
119.2
1208.1
1528.2
7.6
5038.6
51.40
0.016
WIF-1
19.5
11133.9
2970.5
8184.8
15679.3
9207.2
3063.2
4362.8
13964.5
0.83
0.195
WISP-1
68.6
176.1
98.6
48.5
293.8
191.5
121.9
48.5
499.5
1.09
0.763
XEDAR
3.3
BDL
BDL
D t
t d i
/
L ± SD
ith
i
d
l
BDL b l
li
it f d t
ti
(LOD) U
i
d t t
t
ith W l h’
ti
d t Data are presented in pg/mL ± SD with min and max values. BDL, below limit of detection (LOD). Unpaired t-test with Welch’s correction was used to
assess statistical differences between PDR and control patients. Fold changes higher than 3-fold and significant differences (P < 0.05) are indicated in bold. *Adiponectin, levels in PDR patients above the highest standards; IGFBP6, NrCAM, TIMP-1, all levels above the highest standards; PlGF, all control levels
were below LOD and set at LOD/p2. https://doi.org/10.1371/journal.pone.0187304.t003 patients (n = 32) and controls (n = 20) from the University of Newcastle upon Tyne, we mea-
sured protein levels of 5 of these proteins (Ang2, NRG1β1, PDGF-AA, PlGF and VEGF). The
fold changes between PDR patients and controls were comparable with those of the first study
group and the differences were statistically significant, except for NRG1β1 (S3 Table). Next, we attempted to identify differences in protein concentrations between PDR patients
with and without FVMs. Vitreous samples were divided into control, PDR patients without
membranes and PDR patients with membranes. A significant upregulation of Ang2 in PDR
+FVM patients compared to PDR-FVM patients was found (Kruskal-Wallis + Mann-Whitney
U test) (Fig 1). Ang2 protein levels were log10 transformed to obtain a normal distribution. A
similar trend for Ang2 was observed in the independent study group (S1 Fig). No statistically
significant differences in concentrations of the other proteins were found in PDR patients with
or without FVMs. CON
PDR
LOD
mean
SD
min
max
mean
SD
min
max
Fold Change
P-value
Adiponectin*
5.5
9680.0
4869.9
2116.3
17181.5
38271.7
21622.7
6359.8
71881.8
3.95
<0.001
ANG-1
5.4
95.1
28.0
57.0
137.8
259.6
240.4
69.4
863.9
2.73
0.031
ANG-2
5.1
93.5
52.9
28.9
161.0
1802.4
2979.4
117.8
11369.4
19.28
0.061
AR
8.2
BDL
BDL
BDNF
1.0
BDL
BDL
bFGF
32.5
117.0
236.8
23.0
653.7
45.2
19.6
23.0
80.0
0.39
0.454
BMP-2
1.7
59.2
11.5
44.6
73.4
95.9
51.5
27.7
202.6
1.62
0.028
BMP-5
148.6
240.7
111.3
105.1
396.9
281.8
106.4
105.1
513.1
1.17
0.439
b-NGF
2.9
BDL
BDL
COCO
418.2
BDL
BDL
DcR3
242.7
202.1
52.1
171.6
282.1
656.5
655.2
171.6
2487.7
3.25
0.028
ErbB3
13.0
154.5
24.5
126.5
186.6
179.4
98.2
9.2
403.9
1.16
0.401
E-Selectin
50.2
BDL
BDL
Galectin-3
32.5
162.9
284.2
23.0
798.7
353.1
497.5
23.0
1579.9
2.17
0.291
GDF-15
0.8
370.7
273.1
48.1
880.7
975.7
425.8
172.8
1645.4
2.63
0.001
GDNF
3.0
BDL
BDL
GH
29.0
34.1
20.5
20.5
75.2
77.5
53.0
20.5
177.8
2.28
0.018
HGF
4.3
2500.6
1062.3
1618.6
4418.0
7967.1
2151.6
2752.1
10711.5
3.19
<0.001
ICAM-1
76.4
696.0
1031.4
54.0
2707.0
3762.4
3531.0
696.9
13167.3
5.41
0.011
ICAM-3
24.7
BDL
BDL
IGFBP-1
4.3
222.5
242.9
38.6
726.1
2210.9
2132.1
207.7
7383.2
9.94
0.006
IGFBP-2
41.5
16751.2
7003.4
7562.7
30992.2
24718.9
9332.7
13573.6
42543.5
1.48
0.047
IGFBP-3
114.1
1043.7
704.0
319.4
2053.4
19596.4
11034.1
2595.4
37544.3
18.78
<0.001
IGFBP-4
539.2
3463.8
1293.0
2009.4
5910.7
3471.7
1488.8
1156.9
6029.0
1.00
0.990
IGFBP-5
115.4
234.3
125.6
81.6
393.4
389.6
366.1
81.6
1570.1
1.66
0.185
IGFBP-6*
68.3
27483.8
8803.2
22349.7
47174.6
33899.1
10489.7
21769.8
55469.0
1.23
0.168
IGF-I
27.0
51.8
41.1
19.1
129.4
62.3
40.2
19.1
151.5
1.20
0.594
IGF-I R
48.6
BDL
BDL
IGF-II
41.4
BDL
BDL
IGF-II R
18.4
199.2
47.5
148.0
266.4
208.7
190.9
27.2
770.8
1.05
0.867
Insulin
20.1
BDL
BDL
Insulin R
3,115.9
BDL
BDL
NCAM-1
104.1
17561.3
8210.6
5251.9
28434.0
17835.7
9907.3
6488.9
37545.7
1.02
0.948
NGF R
10.3
41.3
27.4
7.3
78.5
74.7
21.7
40.2
105.6
1.81
0.019
Notch-1
2.1
21.9
16.4
12.6
58.3
17.4
18.2
1.5
62.7
0.80
0.590
NOV
5.8
1836.0
458.4
1202.5
2740.6
3206.6
942.9
1627.7
5509.0
1.75
<0.001
NrCAM*
5.0
4872.9
1914.6
2185.5
7215.7
6082.5
3285.2
715.4
12610.8
1.25
0.313
NRG1-b1
10.8
11.9
4.8
7.6
19.9
36.9
27.5
7.6
69.7
3.10
0.007
NT-3
12.1
BDL
BDL
NT-4
5.3
BDL
BDL
PDGF Ra
1,170.8
BDL
BDL
PDGF Rb
10.8
BDL
BDL
PDGF-AA
2.0
99.1
86.8
9.6
263.3
602.2
556.6
91.8
1827.7
6.08
0.007
PDGF-AB
5.0
6.6
1.6
3.5
8.3
16.9
16.2
5.5
67.5
2.54
0.042
PDGF-BB
0.4
0.7
0.5
0.3
1.3
2.9
2.7
0.6
9.3
4.03
0.015
(Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0187304
November 2, 2017 8 / 21 Data are presented in pg/mL ± SD with min and max values. BDL, below limit of detection (LOD). Unpaired t-test with Welch’s correction was used to
assess statistical differences between PDR and control patients. Fold changes higher than 3-fold and significant differences (P < 0.05) are indicated in bold.
*Adiponectin, levels in PDR patients above the highest standards; IGFBP6, NrCAM, TIMP-1, all levels above the highest standards; PlGF, all control levels
were below LOD and set at LOD/p2. https://doi.org/10.1371/journal.pone.0187304.t003 Correlations between vitreous protein concentrations and clinical
features of PDR Correlations between vitreous protein concentrations in PDR patients and clinical features of
fibrosis, neovascularization and vitreous hemorrhage were assessed using Spearman’s rank
correlation coefficients. Moderate correlations were defined as coefficients between 0.40 and
0.59 and strong correlations between 0.60–0.79. PDGF-AA, Ang2 and PlGF showed a strong
correlation with the degree of fibrosis (R2 = 0.638, 0.632 and 0.597, respectively), whereas
IGF-II R showed a strongly negative correlation (R2 = -0.617). Additionally, IGFBP-6, GDF-15
and Ang1 were strongly correlated with neovascularization (R2 = 0.629, 0.623 and 0.599,
respectively). The degree of vitreous hemorrhage was negatively correlated with NrCAM,
NCAM-1 and DcR3 (R2 = 0.709, 0.669 and 0.563, respectively). PLOS ONE | https://doi.org/10.1371/journal.pone.0187304
November 2, 2017 9 / 21 Identification of proteins involved in PDR membrane formation CON
PDR - FVM
PDR + FVM
0
1
2
3
4
5
Log10 Protein levels (pg/ml)
Ang2
<0.0001
0.0103
0.0131
Fig 1. Ang2 protein is increased in vitreous of PDR patients with FVMs. Protein levels of Ang2, as
detected by Quantibody arrays, were log10 transformed to obtain a normal distribution. Differences between
groups were analyzed by a Student’s t-test. CON, non-diabetic control patients; PDR–FVM, PDR patients
without FVMs; PDR + FVM, PDR patients with FVMs. The lines represent the geometric means. https://doi.org/10.1371/journal.pone.0187304.g001 Fig 1. Ang2 protein is increased in vitreous of PDR patients with FVMs. Protein levels of Ang2, as
detected by Quantibody arrays, were log10 transformed to obtain a normal distribution. Differences between
groups were analyzed by a Student’s t-test. CON, non-diabetic control patients; PDR–FVM, PDR patients
without FVMs; PDR + FVM, PDR patients with FVMs. The lines represent the geometric means. https://doi.org/10.1371/journal.pone.0187304.g001 https://doi.org/10.1371/journal.pone.0187304.g001 Effects of bevacizumab on protein concentrations Six of the PDR patients had been treated by intravitreous injection with bevacizumab 3 days
prior to surgery. We investigated whether the use of bevacizumab had effects on the concentra-
tions of proteins other than VEGF. Whereas VEGF concentrations were almost completely
downregulated by bevacizumab, concentrations of PlGF (a family member of VEGF) were not
significantly affected (Fig 2). IGF1 concentrations, however, were significantly higher in IGF1
CON
PDR - B
PDR + B
0
50
100
150
200
Protein levels (pg/ml)
0.6502
0.0155 VEGF-A
CON
PDR - B
PDR + B
0
2000
4000
6000
Protein levels (pg/ml)
0.0177
0.0290
PlGF
CON
PDR - B
PDR + B
0
100
200
300
400
500
Protein levels (pg/ml)
0.0734
0.3832
IGF1
CON
PDR - B
PDR + B
0
50
100
150
200
Protein levels (pg/ml)
0.6502
0.0155
Fig 2. Effect of anti-VEGF therapy on protein levels. Samples were divided in three groups: CON, non-diabetic control patients; PDR—B, PDR patients
that did not receive bevacizumab; PDR + B, PDR patients that received bevacizumab. Differences between groups were analyzed with an unpaired t test
with Welch’s correction. Lines represent mean values. https://doi org/10 1371/journal pone 0187304 g002 PlGF
CON
PDR - B
PDR + B
0
100
200
300
400
500
Protein levels (pg/ml)
0.0734
0.3832 VEGF-A
CON
PDR - B
PDR + B
0
2000
4000
6000
Protein levels (pg/ml)
0.0177
0.0290 Fig 2. Effect of anti-VEGF therapy on protein levels. Samples were divided in three groups: CON, non-diabetic control patients; PDR—B, PDR patients
that did not receive bevacizumab; PDR + B, PDR patients that received bevacizumab. Differences between groups were analyzed with an unpaired t test
with Welch’s correction. Lines represent mean values. Fig 2. Effect of anti-VEGF therapy on protein levels. Samples were divided in three groups: CON, non-diabetic control patients; PDR—B, PDR patients
that did not receive bevacizumab; PDR + B, PDR patients that received bevacizumab. Differences between groups were analyzed with an unpaired t test
with Welch’s correction. Lines represent mean values. https://doi.org/10.1371/journal.pone.0187304.g002 https://doi.org/10.1371/journal.pone.0187304.g002 PLOS ONE | https://doi.org/10.1371/journal.pone.0187304
November 2, 2017 10 / 21 Identification of proteins involved in PDR membrane formation Fig 3. Coregulation of proteins. Pearson correlations between Quantibody protein profiles over all samples
as calculated for all pairs of proteins. Effects of bevacizumab on protein concentrations The proteins are presented in a correlation network in which the nodes
represent proteins and the interconnecting lines represent the correlation between the proteins. The thickness
of the lines represent the strength of the correlation. All correlations <0.7 and disconnected proteins were
removed. h
//d i
/10 1371/j
l
0187304 003 Fig 3. Coregulation of proteins. Pearson correlations between Quantibody protein profiles over all samples
as calculated for all pairs of proteins. The proteins are presented in a correlation network in which the nodes
represent proteins and the interconnecting lines represent the correlation between the proteins. The thickness
of the lines represent the strength of the correlation. All correlations <0.7 and disconnected proteins were
removed. https://doi.org/10.1371/journal.pone.0187304.g003 bevacizumab-treated eyes (p = 0.0155). Significant effects of bevacizumab on other proteins
were not observed. bevacizumab-treated eyes (p = 0.0155). Significant effects of bevacizumab on other proteins
were not observed. Gene expression in idiopathic and PDR-associated membranes In order to identify possible cellular or tissue sources of the proteins in the vitreous of PDR
patients, we determined gene expression in FVMs and compared these data to gene expression
in blood cells from diabetic patients, control retina tissue, retinal pigment epithelial cells
(RPE), human retinal endothelial cells (HRECs), human retinal pericytes (HRPCs) and human
glial cells (Table 4). In total 11 idiopathic ERMs, and 10 PDR membranes were analyzed. In accordance with protein concentrations found in the vitreous of PDR patients, 5 genes
were found to be consistently expressed in all PDR membranes (GDF15, IGFBP3, THBS1,
TIMP1 and VEGFA). In addition, BDNF and GDNF were found to be expressed in all idio-
pathic and 9 of the 10 PDR membranes (Table 4), even though protein concentrations of these
respective proteins in the vitreous were below the LOD (Table 3). Increased vitreous protein concentrations may be due to either increased local production
or due to leakage from the vasculature. Despite showing elevated vitreous concentrations,
angiogenic growth factors such as adiponectin, PDGFA and PDGFB mRNAs were hardly, or Table 4. Gene expression in membranes, retinal tissues and cells, and blood. Correlations between proteins We investigated whether proteins were co-regulated and analyzed this by a pairwise compari-
son of proteins (Fig 3). Visual inspection revealed 3 groups of co-expressed proteins. The larg-
est group contained the most interactions, showing co-expression of various proteins centered
around a PDGF axis, consisting of the PDGF-AA, AB and BB subunits. In another group, co-
regulation was found between neuronal cell adhesion molecule (NrCAM) and neural cell
adhesion molecule (NCAM). Weaker associations were found in the third group between
growth differentiation factor 15 (GDF15), nephroblastoma overexpressed (NOV), hepatocyte
growth factor (HGF), and insulin-like growth factor binding protein 3 (IGFBP3). PLOS ONE | https://doi.org/10.1371/journal.pone.0187304
November 2, 2017 11 / 21 Identification of proteins involved in PDR membrane formation Left side (iERM, PDR): number of membranes in which gene expression is detected. Right side (Relative expression): abundance of gene expression in
arbitrary units. iERM, idiopathic epiretinal membrane; PDR, epiretinal membrane from PDR patient; Retina, whole retina of non-diabetic donor eye; RPE,
primary retinal pigment epithelial cells; Glial cells, U373 cell line; HRECs, human primary retinal endothelial cells; HRPCs, human primary retinal pericytes;
Blood, pooled whole blood from patients with diabetic macular edema. Underlined values show significant difference in gene expression levels between
PDR and iERM membranes (P < 0.05). https://doi.org/10.1371/journal.pone.0187304.t004 Identification of proteins involved in PDR membrane formation VEGFA
CON
PDR
0
2000
4000
6000
8000
10000
mRNA levels
0.0090
p =
NRG1
CON
PDR
0
20
40
60
80
100
mRNA levels
0.0265
p =
ANGPT2
CON
PDR
0
200
400
600
mRNA levels
0.0754
p =
IGFBP3
CON
PDR
0
5000
10000
15000
mRNA levels
0.2186
p =
PGF
CON
PDR
0
200
400
600
800
mRNA levels
0.0138
p =
BDNF
CON
PDR
0
100
200
300
400
500
mRNA levels
0.0310
p =
CTGF
CON
PDR
0
5000
10000
15000
mRNA levels
0.4759
p =
VIM
CON
PDR
0
10000
20000
30000
mRNA levels
0.7989
p =
Fig 4. mRNA levels in idiopathic and PDR membranes. Transcript levels of genes were quantified by real-time quantitative PCR. Differences
between groups were analyzed by a Student’s t-test. Lines represent mean values. https://doi.org/10.1371/journal.pone.0187304.g004 ANGPT2
CON
PDR
0
200
400
600
mRNA levels
0.0754
p =
IGFBP3
CON
PDR
0
5000
10000
15000
mRNA levels
0.2186
p = VEGFA
CON
PDR
0
2000
4000
6000
8000
10000
mRNA levels
0.0090
p =
NRG1
CON
PDR
0
20
40
60
80
100
mRNA levels
0.0265
p =
ANGPT2
CON
PDR
0
200
400
600
mRNA levels
0.0754
p =
IGFBP3
CON
PDR
0
5000
10000
15000
mRNA levels
0.2186
p = IGFBP3
CON
PDR
0
5000
10000
15000
mRNA levels
0.2186
p = VEGFA
CON
PDR
0
2000
4000
6000
8000
10000
mRNA levels
0.0090
p =
NRG1
CON
PDR
0
20
40
60
80
100
mRNA levels
0.0265
p = PGF
CON
PDR
0
200
400
600
800
mRNA levels
0.0138
p =
BDNF
CON
PDR
0
100
200
300
400
500
mRNA levels
0.0310
p = CTGF
CON
PDR
0
5000
10000
15000
mRNA levels
0.4759
p =
VIM
CON
PDR
0
10000
20000
30000
mRNA levels
0.7989
p = Fig 4. mRNA levels in idiopathic and PDR membranes. Transcript levels of genes were quantified by real-time quantitative PCR. Differences
between groups were analyzed by a Student’s t-test. Lines represent mean values. Fig 4. mRNA levels in idiopathic and PDR membranes. Transcript levels of genes were quantified by real-time quantitative PCR. Differences
between groups were analyzed by a Student’s t-test. Lines represent mean values. https://doi.org/10.1371/journal.pone.0187304.g004 not at all, found in membrane tissues. In other ocular tissues studied, adiponectin was only
detected in blood cells and not in any of the ocular tissues including FVMs, whereas PDGFA
was expressed in white blood cells and glial cells and PDGFB was expressed in white blood
cells, RPE cells, glial cells and HRECs. Since PDGF concentrations in the vitreous of PDR
patients were lower than the concentrations of PDGF reported in the plasma of both diabetic
patients [34] and healthy volunteers [35], PDGFA is most likely derived from blood, whereas
PDGFB is probably synthesized by either RPE, HREC or glial cells. TIMP1, which showed the
highest vitreous protein concentrations, also showed the highest abundance in mRNA levels in
all tissues and cells, with HRECs being the major source. When considering differences in mRNA expression levels between non-diabetic ERMs and
PDR membranes, a few observations stand out: expression levels of ANGPT2 were 4.6-fold
higher in PDR membranes as compared to non-diabetic ERMs (Table 4; Fig 4), which is in
agreement with the elevated vitreous protein concentrations in PDR patients (Table 3). Con-
versely, gene expression levels of NRG1, PGF and VEGFA were strikingly lower in PDR mem-
branes when compared to controls (Table 4; Fig 4), whereas proteins concentrations in the
vitreous of PDR patients were increased (Table 3). In addition, mRNA levels of connective tis-
sue growth factor (CTGF), a marker of fibrosis [4], IGFBP3 and vimentin (VIM), a highly
abundant glial cell marker in ERMs, were comparable between non-diabetic ERMs and PDR
membranes (Fig 4). Gene expression in idiopathic and PDR-associated membranes n = 11
n = 10
Relative expression
Gene
iERM
PDR
iERM
PDR*
Retina
RPE
Glial cells
HRECs
HRPCs
Blood
ADIPOQ
0
0
0
0
0
0
0
0
0
2
ANGPT2
7
9
30
135
150
0
4
22
0
13
BDNF
11
9
182
72
117
387
475
1337
550
1015
bNGF
8
6
7
32
13
394
134
30
210
687
GDF15
11
10
35
48
9
14
80
34
411
487
GDNF
11
10
12962
41091
0
0
156
177
0
512
HGF
10
8
136
359
268
3
0
72
857
153
ICAM1
11
9
90
224
780
545
24
279
75
1394
IGFBP1
7
7
26
47
0
1
4
721
7
56
IGFBP3
11
10
4179
2726
282
10095
317
3384
5234
257
NCAM1
10
8
810
392
1006
11
176
241
0
19
NGFR
10
8
1295
1933
1302
0
27
0
0
0
NOV
4
5
5
10
38
151
0
10
5
163
NRG1
10
5
36
12
47
99
649
318
33
17
NRG2
7
6
26
18
69
1
189
5
2
0
NRN1
11
9
288
392
3652
0
1628
169
0
979
NTF3
4
4
9
6
36
940
19
80
0.2
711
NTF4
0
0
0
0
0
0
101
3
5
773
PDGFA
0
2
0
4
0
0
11
0
0
17
PDGFB
0
0
0
0
0
0.5
3
14
0
29
PGF
9
7
242
47
71
0
10
15
0
5913
THBS1
11
10
2479
2011
2
610
88481
2749
7989
1132
TIMP1
11
10
6544
10088
11366
90200
40299
361760
59631
12807
VEGFA
11
10
4434
907
8879
18
8369
165
188
507
Left side (iERM, PDR): number of membranes in which gene expression is detected. Right side (Relative expression): abundance of gene expression in
arbitrary units. iERM, idiopathic epiretinal membrane; PDR, epiretinal membrane from PDR patient; Retina, whole retina of non-diabetic donor eye; RPE,
primary retinal pigment epithelial cells; Glial cells, U373 cell line; HRECs, human primary retinal endothelial cells; HRPCs, human primary retinal pericytes;
Blood, pooled whole blood from patients with diabetic macular edema. Underlined values show significant difference in gene expression levels between
PDR and iERM membranes (P < 0.05). Table 4. Gene expression in membranes, retinal tissues and cells, and blood. PLOS ONE | https://doi.org/10.1371/journal.pone.0187304
November 2, 2017 12 / 21 Discussion In the present study, vitreous samples were screened for 507 proteins, including various neural
and glial growth factors, leading to the identification of 55 vitreous proteins elevated in PDR. A search in the Embase and Pubmed databases indicates that 8 of these proteins (BMP2,
DcR3, GDF15, IGFBP4, NGFR, NOV, NRG1β1, UBB+1) were not previously reported to be
associated with PDR. A serious sight threatening complication of PDR is the formation of FVMs, which may
result in retinal traction and retinal detachment. An initial step in the formation of these PLOS ONE | https://doi.org/10.1371/journal.pone.0187304
November 2, 2017 13 / 21 Identification of proteins involved in PDR membrane formation membranes may be neurite outgrowth from neurons and Mu¨ller cells, which not only secrete
neurotrophic and pro-angiogenic growth factors, but may also serve as a template for the
growth of other cell types and new blood vessels [4,18,19]. We identified Ang2 as being the
most strongly correlated protein to the degree of fibrosis and the presence of FVMs in patients
with PDR. Furthermore, the PDGFs seem to play a role in fibrosis and in neurite outgrowth as
well, based on our observations in all membranes. Proteins related to neurite outgrowth Many of the tested neurotrophins, such as NT3, NT4, bNGF, BDNF and GDNF were below
the LOD in both control samples and PDR samples, suggesting that these proteins are not
involved in retinal-vitreous neurite formation. Other studies confirmed the absence of bNGF
and BDNF [36], but showed detectable levels of NT3 and NT4 [36] and GDNF [37], suggesting
differences in sensitivity or specificity of the detection methods. While GDNF was found to be
undetectable in the vitreous of PDR patients, we did find active mRNA transcription of GDNF
in all PDR-associated membranes, as well as in the non-diabetic macular puckers. Further
studies to clarify the possible role that GDNF may play in retinal neurite growth are
warranted. Other neurotrophic factors such as Neuregulin1/Heregulin1-b1 (NRG-1) exhibited a >3
fold upregulation in the vitreous of PDR patients, implicating their possible involvement in
neurite outgrowth. Indeed, previous reports show that NRG1 can elicit neurite outgrowth in
dorsal root ganglia explants [38] and developing rat retina [39]. Additionally, there may be
increased sensitivity for neurotrophic factors as evidenced by the elevated concentrations of
nerve growth factor receptor (NGFR) found in the vitreous of PDR patients. However, in the
independent study group, the increased NRG1β1 protein levels were not confirmed, thus mak-
ing the involvement of NRG1β1 in neurite outgrowth dubious. Another possibility is that PDGF drives neurite growth, in addition to its angiogenic effects
[40]. Indeed, PDGF has been shown to induce neuronal [41,42] and Mu¨ller cell proliferation
in vitro [43], and outgrowth of neurites in primary rat brain cultures [44]. Additionally, trans-
genic mouse models overexpressing PDGFA have increased retinal glial cell proliferation [45],
which can in turn drive neurite outgrowth via purinergic G protein-coupled receptor activa-
tion [46]. A role for PDGFs in fibrosis Although PDGF is known to be a strong angiogenic stimulus, we also found a strong correla-
tion between PDGFAA concentrations and retinal fibrosis. In addition, PDGF was correlated
with a number of other proteins known to be involved in fibrosis and neovascularization such
as angiopoietin-1 (Ang1, ANGPT1) and -2 (Ang2, ANGPT2), PlGF and TGFβ [47]. This raises
the possibility that PDGF may be one of the driving forces behind pathological angiogenesis
and fibrosis in patients with PDR. Recent studies have shown that PDGF has a role in the pro-
duction of FVMs in patients with a related condition, proliferative vitreoretinopathy (reviewed
by Lei et al., 2010) [48]. In this condition, in contrast to PDR, fibrosis rather than a mixture of
angiogenesis and fibrosis is the main pathological process. Angiopoietins Ang1 and Ang2 are known to exert their biological effects by competitively binding to endo-
thelial cell-specific tyrosine kinase with immunoglobulin and epidermal growth factor homol-
ogy domains 2 (Tie-2) receptors [49]. The angiopoietin/Tie2 system is a context-dependent
system with opposing effects. Binding of Ang1 leads to Tie2 phosphorylation and endothelial PLOS ONE | https://doi.org/10.1371/journal.pone.0187304
November 2, 2017 14 / 21 Identification of proteins involved in PDR membrane formation cell stabilization, but Ang2 binding leads to robust angiogenesis in the presence of VEGF [50]. A decreased ratio of Ang1 and Ang2 seems to be a critical switch for the development of vascu-
lar pathology, including blood-retinal barrier breakdown [51] and pericyte migration [52]. Here, we confirm reports that PDR is characterized by a disturbed balance of Ang1 and Ang2,
since the concentrations of Ang2 were found to be increased almost 20-fold in PDR, whereas
Ang1 concentrations only increased 3-fold. In addition, Ang2 was found to be correlated to the degree of retinal fibrosis. ANGPT2 gene
expression was found in all PDR membranes and in the majority of non-diabetic ERMs. Fur-
thermore, vitreous samples of PDR with FVMs had increased concentrations of Ang2 com-
pared to PDR without FVMs. This could be accounted for by increased production of Ang2
from highly activated FVM endothelial cells [53]. It seems that, in addition to playing a role in
active angiogenesis [50,54], Ang2 may well be involved in retinal fibrosis in PDR. This is sup-
ported by increased intravitreal Ang2 concentrations related to rhegmatogenous retinal
detachment [55] and formation of FVMs in patients with retinopathy of prematurity [56]. Also, Ang2 was shown to be causative in the formation of liver fibrosis in rats [57] and cardiac
fibrosis in db/db mice [58]. Thrombospondin We observed high protein concentrations of thrombospondin-1 in vitreous of PDR patients,
10-fold higher than in control vitreous samples. In conjunction, gene expression of THBS1
was observed in all membranes in comparable quantities in non-diabetic ERMs and PDR
membranes. Thrombospondin-1 is a matricellular glycoprotein that has anti-angiogenic prop-
erties and is involved in wound healing and fibrosis in the eye [59]. A possible mechanism is
the activation of latent TGFβ, either directly [60], or indirectly by induction of MMP-2 and
MMP-9 [61,62]. It is detected as an abundant protein on platelets and is secreted by endothe-
lial cells, fibroblasts, smooth muscle cells, and many other cells of the retina, including glial
cells (summarized by Masli et al., 2014 [59]). Indeed, we observed high transcript levels in
these cell types, especially in glial cells, endothelial cells and pericytes. Thrombospondin-1 pro-
tein in vitreous has not been widely studied thus far. One study reports undetectable levels
[63], whereas another study reports detectable levels by western blotting in human postmor-
tem donor eyes and rat eyes [64]. In contrast to our results, vitreous samples from diabetic rats
showed decreased thrombospondin-1 levels as compared to controls in the prior study. More
research is needed to find out whether these differences are caused by technical or species-
dependent factors. Considering the contribution to anti-angiogenesis, thrombospondin-1 may
be upregulated in response to pro-angiogenic factors to counteract and balance the angiogenic
switch in PDR. Interestingly, thrombospondin-1 and Ang2 were both clustered around the
PDGF-axis in the correlation network analysis, which suggests that these proteins are co-regu-
lated within our samples. This may mean that PDGFs, thrombospondin-1 and Ang2 work
together in the formation of FVMs in PDR. More research is needed to clarify the relationship
between these proteins. IGFBPs Two insulin-like growth factor binding proteins, IGFBP-1 and -3, stood out with respect to
their increased vitreous protein concentrations in PDR patients as compared to controls, with
an increase of 10- and 19-fold respectively. Of these, IGFBP3 mRNA was also abundantly
expressed in non-diabetic ERMs and PDR membranes and several control tissues, with RPE
cells showing the most abundant expression. Others have reported increased IGFBP1 [65] and
IGFBP3 [66,67] levels in the vitreous of PDR patients in the same range. IGFBPs bind IGFs in PLOS ONE | https://doi.org/10.1371/journal.pone.0187304
November 2, 2017 15 / 21 Identification of proteins involved in PDR membrane formation the serum to protect them from degradation and increase their bioavailability [68]. IGFBP3 is
described to have both pro-angiogenic and anti-angiogenic effects, probably dependent on the
context and the presence of certain other growth factors. IGF1- and VEGF-induced prolifera-
tion and survival of human umbilical vein endothelial cells were both inhibited by IGFBP3
[69], whereas IGFBP3 supplementation to mouse Matrigel implants increased vascular
ingrowth as compared to control animals [70]. In addition, IGFBP3 has been reported to be a
protector of blood-retinal barrier breakdown and a stimulator of vasorelaxation by mediating
NO levels [71]. Because of complex interactions with other serum proteins, the mechanism of
IGFBP3 involvement in the pathophysiology of PDR may be difficult to unravel. On the other
hand, IGFBP3 levels may serve as a useful biomarker of disease severity in PDR. Effect of bevacizumab Because some of the patients received bevacizumab before surgery, we were able to analyze the
effects of anti-VEGF treatment on protein concentrations. There have been concerns that inhi-
bition of VEGF may upregulate other pro-angiogenic proteins, which could explain resistance
to anti-VEGF therapy. One such protein is PlGF, a family member of VEGF, which has been
reported to be iatrogenically upregulated by anti-VEGF therapy [72,73]. In the present study,
we did not observe an upregulation on PlGF following bevacizumab treatment, whereas con-
centrations of IGF1 were significantly upregulated. These observations need to be carefully
interpreted since the number of patients was rather low and need further investigations. IGF1
upregulation may be a reaction to the absence of VEGF, since IGF1 is known to induce VEGF
synthesis [74]. It is worth noting that mRNA levels of VEGF were also significantly lower in
the membranes of PDR patients as compared to controls. It is possible that this may due to the
anti-VEGF treatment since all except two of the PDR patients received bevacizumab before
surgery. It has previously been reported that anti-VEGF treatment may target endogenous
VEGF levels, which is known to regulate its own transcription through VEGFR2 [75]. Besides
VEGF, concentrations of 3 other protein were decreased in ERMs of PDR patients as com-
pared to controls: PlGF, NRG1 and BDNF. Decreased PlGF may be explained by the transcrip-
tional regulation by VEGF in microvascular endothelial cells [76], but the relation of VEGF
with NRG1 and BDNF is less clear. In one study VEGF was found to work upstream from
BDNF produced by endothelial cells in neurogenesis in songbirds [77] and in another study
BDNF was found to promote the expression of NRG1 in neurons [78]. These observations
warrant further investigation on larger patient groups. PLOS ONE | https://doi.org/10.1371/journal.pone.0187304
November 2, 2017 Limitations of the study This study was limited by the small number of samples that we were able to obtain. Future
studies using a larger patient population will enhance the statistical power of the correlations
obtained in this study. Additionally, the use of a non-proliferative diabetic group of patients
will allow more accurate assessment which growth factors are specific to PDR. The cross-sectional acquisition of vitreous fluid only allowed protein measurements to be
taken at a single time point in the course of disease. This makes inferences regarding causality
difficult since the sequence of fluctuations in protein concentrations over time could not be
obtained. Furthermore, we were unable to distinguish between proteins produced in the retina
and proteins derived from the blood via a compromised blood-retina barrier or vitreous hem-
orrhage [79]. Our selection of proteins analyzed in the antibody arrays was limited by commercial avail-
ability. In this way, we regret that it was not possible to include measurements of CTGF, a pro-
tein previously reported to be integral in the relationship between retinal fibrosis and PLOS ONE | https://doi.org/10.1371/journal.pone.0187304
November 2, 2017 16 / 21 Identification of proteins involved in PDR membrane formation angiogenesis [4]. Further studies investigating the changes, origin, and sites of action of the
proteins identified here will yield further insight into the pathophysiology of FVM formation
in PDR. Conclusions In conclusion, we found 8 novel proteins in the vitreous of PDR patients, as well as novel cor-
relations between vitreous proteins and retinal fibrosis and angiogenesis. Ang2 was not only
strongly related to the degree of fibrosis, but was also related to the occurrence of FVMs, sug-
gesting that it may have a causative role in the formation of these membranes. In addition, the
elevated concentrations of the neurotrophic factors NRG1 and PDGF in vitreous, and high
gene expression levels of GDNF and BDNF in PDR membranes present potential proteins
responsible for the retinal neurite growth that is displayed in all FVMs. The large amount of
proteins that have been screened in our study may serve as a basis for more detailed analysis in
larger study groups, eventually leading to a better understanding of molecular mechanisms of
PDR pathology. Supporting information S1 Table. Significantly modulated human vitreous proteins. Proteins in vitreous detected by
the RayBio1 L-Series 507 Biotin Label-based Antibody Array that were significantly modu-
lated in a subset of PDR patients (n = 7) relative to non-diabetic controls (n = 5). (PDF) S2 Table. Primer details. Gene nomenclature, Gen bank accession code, primer sequences,
and size and predicted Tm of the amplified products. (PDF) S3 Table. Protein concentrations in vitreous of control and PDR patients in independent
study group. Protein concentrations were determined by Quantibody arrays in patients with
macular hole (CON) and patients with proliferative diabetic retinopathy (PDR) that under-
went vitreoretinal surgery. (PDF) S1 Fig. Ang2 protein is increased in vitreous of PDR patients with FVMs. In an indepen-
dent study group protein levels of Ang2, as detected by Quantibody arrays, were log10 trans-
formed to obtain a normal distribution. Differences between groups were analyzed by a
Student’s t-test. CON, non-diabetic control patients; PDR–FVM, PDR patients without FVMs;
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English
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Industrial Fishing Near West African Marine Protected Areas and Its Potential Effects on Mobile Marine Predators
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Frontiers in marine science
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cc-by
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ORIGINAL RESEARCH
published: 04 March 2021
doi: 10.3389/fmars.2021.602917 ORIGINAL RESEARCH
published: 04 March 2021
doi: 10.3389/fmars.2021.602917 Industrial Fishing Near West African
Marine Protected Areas and Its
Potential Effects on Mobile Marine
Predators Guido Leurs1,2*, Karin J. van der Reijden1, Sidi Yahya Cheikhna Lemrabott1,3, Iça Barry4,
Diosnes Manuel Nonque4, Han Olff1, Samuel Ledo Pontes5, Aissa Regalla5 and
Laura L. Govers1,2 Guido Leurs1,2*, Karin J. van der Reijden1, Sidi Yahya Cheikhna Lemrabott1,3, Iça Barry4,
Diosnes Manuel Nonque4, Han Olff1, Samuel Ledo Pontes5, Aissa Regalla5 and
Laura L. Govers1,2 1 Conservation Ecology Group, Groningen Institute for Evolutionary Life Sciences, University of Groningen, Groningen,
Netherlands, 2 Department of Coastal Systems, Royal Netherlands, Institute for Sea Research (NIOZ), Den Burg,
Netherlands, 3 Institut Mauritanien de Recherches Océanographiques et de Pêches, Nouadhibou, Mauritania, 4 Centro
de Investigac" ão Pesqueira Aplicada, Bissau, Guinea-Bissau, 5 Instituto da Biodiversidade e das Áreas Protegidas, Bissau,
Guinea-Bissau Edited by: Swimmer,
Pacific Islands Fisheries Science
Center (NOAA), United States *Correspondence:
Guido Leurs
g.h.l.leurs@rug.nl Specialty section:
This article was submitted to
Marine Conservation
and Sustainability,
a section of the journal
Frontiers in Marine Science
Received: 04 September 2020
Accepted: 11 February 2021
Published: 04 March 2021 Specialty section:
This article was submitted to
Marine Conservation
and Sustainability,
a section of the journal
Frontiers in Marine Science Specialty section:
This article was submitted to
Marine Conservation
and Sustainability,
a section of the journal
Frontiers in Marine Science Received: 04 September 2020
Accepted: 11 February 2021
Published: 04 March 2021 Edited by: Edited by:
Annette Breckwoldt,
Leibniz Centre for Tropical Marine
Research (LG), Germany Marine Protected Areas (MPAs) are increasingly implemented to facilitate the
conservation of marine biodiversity and key habitats. However, these areas are often
less effective to conserve mobile marine species like elasmobranchs (i.e., sharks and
rays). Industrial fishing near MPA borders possibly impacts vulnerable species utilizing
these protected areas. Hence, we aimed to study spatiotemporal patterns of industrial
fisheries near MPAs, in relation to the bycatch of elasmobranchs. Specifically, we
analyzed the spatiotemporal fishing effort within the West African region, mapped
fishing effort in the direct vicinity of the Parc National du Banc d’Arguin (PNBA,
Mauritania) and the Bijagós Archipelago (BA, Guinea Bissau), and compared the
seasonal overlap between elasmobranch bycatch and fishing effort near these MPAs. We combined Automatic Identification System (AIS) data and local fisheries observer
data, and determined fishing effort for each gear type and compared this with bycatch
of elasmobranchs. We found that industrial fishing effort was dominated by trawling,
drifting longlines, and fixed gear types. Although no industrial fishing was observed
within both MPAs, 72 and 78% of the buffer zones surrounding the MPAs were fished
for the Banc d’Arguin and Bijagós, respectively. Within the Banc d’Arguin buffer zone,
trawling and drifting longlines dominated, with longlines mainly being deployed in fall. In the Bijagós buffer zone, trawling and fixed gears were most prevalent. Fisheries
observer data for Mauritania showed that elasmobranch catches increased during the
most recent sampling years (2016–2018). Elasmobranch catches within the waters of
Guinea Bissau peaked in 2016 and decreased in the following two years. Seasonal
patterns in elasmobranch bycatch within the waters of both countries are likely caused
by increased catches of migratory species. Catches of rays peaked in May and June for Reviewed by:
Kristian Metcalfe,
University of Exeter, United Kingdom
Yonat B. INTRODUCTION home
ranges,
which
challenges
effective
conservation
of
this species group (Dulvy et al., 2014; MacKeracher et al.,
2018). To halt the degradation of marine ecosystems and to counter
overexploitation of marine resources, an increasing number of
Marine Protected Areas (MPAs) have been implemented over the
last two decades (Watson et al., 2014; McDermott et al., 2018). The majority of these implemented MPAs cover coastal areas, like
vegetated wetlands and coastal reefs, which can be important for
marine megafauna species (Fox et al., 2012; Sievers et al., 2019). Megafaunal species (e.g., sharks, rays, sirenians, cetaceans, and
sea turtles) frequently utilize coastal areas as nursery grounds
in early life stages (e.g., Bangley et al., 2018), or as breeding
areas (e.g., Van Waerebeek and Read, 2014), foraging areas (e.g.,
Eckert et al., 2006; Sievers et al., 2019), and as predator-free
refuge areas later in life (e.g., Heithaus et al., 2009). However,
megafauna species generally have large home ranges and are often
migratory (Lewison et al., 2014). They therefore only spend a
limited, but essential proportion of their life cycle in such areas. Within these coastal areas, megafaunal species exhibit essential
ecological roles, including as (top) predators (Ferreira et al.,
2017). In addition, due to their migratory nature, these species
form important functional links (e.g., transferring nutrients)
between coastal areas and other systems, such as the pelagic zone
(Williams et al., 2018; Sievers et al., 2019). As a consequence of stricter fishing regulations in many
developed countries, distant-water fleets of these nations moved
to the territorial waters of developing countries, including
many
countries
in
West
Africa
(Balmford
et
al.,
2004;
Worm et al., 2009). The high productivity of these waters,
caused by the upwelling of the Canary current, attracts
fishing fleets from nations all over the world (Belhabib
et al., 2019). Consequently, fishing effort within this region
is among the highest in the world (Pauly and Christensen,
1995; Grecian et al., 2016). The region also contains highly
diverse marine ecosystems which are threatened by habitat
degradation, overexploitation, and pollution (Tittensor et al.,
2010; Stuart-Smith et al., 2013). Furthermore, the West African
region is known for its data deficiency and high prevalence
of endangered marine species, in particular species like
hammerhead sharks (Sphyrna spp.), Lusitanian cownose rays
(Rhinoptera marginata), and blackchin guitarfishes (Glaucostegus
cemiculus). INTRODUCTION There are two large intertidal MPAs of high ecological
importance within the region: Parc National du Banc d’Arguin
(PNBA) in Mauritania and the Bijagós Archipelago (BA) in
Guinea Bissau (Figure 1). Both areas are considered to play an
important role as spawning and nursery area for commercial
fish species, and for migratory species, including elasmobranchs
(Jager, 1993; Valadou et al., 2006). Declines of the annual
catch per unit effort of rays and sharks within the boundaries
of these MPAs have sparked concerns among park managers,
conservationists, scientists, and the local communities about
the status of these species groups within the region (Cheikna
Lemrabott et al., unpub. data; Leurs, pers. obs.). Although fishing
pressure through artisanal practices and bycatch rates within the
MPAs are also substantial (Campredon and Cuq, 2001; Valadou
et al., 2006; Diop and Dossa, 2011), fishing effort of industrial
fleets at the borders of these MPAs could potentially have
negative effects on the population status of marine megafauna
utilizing these coastal areas (Guénette et al., 2014; Di Lorenzo
et al., 2016). Herein we describe the industrial fishing activity
within the West African region between 2012 and 2018 with
three main objectives: (1) to analyze the spatiotemporal extent
of gear-specific fishing efforts within the region, (2) to map
fishing activity in the direct vicinity of the two largest West
African MPAs, PNBA and the BA, and (3) to link the industrial
fishing effort with seasonal bycatch of elasmobranchs (i.e., sharks Coastal areas like seagrass meadows, rocky shores, tidal flats,
and mangroves also provide an essential nursery habitat for
pelagic and commercial fish species (Stål et al., 2008; Binet
et al., 2013; Honda et al., 2013). Designating such vital areas
as MPAs can result in increased species richness and biomass
of commercial fish species in surrounding areas; the so-called
spillover effects (Polunin and Roberts, 1993; Stobart et al., 2009). Consequently, fisheries might be attracted to the borders of MPAs
(Di Lorenzo et al., 2016). Although this phenomenon may not be
problematic for highly productive species with small home ranges
(i.e., small teleosts), concentrated fishing activities might pose
threats to vulnerable species with large home ranges, migratory
behavior, or species that only utilize the protected areas during a
certain life stage (Burgess et al., 2013; Dulvy et al., 2014; Lewison
et al., 2014). Citation: Leurs G, van der Reijden KJ,
Cheikhna Lemrabott SY, Barry I,
Nonque DM, Olff H, Ledo Pontes S,
Regalla A and Govers LL (2021)
Industrial Fishing Near West African
Marine Protected Areas and Its
Potential Effects on Mobile Marine
Predators. Front. Mar. Sci. 8:602917. doi: 10.3389/fmars.2021.602917 March 2021 | Volume 8 | Article 602917 1 Frontiers in Marine Science | www.frontiersin.org Leurs et al. Industrial Fishing Near West African MPAs Mauritania, and in October for Guinea Bissau. Shark catches were highest in February
and July in Mauritanian waters, and in May and October in the waters of Guinea Bissau. Our study indicates that industrial fisheries near the border of ecologically important
MPAs may have potentially major implications for ecosystem functioning by the removal
of (migratory) predatory species. Keywords: fisheries, threatened species, coastal ecosystems, marine conservation, elasmobranchs, fisheries
ecology Frontiers in Marine Science | www.frontiersin.org INTRODUCTION Elasmobranchs (i.e., sharks and rays) are a species group
susceptible
to
bycatch,
and
with
their
low
recruitments
rates, high maturity ages, and other K-selected life history
characteristics, many species of this group are particularly
vulnerable to any non-natural mortality rates (MacKeracher
et al., 2018). In addition, the status of many elasmobranch
species
remains
unknown
and
many
species
have
wide March 2021 | Volume 8 | Article 602917 2 Industrial Fishing Near West African MPAs Leurs et al. FIGURE 1 | Defined study area indicating the Exclusive Economic Zones (EEZs; dashed lines) and Marine Protected Areas (MPAs; green lines) within the West
African region. The inner gray border represents the northern and southern edges of the study area. The two focal MPAs, the Parc National du Banc d’Arguin
(Mauritania) and the Bijagós Archipelago (Guinea Bissau), are specifically indicated. FIGURE 1 | Defined study area indicating the Exclusive Economic Zones (EEZs; dashed lines) and Marine Protected Areas (MPAs; green lines) within the West
African region. The inner gray border represents the northern and southern edges of the study area. The two focal MPAs, the Parc National du Banc d’Arguin
(Mauritania) and the Bijagós Archipelago (Guinea Bissau), are specifically indicated. the territorial waters of the Democratic Republic of Congo in the
south (Figure 1). Geographical data on the EEZs of all nations
within this region were extracted from the “MarineRegions”
dataset (Lonneville et al., 2019). Areas outside of any EEZ were
classified as the high seas. and rays) to estimate its effect on nature conservation goals of
coastal MPAs. MATERIALS AND METHODS Within our study area, we focused on two large MPAs: PNBA
(N20◦14′5′′, W16◦6′32′′) and the BA (N11◦15′0′′, W16◦5′0′′)
(Figure 1), for which spatial delineation was obtained from
the World Database on Protected Areas (UNEP-WCMC and
IUCN., 2019). The PNBA is the largest marine park in West
Africa, and was designated as a RAMSAR site in 1982 and as Data Processing A 0.1◦grid (±11 × 11 km near the equator) was superimposed
on the study area, and industrial fishing effort was calculated
per grid cell. Fished extent was determined as the proportion
of fished grid cells relative to the total number of grid cells
(n = 224,926). To determine and visualize the annual, gear-
specific fishing effort in direct vicinity of both MPAs, we
created two buffer zones around each MPA of 1.5 and 2.0
times the surface area of the MPA. We also calculated the
cumulative fishing effort over increasing distance from each
MPA of each gear type specifically. Fishing effort based on
the AIS data was not compared between years, as the number
of vessels detected by the GFW algorithms increased every
study year due to technological enhancements. For this reason,
2018 is reported for the most recent fishing effort calculations. For annual trends in fishing effort, we used the fishery-
dependent data. In total, 15 different gear categories within West African
waters were identified, which we reclassified into six more
general categories (Table 1). In addition, the GFW linked
Maritime Mobile Service Identity (MMSI) information to the
AIS transmissions, providing the flag state of registration for
each vessel. Fishing effort, as the total number of fishing hours
(in kilohours, kh), was then determined per vessel, flag state,
gear type, and year for every 0.1◦longitude/latitude grid cell
over 2012–2018. Fishery-dependent data were collected as part of fisheries
observer programs by the national fisheries institutes Institut
Mauritanien de Recherches Océanographique et de Pêches The fishery-dependent observer data contained information
on both catches (in tons) and fishing effort (in fishing days). Catches were classified into functional groups, as limited
information on species identification was available. From 2012 to
2015, both focal countries reported elasmobranch catches as part
of diverse groups like, “Diverse pelagic” or “Diverse demersal.”
Since 2016, catches of sharks and rays were reported separately
(i.e., catches were not grouped together as elasmobranchs or
grouped into other functional groups). Our data analysis only
includes those catches reported as elasmobranchs, resulting in
a conservative estimate of catches. Rays included all species
labeled as “Raia,” and sharks included all species of hammerhead
sharks (Sphyrna spp.), or species labeled as “Elasmobranchii” or
“Caudo.” Fishing effort was registered as the number of hours
that a vessel was actively fishing during a fishing expedition,
separated per gear type. Data Collection Fishing effort data (2012–2018) were obtained from the
Global Fishing Watch (GFW1), based on processed Automatic
Identification System (AIS) transmissions of large vessels
(Kroodsma et al., 2018). The GFW applied artificial neural
network algorithms to the AIS-data, which determined fishing
activity and gear type used based on the speed and movement
pattern of the vessel. As AIS is mandatory for all vessels
above 300 gross tonnage, the dataset only includes large
industrial vessels. Study Area We focused on the Eastern Central Atlantic (major fishing area
34 as defined by the Food and Agriculture Organization of the
United Nations, FAO) as our main study area. This study site
ranges from the territorial waters of Morocco in the north to March 2021 | Volume 8 | Article 602917 Frontiers in Marine Science | www.frontiersin.org 3 Industrial Fishing Near West African MPAs Leurs et al. a UNESCO World Heritage site in 1989. The entire national
park is 12,000 km2, of which 5,600 km2 marine area (Binet
et al., 2013). The area comprises of a large variety of habitats,
from bare tidal flats and intertidal seagrass meadows to extensive
subtidal areas. The BA covers a 12,958 km2 archipelago consisting
of 88 islands and islets. The archipelago was designated as a
UNESCO Biosphere Reserve in 1996 and as a RAMSAR site in
2014. The Bijagós contains dense mangrove forests, tidal flats,
complex gully systems, and extensive subtidal areas. Within the
Bijagós Biosphere Reserve, the islands of Formosa, Orango, and
Joao Vieira are designated as MPAs. Both MPAs are considered
to be important for a large variety of (commercial) fish species,
elasmobranchs, and migratory shorebirds. (IMROP) and Centro de Investigac"
ão Pesqueira Aplicada (CIPA),
for Mauritania and Guinea Bissau, respectively. The data from
the Mauritanian EEZ are based on logbook data documented
and curated by the national fisheries institute. Data for this area
were reported in the total catch per functional group and the
fishing effort was documented from 2012 to 2018. The data from
Guinea Bissau were collected by observers, who recorded the
catch (in kg) per functional group (e.g., “Rays,” “Sharks,” and
“Diverse pelagics”). Observers also recorded the effort (in hours)
for each vessel. The total catch per functional group and the
total fishing effort were collected from 2012 to 2016 (Centro
De Investigação Pesqueira Aplicada (CIPA), 2012, 2013, 2014,
2015, 2016). Vessel-based observer data were combined with
fleet-wide landing data to extrapolate bycatch observations to
fleet level. No information on the survey effort were recorded for
these data. Presented data thus reflect non-standardized survey
efforts per month. 1globalfishingwatch.org RESULTS longlines mainly operated on the high seas (80.3% of the total
effort by longliners). Trawlers were concentrated within the
coastal zones and only covered 1.2 ± 0.3% of the entire region. Over the entire study period, vessels from 60 flag states were
observed within the West African region, although only 10 flag
states were responsible for 88% of the total fishing effort. The five
most active flag states within the region were Spain (24%), China
(15%), Japan (12%), Morocco (11%), and Ghana (6%). Data Processing Seasonality of elasmobranch catches
was investigated using catch recordings, for both countries
separately. In addition, total fishing effort was determined from
the registered fishing effort and was subsequently compared to
the AIS-based fishing effort of the GFW. For this, seasons were
determined as winter (December–February), spring (March–
May), summer (June–August), and fall (September–November). TABLE 1 | New categories based on categories assigned by the Global
Fishing Watch (GFW). Category
GFW label
Trawlers
“Trawlers”
Drifting longlines
“Drifting longlines”
Fixed gear
“Set longlines”
“Pots and traps”
“Set gillnets”
“Other fixed gears”
Purse seines
“Tuna seines”
“Purse seines”
“Other seines”
Other gear
“Pole and line”
“Dredge”
“Squid jiggers”
“Trollers”
“Other gears”
Unknown gear
“Fishing”
Frontiers in Marine Science | www.frontiersin.org TABLE 1 | New categories based on categories assigned by the Global
Fishing Watch (GFW). Category
GFW label
Trawlers
“Trawlers”
Drifting longlines
“Drifting longlines”
Fixed gear
“Set longlines”
“Pots and traps”
“Set gillnets”
“Other fixed gears”
Purse seines
“Tuna seines”
“Purse seines”
“Other seines”
Other gear
“Pole and line”
“Dredge”
“Squid jiggers”
“Trollers”
“Other gears”
Unknown gear
“Fishing”
Frontiers in Marine Science | www.frontiersin.org TABLE 1 | New categories based on categories assigned by the Global
Fishing Watch (GFW). March 2021 | Volume 8 | Article 602917 4 Industrial Fishing Near West African MPAs Leurs et al. Spatiotemporal Fishing Activity off West
Africa A total of 5,449 kh (0.39 h−1 km−2) of fishing effort by AIS-
operating vessels was observed within the entire West African
region, including the high seas, between 2012 and 2018 (Figure 2
and Supplementary Table S1), with an average annual effort of
778 ± 466 kh (mean ± sd). Over the 6-year study period, at
least 42.2% of the West African region (5.9 × 106 km2) was
fished at least once (at our 0.1◦resolution), with a mean annual
extent of 21.9 ± 6.7% (3.9 ± 0.9 × 106 km2) (Supplementary
Figure S1). Fishing effort concentrated in coastal waters (70%
in EEZs compared to 30% in high seas), with the EEZs of
Mauritania (10%), Western Sahara (8%), Morocco (8%), and
Guinea Bissau (7%) together containing over 36% of the total
fishing effort (Supplementary Table S1). The spatial distribution
of the fishing effort peaked between the longitudes −18.45 and
−15.45 (70.3 ± 56.6 kh), and offSierra Leone between the
latitudes 3.15 and 5.65 (27.2 ± 19.6 kh) (Figure 2). From the
six gear types observed within the study area, trawlers (2,625 kh;
48.2%) and drifting longlines (1,901 kh; 34.9%) were the most
deployed gears. Fishing effort of other gear types was relatively
low (∼200 kh combined; Supplementary Table S1). Drifting DISCUSSION In this study, we provide new insights in the recent (2012–2018)
effort and spatiotemporal distribution of industrial fisheries in
West Africa. In addition, we focused on fishing effort in the
vicinity of two large, coastal MPAs. AIS records demonstrated
that fishing activity is concentrated near the borders of MPA:
PNBA (Mauritania) and the Bijagós Biosphere Reserve (BA,
Guinea Bissau). Fishing effort within the Mauritanian EEZ
was relatively stable, whereas effort within the EEZ of Guinea
Bissau increased significantly with 12 fishing days a month. Industrial fishing activity was mainly dominated by trawlers,
drifting longlines, and fixed gears. These gears mainly target
mackerel (Scomber spp.), sardinella (Sardinella spp.), horse
mackerels (Trachurus spp.), and cephalopods (Belhabib et al.,
2013; Belhabib and Pauly, 2015; FAO, 2019), but have bycatches
of sharks and rays. In the waters from both Mauritania and
Guinea Bissau, the catches of elasmobranchs peaked in the most
recent years of the study period. Seasonal peaks in industrial
shark and ray catches were observed as well, but these did
not coincide with seasonal maxima in industrial fishing effort. We showed that industrial fisheries (especially trawlers) are
concentrated within a thin belt surrounding both MPAs. This
concentrated fishing effort could have potential effects on mobile
marine predators such as elasmobranchs and other species that
utilize coastal MPAs for a part of their life cycle only. Hence,
fishing concentrations near MPA borders may impair the role
of coastal MPAs for the protection of endangered highly mobile
marine megafauna. Inclusion of seasonal migration patterns and
seasonal fishery bans near MPAs could aid in the conservation of
mobile marine megafauna. Traceable catches of sharks and rays were only documented
in 2016, 2017, and 2018. Elasmobranch catches peaked with 85.8
tons in 2018, of which 55.5 tons were rays (64.7%) and 30.3
tons were sharks (35.3%) (Figure 3A). Ray catches were highest
from April to July (8.4 ± 3.3 tons; mean ± se), whereas shark
catches peaked in February (7.3 ± 3.4 tons) and July (6.0 ± 2.3
tons) (Figure 3B). Fishing Activity Near MPAs Fishing effort
increased over short distances from the PNBA, with trawlers
showing the highest increase in efforts near the MPA and within
the buffer zones (Supplementary Figure S2). Fishing effort
within the 2.0x buffer zone around the PNBA was 117.5 kh in
2018, with no industrial fishing observed within the boundaries
of the PNBA. In 2018, 42.0% of the grid cells within the buffer
zone were fished at least once, with trawlers dominating in both
effort (89.3 kh) and extent (33.2%). and are therefore not included here. Reported catches were
highest in 2016, with 262.92 tons, of which 18.97 tons (7.2%)
were ray species and 243.95 tons (92.8%) were shark species. In the most recent year of the study (2018), total elasmobranch
catches were 39.46 tons, with catches existing of 35.79 tons of rays
(90.7%) and 3.68 tons of sharks (9.3%). Ray catches were highest
in April and May with 7.95 ± 3.04 (mean ± se) and 6.80 ± 1.13
tons, respectively (Figure 5B). Shark catches were also highest in
October with a mean weight of 23.74 ± 17.86 tons and in May
(23.49 ± 10.42 tons). Spatial distribution of trawlers was relatively constant
throughout the year, while effort was highest in July (4.2 ± 3.8 kh)
and December (4.4 ± 2.8 kh). There was a clear seasonal change
in the spatial distribution of drifting longlines and fixed gears
within the Mauritanian EEZ. Drifting longlines were constantly
present, but gradually increased from spring (3.3 kh) to fall
(8.4 kh). Fixed gear types showed higher fishing effort in fall
and winter (Figure 4). Overall fishing effort within the 2.0x-
buffer zone peaked in the months July, August, and December
(Figure 3C). Seasonal patterns in fishing effort between the
AIS data (2.0x buffer zone) and the fishery-dependent data
(Mauritanian EEZ) showed similar patterns (Figure 5C). Frontiers in Marine Science | www.frontiersin.org Bijagós Archipelago (BA) Fishing effort within the EEZ of Guinea Bissau totaled to 386.0 kh
(3.4 h−1 km−2) in the study period, with a total fished extent
of 73.5%. Based on fishery-dependent data, the fishing effort
significantly increased (ß = 12.39, t = 5.05, p < 0.01) with
12.4 days per month from 10.4.103 days in 2013 to 27.8.103
fishing days in 2016 (Figure 5A). A total of 21 flag states were
active within the EEZ, dominated by mainly Spain (34.3%),
China (28.8%), and Senegal (9.8%) (Supplementary Table S1). During the study period, all six gear types (Table 1) were
observed. Trawlers showed highest effort (374 kh; 96.9%), and
were concentrated near the coast (48.4% of EEZ) (Figure 6). Unidentified gear types were the second most dominant with a
fishing activity of 8.7 kh (2.3%). Although fishing effort near the PNBA and BA showed a
seasonal pattern, a similar pattern was not visible in reported
elasmobranch catches from both EEZs. The observed peaks
are probably explained by temporal higher abundances of
these species, indicating migratory behavior of these species. In Mauritania, sharks were caught most in February and July. These observations are congruent with Zeeberg et al. (2006),
who report highest catches in August for hammerhead sharks
and February for other shark species. The scalloped hammerhead
shark (Sphyrna lewini), for instance, utilizes shallow coastal
habitats during early life stages (e.g., mangrove areas), before
it moves to more pelagic and deeper habitats (Hoyos-Padilla
et al., 2014; Coiraton et al., 2020). The species migrates back
to coastal, shallow habitats for parturition during the boreal No industrial fishing effort was observed within the BA
boundaries, but high effort was observed near the MPA borders
(Supplementary Figure S2). Within the 2.0x buffer zone, fishing
effort was 88.3 kh in 2018 with an extent of 42.9%. Trawlers
were dominant in both effort (65.4%) and extent (41.2%) in
2018 based on AIS data. Fished extent within the buffer zone
remained relatively constant throughout the year for all gear
types, but fishing effort peaked in spring (Figures 5C, 6). Seasonal
patterns in fishing effort between the AIS data (2.0x buffer zone)
and the fishery-dependent data (entire EEZ) showed similar
patterns (Figure 5C). Elasmobranch catches within the EEZ of Guinea Bissau were
reported separately in 2012 and from 2014 to 2018 (Figure 5A). Fishing Activity Near MPAs a c
a o a du
a c d
gu
(
)
Automatic Identification System-registered vessels showed a
total of 560.7 kh fishing effort (3.2 h−1 km−2) within the
Mauritanian EEZ over the study period, covering 95.3% of the
EEZ. Based on the fishery-dependent data, fishing effort of the
entire fleet operated within the Mauritanian EEZ ranged between
26.7.103 days in 2013 and 54.1.103 fishing days in 2018 (Figure
3A). No significant increase in fishing effort was found for the
Mauritanian EEZ. In total, 41 flag states operated within this EEZ
during the study period, with Spain (36.4%), China (30.4%), and
Mauritania (7.7%) being the dominant fleets (Supplementary
Table S1). Fishing vessels deployed all gear types, with trawlers
as the most dominant gear type (353.3 kh; 63.0%). Because these
trawlers mainly operated in coastal waters (Figure 4), the fished FIGURE 2 | Total fishing effort off West Africa from 2012 to 2018. Color scale indicates the total hours of fishing within each grid cell (low = blue, moderate =
yellow/orange, and high = purple). Histograms on the axis show the total fishing effort in hours over the longitudinal and latitudinal range of the region. The
longitudinal and latitudinal ranges of both MPAs are indicated with green lines. FIGURE 2 | Total fishing effort off West Africa from 2012 to 2018. Color scale indicates the total hours of fishing within each grid cell (low = blue, moderate =
yellow/orange, and high = purple). Histograms on the axis show the total fishing effort in hours over the longitudinal and latitudinal range of the region. The
longitudinal and latitudinal ranges of both MPAs are indicated with green lines. March 2021 | Volume 8 | Article 602917 5 Frontiers in Marine Science | www.frontiersin.org Industrial Fishing Near West African MPAs Leurs et al. extent was relatively small (35.1% of the EEZ). Fishing effort
increased over short distances from the PNBA, with trawlers
showing the highest increase in efforts near the MPA and within
the buffer zones (Supplementary Figure S2). Fishing effort
within the 2.0x buffer zone around the PNBA was 117.5 kh in
2018, with no industrial fishing observed within the boundaries
of the PNBA. In 2018, 42.0% of the grid cells within the buffer
zone were fished at least once, with trawlers dominating in both
effort (89.3 kh) and extent (33.2%). extent was relatively small (35.1% of the EEZ). Bijagós Archipelago (BA) In other years, catches were integrated in other functional groups March 2021 | Volume 8 | Article 602917 Frontiers in Marine Science | www.frontiersin.org 6 Industrial Fishing Near West African MPAs Leurs et al. FIGURE 3 | Total elasmobranch catches (bars) and fishing effort (line) within the Mauritanian EEZ, with no-data periods for elasmobranchs indicated in gray (A); with
a close-up of the monthly mean catches, separated for sharks (black) and rays (gray), over the 2016–2018 period (B), in relation to fishing effort within the PNBA 2x
buffer zone based on the AIS data (gray; in kh), and the total fishing effort in the Mauritanian EEZ as reported by the fisheries institute (black; in fishing days, FD) (C). FIGURE 3 | Total elasmobranch catches (bars) and fishing effort (line) within the Mauritanian EEZ, with no-data periods for elasmobranchs indicated in gray (A); with
a close-up of the monthly mean catches, separated for sharks (black) and rays (gray), over the 2016–2018 period (B), in relation to fishing effort within the PNBA 2x
buffer zone based on the AIS data (gray; in kh), and the total fishing effort in the Mauritanian EEZ as reported by the fisheries institute (black; in fishing days, FD) (C). summer (Capapé et al., 1998; Hazin et al., 2001). Recent findings
suggest that scalloped hammerhead sharks are more dependent
on coastal habitats than previously hypothesized (Coiraton et al.,
2020). The PNBA is also hypothesized to be an important
feeding and parturition site for the Lusitanian cownose ray
(R. marginata). Within the PNBA, ray catches by artisanal
fishermen peak from November to the end of February (Cheikna
Lemrabott, in prep.). A similar season (September to December)
is reported for industrial fisheries and scientific surveys outside
the PNBA (Hofstede, 2001; Krakstad et al., 2004, 2005). Our
study, on the other hand, shows that the catches of rays peak in
April and July within the Mauritanian EEZ. Differences might
be caused by the fact that temporal scales of these studies do
not overlap with the temporal scale of this study. Alternatively,
annual differences in coastal upwelling events might cause
changes in catches. However, little information is available on elasmobranch
abundance and habitat use. The scientific reports, based on
observer data, additionally comprise limited species-specific
information and have little consistence in registration. The
actual numbers thus may be uncertain. Bijagós Archipelago (BA) However, reported
bycatch of elasmobranches are supported by other studies
(Belhabib and Pauly, 2015), sometimes showing much higher
catch rates. We therefore argue that our estimates probably
underestimate actual catches. We demonstrated that trawlers were present during the
whole year and dominated both fishing effort and spatial
extent near the PNBA and BA. Drifting longlines were absent
near BA, but peaked near the PNBA in fall. Both gears
generally have high bycatch of sharks and rays (Zeeberg
et al., 2006; Oliver et al., 2015). Drifting longlines were not
present near BA, but the presence of this gear type near
the PNBA peaked in fall. Trawlers have reported bycatch to
mainly consist of pelagic teleosts (31%), hammerhead sharks For Guinea Bissau, we demonstrate increased catches
of
sharks
and
rays
in
May,
October,
and
November. March 2021 | Volume 8 | Article 602917 Frontiers in Marine Science | www.frontiersin.org 7 Industrial Fishing Near West African MPAs Leurs et al. FIGURE 4 | Fishing effort in the direct vicinity of PNBA (green) in Mauritania. Grid cell colors indicate seasonal mean fishing effort over the 2012–2018 period. Orange
and red dashed lines represent 1.5x and 2.0x buffer zones of the PNBA. Exclusive Economic Zones (EEZ) are indicated as gray dashed lines. FIGURE 4 | Fishing effort in the direct vicinity of PNBA (green) in Mauritania. Grid cell colors indicate seasonal mean fishing effort over the 2012–2018 period. Orange
and red dashed lines represent 1.5x and 2.0x buffer zones of the PNBA. Exclusive Economic Zones (EEZ) are indicated as gray dashed lines. FIGURE 4 | Fishing effort in the direct vicinity of PNBA (green) in Mauritania. Grid cell colors indicate seasonal mean fishing effort over the 2012–2018 period. Orange
and red dashed lines represent 1.5x and 2.0x buffer zones of the PNBA. Exclusive Economic Zones (EEZ) are indicated as gray dashed lines. March 2021 | Volume 8 | Article 602917 8 Frontiers in Marine Science | www.frontiersin.org Industrial Fishing Near West African MPAs Leurs et al. Bijagós Archipelago (BA) FIGURE 5 | Total elasmobranch catches (bars) and fishing effort (line) within the Guinea-Bissau EEZ, with no-data periods for elasmobranchs indicated in gray (A),
with a close-up of the monthly mean catches, separated for sharks (black) and rays (gray), over the 2014–2016 period (B), in relation to fishing effort within the BA 2x
buffer zone based on the AIS data (gray; in kh), and the total fishing effort in the EEZ of Guinea Bissau as reported by the fisheries institute (black; in fishing
days, FD) (C). FIGURE 5 | Total elasmobranch catches (bars) and fishing effort (line) within the Guinea-Bissau EEZ, with no-data periods for elasmobranchs indicated in gray (A),
with a close-up of the monthly mean catches, separated for sharks (black) and rays (gray), over the 2014–2016 period (B), in relation to fishing effort within the BA 2x
buffer zone based on the AIS data (gray; in kh), and the total fishing effort in the EEZ of Guinea Bissau as reported by the fisheries institute (black; in fishing
days, FD) (C). FIGURE 5 | Total elasmobranch catches (bars) and fishing effort (line) within the Guinea-Bissau EEZ, with no-data periods for elasmobranchs indicated in gray (A),
with a close-up of the monthly mean catches, separated for sharks (black) and rays (gray), over the 2014–2016 period (B), in relation to fishing effort within the BA 2x
buffer zone based on the AIS data (gray; in kh), and the total fishing effort in the EEZ of Guinea Bissau as reported by the fisheries institute (black; in fishing
days, FD) (C). (28%), and other shark species (19%) (Hofstede and Dickey-
Collas, 2006). Similarly, Zeeberg et al. (2006) reported that
42% of all bycatch for trawlers operating offMauritania was
hammerhead sharks, with other bycatch including large teleosts
(i.e., sunfish Mola mola and billfishes; 26%), reef manta rays
(Manta birostris; 9%), other sharks (9%), cetaceans (8%),
benthic rays (5%), and sea turtles (1%). Bycatch of longline
gear types within the region is characterized by species such
as the Atlantic blue marlin (Makaira nigricans), blue sharks
(Prionace glauca), and smooth hammerhead sharks (Sphyrna
zygaena) (Fernandez-Carvalho et al., 2015). Hence, trawlers and
longliners surrounding the MPAs pose a conservation threat to
elasmobranchs within the MPAs. (28%), and other shark species (19%) (Hofstede and Dickey-
Collas, 2006). Similarly, Zeeberg et al. Bijagós Archipelago (BA) (2006) reported that
42% of all bycatch for trawlers operating offMauritania was
hammerhead sharks, with other bycatch including large teleosts
(i.e., sunfish Mola mola and billfishes; 26%), reef manta rays
(Manta birostris; 9%), other sharks (9%), cetaceans (8%),
benthic rays (5%), and sea turtles (1%). Bycatch of longline
gear types within the region is characterized by species such
as the Atlantic blue marlin (Makaira nigricans), blue sharks
(Prionace glauca), and smooth hammerhead sharks (Sphyrna
zygaena) (Fernandez-Carvalho et al., 2015). Hence, trawlers and
longliners surrounding the MPAs pose a conservation threat to
elasmobranchs within the MPAs. Our results show that overall fishing effort was mainly
concentrated near the borders of both MPAs. MPAs are
known to increase local fish biomass, drawing fishing vessels
to their borders to target the “spillover” from these areas
(Di Lorenzo et al., 2016). Another possible explanation for
the concentrated fishing in this area is the local upwelling of
the Canary Current, which makes the coast offthe Western
Sahara and Mauritania one of the richest fishing areas in
the world (Goffinet, 1992). However, this does not explain
why fishing effort is also concentrated near the BA, as it is
located south of the upwelling’s boundary (Goffinet, 1992). This upwelling is strongest during the short period from
December to March (Cushing, 1971), which could result in March 2021 | Volume 8 | Article 602917 Frontiers in Marine Science | www.frontiersin.org 9 Leurs et al. Industrial Fishing Near West African MPAs FIGURE 6 | Fishing effort in the direct vicinity of the BA in Guinea Bissau (in green). Grid cell colors represent seasonal mean fishing effort over the 2012–2018
period. Orange and red dashed lines indicate 1.5 and 2.0 buffer zones, respectively. Exclusive Economic Zones (EEZ) are indicated as gray dashed lines. FIGURE 6 | Fishing effort in the direct vicinity of the BA in Guinea Bissau (in green). Grid cell colors represent seasonal mean fishing effort over the 2012–2018
period. Orange and red dashed lines indicate 1.5 and 2.0 buffer zones, respectively. Exclusive Economic Zones (EEZ) are indicated as gray dashed lines. areas for (parts) of their lifecycle and migrate between multiple
habitats. For instance, American cownose rays (Rhinoptera
bonasus) can migrate over distances of more than 1,500 km
and scalloped hammerhead shark movements could be traced elevated fishing activity due to higher local production. FUNDING This study was funded by the MAVA Foundation through the
“Waders of the Bijagós” project. LG was funded by the Dutch
Research Council (NWO016.VENI.181.087). KR was funded
through a grant from the Dutch Gieskes-Strijbis Fund. This study was funded by the MAVA Foundation through the
“Waders of the Bijagós” project. LG was funded by the Dutch
Research Council (NWO016.VENI.181.087). KR was funded
through a grant from the Dutch Gieskes-Strijbis Fund. DATA AVAILABILITY STATEMENT The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fmars. 2021.602917/full#supplementary-material The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation. ACKNOWLEDGMENTS Many thanks to the Global Fishing Watch for the open access
data that provide a valuable insight into these remote waters. Specifically, thanks to Tyler Clavelle and David Kroodsma for
the advice and help with the newest version of the dataset. We
would like to thank all fisheries observers, statisticians, and all
other stafffrom IMROP (Mauritania) and CIPA (Guinea-Bissau)
for collecting and providing the fishery-dependent data used in
this study. Finally, we would like to thank Fábio Barroso and
Tommaso Saccà. Bijagós Archipelago (BA) Indeed,
it partly coincides with elevated fishing effort within the
Mauritanian EEZ, but not with peaks in fishing effort in the
waters of Guinea Bissau, as migratory species utilize coastal March 2021 | Volume 8 | Article 602917 Frontiers in Marine Science | www.frontiersin.org 10 Industrial Fishing Near West African MPAs Leurs et al. AUTHOR CONTRIBUTIONS at 684 km from coastal areas (Diemer et al., 2011; Ogburn
et al., 2018). Our results from the 2.0x buffer zones around
the PNBA and BA could indicate that this concentrated fishing
activity might interfere with the migratory nature of these marine
megafauna species. GL, KR, SCL, HO, and LG outlined and drafted the study. GL coordinated the study and wrote the first draft. GL, KR,
and SCL conducted data analyses. GL, KR, SCL, and LG
wrote consecutive draft versions of the manuscript. GL, KR,
SCL, IB, and DN collected and processed data used in this
study. SLP, AR, and HO provided changes and feedback on
later versions of the manuscript. All authors approved the
submitted version. g
In this study, we revealed spatiotemporal patterns of industrial
fisheries in West Africa. We showed seasonal fluctuations but
overall high concentrations of effort near the borders of the
Banc d’Arguin National Park and the BA MPAs. We furthermore
showed seasonal patterns in elasmobranchs bycatch recordings
within the EEZs of the corresponding countries, illustrating
the migratory behavior of these species. We therefore conclude
that the high concentration of fishing effort surrounding these
important coastal areas conflicts with the migratory nature and
vulnerability of elasmobranch species using these areas. This
may lead to a further decrease of these vulnerable species in
both pelagic and coastal habitats, and their associated ecological
role in linking these habitats. The increasing removal of
predatory species from marine ecosystems can cascade through
the ecosystem, with consequences for (both ecological and
economic) ecosystem services (Barbier et al., 2011; Estes et al.,
2011). For example, the removal of top predators like cod
(Gadus morhua) is assumed to be the most likely explanation
for the observed increase in mid-sized fishes, which in turn
has caused increases in macro-algae recruitment (ecologic) or
a weakening of the biological pump of nutrients from great
depths, possibly negatively influencing productivity of fisheries
(economic) (Sieben et al., 2011; Hammerschlag et al., 2019).The
densely concentrated fishing activity near the border of such
protected areas therefore not only undermines the conservation
value of these areas for these megafauna species, but might
cascade into reduced functioning of coastal ecosystems and
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Ledo Pontes, Regalla and Govers. This is an open-access article distributed under the
terms of the Creative Commons Attribution License (CC BY). The use, distribution
or reproduction in other forums is permitted, provided the original author(s) and
the copyright owner(s) are credited and that the original publication in this journal
is cited, in accordance with accepted academic practice. No use, distribution or
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reproduction of dusky dolphins, Lagenorhynchus obscurus, from Coastal Peru. J. Mamm. 75, 1054–1062. Watson, J. E. M., Dudley, N., Segan, D. B., and Hockings, M. (2014). The
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Planning of Optimal Capacity for the Middle-Sized Storage Using a Mathematical Model
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1 Introduction Due to the ongoing globalisation especially small and mid-
size companies are affected by the immense pressure to re-
duce cost wherever they can. Nowadays almost no owner
of a mid-size company could afford to spend more invest
in stocking spare parts as minimum required in order not
to endanger the production flow [1]. According to the view
of the interviewed entrepreneurs, this difficult task was
named as one of the major challenges they face in optimis-
ing their warehouse business. One common problem here
is very often a lack of knowledge which spare parts or raw
material in which quantity is important to stock and which
sort of material does not necessarily needed to be stocked. *Corresponding Author: Jozef Kuľka: Technical University of
Kosice, Faculty of Mechanical Engineering, Department of Design
and Transport Engineering, Letna 9, 042 00 Kosice; Slovakia;
Email: jozef.kulka@tuke.sk
Martin Mantič: Technical University of Kosice, Faculty of Mechani-
cal Engineering, Department of Design and Transport Engineering,
Letna 9, 042 00 Kosice; Slovakia; Email: martin.mantic@tuke.sk
Melichar Kopas: Technical University of Kosice, Faculty of Mechan-
ical Engineering, Department of Design and Transport Engineering,
Letna 9, 042 00 Kosice; Slovakia; Email: melichar.kopas@tuke.sk
Eva Faltinová: Technical University of Kosice, Faculty of Mechani-
cal Engineering, Department of Design and Transport Engineering,
Letna 9, 042 00 Kosice; Slovakia; Email: eva.faltinova@tuke.sk
Marián Siman: Technical University of Kosice, Faculty of Mechani-
cal Engineering, Department of Design and Transport Engineering,
Letna 9, 042 00 Kosice; Slovakia; Email: marian.siman@tuke.sk *Corresponding Author: Jozef Kuľka: Technical University of
Kosice, Faculty of Mechanical Engineering, Department of Design
and Transport Engineering, Letna 9, 042 00 Kosice; Slovakia;
Email: jozef.kulka@tuke.sk Research Article
Jozef Kuľka*, Martin Mantič, Melichar Kopas, Eva Faltinová, and Marián Siman
Planning of Optimal Capacity for the Middle-Sized
Storage Using a Mathematical Model
https://doi.org/10.1515/eng-2019-0077
Received Sep 19, 2019; accepted Oct 02, 2019
According to the interviewed companies the entrepreneurs
mainly report the following problems: https://doi.org/10.1515/eng-2019-0077
Received Sep 19, 2019; accepted Oct 02, 2019 According to the interviewed companies the entrepreneurs
mainly report the following problems: 1. Stocking is difficult to calculate and often done by
employees without transparency for the manage-
ment. Abstract: The article deals with the issue of midsize busi-
nesses, their position within the association, and specifi-
cally determining the size and range of stocks in their ware-
houses. A mathematical model is used and several exam-
ples explain the issue of calculating warehouse items. 2. Lack of modern stock calculation methods [2–4]. Providing a solution for these difficulties is not easy
due to the fact that they all have various and different pa-
rameters just as size, geographic, industry sector etc [5]. Certainly there are many expensive tools and products to
streamline the set up of warehouses available on the mar-
ket. However for those of the companies who could not af-
ford or are incapable to incorporate they could make use
of the help of a mathematical approach model which is ca-
pable to identify the optimised amount of spare parts on
basis of key driving parameters tailored to each of the indi-
vidual circumstances [6]. This provisioning model is used
successfully in the Aviation industry and prevents Airlines
to run out of spare parts which could course an expensive
downtime of one or more aircrafts. As spare parts in the
aviation industry are mostly very expensive a lot of efforts
have been done to find a model which guaranties that the
right spare parts are available whenever they are immedi-
ately needed. With a few modifications on this model, it
can be adapted to the individual needs of mid-size com-
panies regardless their business and specification. Compa-
nies who have to run production machineries all have one
thing in common. They must avoid a situation where there
production flow is stopped due to a shortage or unavailabil-
ity of the needed spare part. On the other hand they have
the pressure not to over stock and invest too much on po-
tential unused spare parts. To cope with these complex re-
quirements the use of an adjusted mathematical approach
model seems to be promising solution [7–9]. Keywords: midsize business, store-house, replacement
part, mathematical model This work is licensed under the Creative Commons Attribution 4.0 Open Eng. 2019; 9:650–654 3 Poisson Distribution Table Recommend quantities (m) and Protection Levels (PL)
have to be assessed assuming a Poisson distribution of
spare part failure across time. In a Poisson process m can-
not be calculated straightforwardly. It requires an iterative
process of assuming m testing it against the PL require-
ment and incrementing m to test it again, etc.i Annual Demand: Annual Demand: Dann = UH × QM × QPM
MTBUR
(1) Dann = UH × QM × QPM
MTBUR (1) (1) Expected demand during Re-Supply Time
Expendables: Drst =
Dann
1 + MRF ×
(︂LTM + AT
365
) The Dann versus MAD test is needed to find out
whether a spare part is qualified for stocking or not. Then
the formula describes the probability to have never more
removals than spare parts on stock during the re-supply
time. (2) ︂︂
Rotable / Repairable:
the formula describ
removals than spar Rotable / Repairable: Rotable / Repairable: Drst =
Dann
1 + MRF ×
[︃(︂MST × TT
365
)︂
(3)
×
(︂
1 −SCR
100
)︂
+
(︂SCR
1000
)︂
×
(︂LTM + AT
365
)︂]︃ (3) Part and machine specific items:
QPM – Part quantity per machine
SCR – Scrap rate
LTM – Lead time
MST – Max/Mean shop processing time
TAT – Turn-Around time
MTBUR – Mean time between unscheduled removals
Additional variables used: user’s warehouse. With this mathematic model it can be
determined which items are essential to stock in order not
to risk a potential production stop caused by a no stock
situation. This model also allows to individual choose
the most suitable protection level for different items. (De-
pending on their importance for the ongoing production
flow) The further aim is to analysis and quantify the ex-
pected demand of items to be stocked by using mathematic
principles to obtain the most efficient quantity and there-
fore minimising the investment in stockings. This model
should be used for both: Additional variables used: Dann – Annual Demand, Drst – Expected demand during re-supply time, m – Recommended quantity, 1. Inventory of products or raw material PL – Spares Protection level (may vary between 80%
and 90% and may be selected as a function of the essen-
tiality. For example category 1 spare part means the ma-
chine cannot run without this spare part and category
2 classifies spare parts which are needed but could not
turn out to stop production), 2. Inventory of spare parts needed to maintain the pro-
duction machines. The following examples demonstrate the calculation
of spares parts by using the mathematic model. The used
parameters can be modified and adjusted as required to
the individual prerequisites.ii CRF – Customised Redundancy Factor - can vary from
0.0 to 1.0 (0% to 100%) with category 2 parts when n > 1. Capture the amount of system redundancy the company
owner intends to consider in his planning
CRF= 0 for category 1 spare parts. The aim of the first calculation is to generically find
out whether a spare should be stocked by analysis the risk
of failure via testing the expected annual demand (Dann)
against the minimum annual demand (MAD). If (Dann) ≥
MAD the first spare part should be allocated to the stock. The risk of out of stock during the re-supply chain delay
could be mitigated by adding extra spares to obtain better
protection in line with the expected demand (Drst) during
the re-supply time. 2 The Mathematic Model Approach The aim of the usage of the mathematic model is to find out
which items/ spare parts and quantities should be stocked
by taking into account the individual circumstances of the 651 Planning of Optimal Capacity for the Middle-Sized Storage Using a Mathematical Model
| 5 Optimization of Stocks in
Storehouses One major impact of the ongoing globalization for mid-size
companies is the pressure to reduce cost where ever they
can. The implementation of an automated end to end lo-
gistics solution was over the last decade only affordable by
mostly global, big size companies. Today with the contin-
uously developments in IT solution even small and mid-
size companies can benefit from such solutions. The aim
for these companies should be to automate manual, time
consuming processes as much as possible. Example 3: Same as example 2, but PLT = 1% PL (m=3) = 0.974 = 97.4% which is above PL-PLT = 97% ⇒
m = 3 The tolerance reduces stocks by 1 spare part! The tolerance reduces stocks by 1 spare part! Example 4: Same as example 3, but CRF = 100% and
parts is considered as category 2 In all of the visited mid -size companies’: In all of the visited mid -size companies’: • The ordering of frequently used parts were done
mostly manual, The Drst calculation has to take this into account and need
to be re-calculated. The Drst calculation has to take this into account and need
to be re-calculated. • The control of the inventory is monitored by employ-
ees on daily or weekly routine checks, The new Drst = 0.55. PL (m=1) = 0.894 = 89.4% which is below PL-PLT ⇒incre-
ment by 1 The new Drst = 0.55. PL (m=1) = 0.894 = 89.4% which is below PL-PLT ⇒incre-
ment by 1 • The follow up of ordered material and spare parts is
done manual. PL (m=2) = 0.982 = 98.2% which is above PL-PLT ⇒m=2
The redundancy reduces stock by 2 spare parts! PL (m=2) = 0.982 = 98.2% which is above PL-PLT ⇒m=2
The redundancy reduces stock by 2 spare parts! These are only three examples of time and cost inten-
sive manual processes within their supply chain. In order
to compete with sometimes global competitor’s owner of
mid-size companies are struggling with e.g. high salaries
of their employees and the amount of people they have
to employ to full fill these routine tasks. All of the inter-
viewed mid-size companies owner confirmed that they are
very much looking forward to any practical solution which
would allow them to automate these processes and help
them to reduce their fix costs. Figure 1: Poisson Distribution Table. Example 2: Spare part category 1; MAD = 1, Dann = 2 , PL
= 98% , Drst = 1.3,
Dann ≥MAD – Spare part gets qualified as stocking spare
part ⇒m = 1;
PL (m=1) = 0.699 = 69.9% which is below PL ⇒increment
by 1
PL (m=2) = 0.900 = 90% which is below PL ⇒increment
by 1
PL (m=3) = 0.974 = 94.7% which is below PL ⇒increment
by 1
PL (m=4) = 0.966 = 99.6% which is above PL ⇒m = 4. P = Probability,
R = number of removals
5 Optimization
Storehouses
One major impact of the on
companies is the pressure
can. The implementation
i i
l
i
h Example 2: Spare part category 1; MAD = 1, Dann = 2 , PL
= 98% , Drst = 1.3,
Dann ≥MAD – Spare part gets qualified as stocking spare
part ⇒m = 1;
PL (m=1) = 0.699 = 69.9% which is below PL ⇒increment
by 1
PL (m=2) = 0.900 = 90% which is below PL ⇒increment
by 1
PL (m=3) = 0.974 = 94.7% which is below PL ⇒increment
by 1
PL (m=4) = 0.966 = 99.6% which is above PL ⇒m = 4. 4 Application of the Poisson
Distribution Formula on Concrete
Examples Warehouse Parameters:
Distributio
Examples Warehouse Parameters: Warehouse Parameters: AT – Administration Time
MRF – Machine Redundancy Factor
UH – Usage Hours per Machine per Year
MAD – Minimum Annual Demand
QM – Quantity of Machine in usage
TT – Transit Time. AT – Administration Time
MRF – Machine Redundancy Factor
UH – Usage Hours per Machine per Year
MAD – Minimum Annual Demand
QM – Quantity of Machine in usage
TT – Transit Time. Example 1: Spare part category 1, MAD = 1; Dann = 0.5
Dann < MAD – Spare part does not qualify as stocking
spare part ⇒m = 0. Probability of failure is considered to low! Example 1: Spare part category 1, MAD = 1; Dann = 0.5
Dann < MAD – Spare part does not qualify as stocking
spare part ⇒m = 0. Probability of failure is considered to low! Example 1: Spare part category 1, MAD = 1; Dann = 0.5 Example 1: Spare part category 1, MAD = 1; Dann = 0.5
Dann < MAD – Spare part does not qualify as stocking
spare part ⇒m = 0. Probability of failure is considered to low! 652
|
J. Kuľka et al. 652
|
J. Kuľka et al. 652 5 Optimization of Stocks in
Storehouses Guideline how to use the Poisson Distribution Table (Ac-
cording to the above examples) Guideline how to use the Poisson Distribution Table (Ac-
cording to the above examples) Whenever the companies It-situation allows it the
above explains manual method to obtain the right stock-
ing quantity the below described formula could be inte-
grated and the entire process can be conducted automat-
ically. P{R ≤m} = e−Drst ×
m
∑︁
0
(Drst)m
m P{R ≤m} = e−Drst ×
m
∑︁
0
(Drst)m
m
(4) (4) Planning of Optimal Capacity for the Middle-Sized Storage Using a Mathematical Model
| 653 . he
ut
e-
p-
m
n-
e-
is
me
ly. d
Figure 3: Comparison of traditional manual order cycles vs. an
automated replenishment process re 2: Process model of an It supported replenishment systemv. With the availability of SAP based IT-solutions in the
ly 90s it was for the first time possible to think about
omation of the supply chain. With the continuously de-
opment of advanced logistic solution from various sup-
ers it is meanwhile possible to connect different system
h each other. The huge cost saving potential of an automated replen-
ment process is that it actually allows companies to de-
ase their level of safety stock because the stock level is
nitored automatically and therewith the response time
d therewith replenishment time shrinks significantly. l i
i
l
h
k
f
l
l
b d
d
Figure 3: Comparison of traditional manual order cycles vs. an
automated replenishment process Figure 2: Process model of an It supported replenishment systemv. Figure 2: Process model of an It supported replenishment systemv. Figure 3: Comparison of traditional manual order cycles vs. an
automated replenishment process With the availability of SAP based IT-solutions in the
early 90s it was for the first time possible to think about
automation of the supply chain. With the continuously de-
velopment of advanced logistic solution from various sup-
pliers it is meanwhile possible to connect different system
with each other. The huge cost saving potential of an automated replen-
ishment process is that it actually allows companies to de-
crease their level of safety stock because the stock level is
monitored automatically and therewith the response time
and therewith replenishment time shrinks significantly. The result is simple, the stock safety level can be decreased
and the investment in stocks therewith as well. Experience
in the airline sector had proven that most of the customer
could melt down their stockings of goods and raw material
by sometimes more than 50%. 654
|
J. Kuľka et al. J. Kuľka et al. number range ⇒employees could be used for new
tasks; References 3. No longer risk of out of stocking due to IT-monitored
stock level. [1]
Radermacher, F.J. (2003). Globalisierung – Herausforderung für
den Mittelstand, In. Schmeisser, W. Handbuch Unternehmen-
snachfolge, Stuttgart. [1]
Radermacher, F.J. (2003). Globalisierung – Herausforderung für
den Mittelstand, In. Schmeisser, W. Handbuch Unternehmen-
snachfolge, Stuttgart. Certainly it is true that the efforts needed to spend on
connecting different It- systems could be a huge challenge
and shout of course not be underestimated but one it is
successfully done the advantages will make it up. The nec-
essary support on doing so shouldn’t turn out as a problem. With the status of October 2011 even in Germany more than
two thousand SAP-IT consultants were offering their help. [2]
Klein,S. (2003). Der strategische Wandel während des Nachfol-
geprozesses im Familienunternehmen (pp. 23ff). In. Schmeisser
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ternehmensnachfolge (Successful corporate succession), Wies-
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Aspekte
des
Führungswechsels, Wiesbaden. Guideline how to use the Poisson Distribution Table (Ac-
cording to the above examples) As also mid-size companies
keep sometimes stockings worth Millions of Euro, a poten-
tial 50% onetime saving could have a huge impact on the
survival of the company. Due to the melt down of the stock
level, the one time savings are only one of its benefits. Also
the time consuming reoccurring tasks of purchasing and
controlling routine material are no longer needed as the
It-system takes care of it. Figure 3: Comparison of traditional manual order cycles vs. an
automated replenishment process Depending on the nominated forwarder the lead time
of the purchased parts an analysis of the right safety level
can be conducted. The aim of this is to purchase only the
quantity needed to maintain the production with a mini-
mum of safety level on top to avoid falling in no stock situ-
ation. Another advantage of an automated managed inven-
tory is that most of the suppliers can guarantee their cus-
tomer that they always will have enough material on side
as the level of stocks it is electronically monitored by the
supplier. All the manual efforts spend in avoiding any po-
tential production stop due to non-availability of the re-
quired material are therewith history. Resources originally
dedicated to watch the stock level could also be freed and
used for different tasks.i The main challenge here is to establish a connection
and exchange of simple xml-messages to the logistic sys-
tem of the main supplier e.g. (as displayed below). Once the connection is set up, the companies should
think about which part numbers it makes sense to incorpo-
rate. The authors suggest to start only with parts which are
quite frequently consumed and low value items. For high
value material only consumed from time to time the expe-
riences shows that these parts should be ordered manual
as usual as it makes no sense to stock parts which are too
valuable and eventually not consumed. Concrete benefits of an automated replenishment in
mid-size companies: 1. Faster re-supplier time due to direct connection to
the supplier ⇒lower invest in stockings; 2. Reoccurring, time-consuming tasks are automated. 2. Reoccurring, time-consuming tasks are automated. No manual purchasing anymore for the agreed part manual purchasing anymore for the agreed part 654 6 Conclusion [5]
Fromm,R. et al. (2002). Die richtige Unternehmernachfolge
im Mittelstand (The correct corporate succession), BBE-Praxis-
Leitfaden (BBE-Praxis Guide), Köln. [5]
Fromm,R. et al. (2002). Die richtige Unternehmernachfolge
im Mittelstand (The correct corporate succession), BBE-Praxis-
Leitfaden (BBE-Praxis Guide), Köln. Considering the ongoing globalization are mainly small
and medium-sized enterprises affected by the enormous
pressure to reduce costs. The article explains the mathe-
matical model for the Poisson distribution of the probabil-
ity of determining the size of stocks and the calculation
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Modeling and forecasting the spread of COVID-19 with stochastic and deterministic approaches: Africa and Europe
|
Advances in difference equations
| 2,021
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cc-by
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© The Author(s) 2021. 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/. R ES EA RCH Open Access Abdon Atangana1,2* and Seda ˙I˘gret Araz3 Abdon Atangana1,2* and Seda ˙I˘gret Araz3 Abdon Atangana1,2* and Seda ˙I˘gret Araz3 *Correspondence:
AtanganaA@ufs.ac.za
1Institute for Groundwater Studies,
Faculty of Natural and Agricultural
Sciences, University of the Free
State, Bloemfontein, South Africa
2Department of Medical Research,
China Medical University Hospital,
China Medical University, Taichung,
Taiwan Abstract Using the existing collected data from European and African countries, we present a
statistical analysis of forecast of the future number of daily deaths and infections up to
10 September 2020. We presented numerous statistical analyses of collected data
from both continents using numerous existing statistical theories. Our predictions
show the possibility of the second wave of spread in Europe in the worse scenario
and an exponential growth in the number of infections in Africa. The projection of
statistical analysis leads us to introducing an extended version of the
well-blancmange function to further capture the spread with fractal properties. A mathematical model depicting the spread with nine sub-classes is considered, first
converted to a stochastic system, where the existence and uniqueness are presented. Then the model is extended to the concept of nonlocal operators; due to
nonlinearity, a modified numerical scheme is suggested and used to present
numerical simulations. The suggested mathematical model is able to predict two to
three waves of the spread in the near future. Full list of author information is
available at the end of the article Keywords: Statistical analysis; Extended blancmange function; Stochastic model;
COVID-19 spread with waves; Modified numerical scheme ( 2021) 2021:57 ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations
https://doi.org/10.1186/s13662-021-03213-2 1 Introduction Interdisciplinary research is the way forward for mankind to be in control of its environ-
ment. Of course they will not be able to have total control since the nature within which
they live is full of uncertainties, many complex phenomena that have not been yet under-
stood with the current collections of knowledge and technology. For example, we cannot
explicitly and confidently explain what is happening at the Bermuda Triangle, although
many studies have been done around this place, some believe it is a devil’s triangle. There
are many other natural occurrences that could not be explained so far with our knowledge. But it has been proven that putting together several concepts from different academic
fields could provide better results. COVID-19 is an invisible enemy that left humans with
no choice than to put all their efforts from all backgrounds with the aim to protect the
survival of their kind. Many souls have been taken, many humans have been infected and
some recovered, but still the spread has not yet reached its peak in many countries. While Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations Page 2 of 107 ( 2021) 2021:57 in some countries the curve of daily new infected has nearly reached zero, in others the
spread is increasing exponentially. For some statistical analysis, we investigated daily cases
of infections and deaths due to the COVID-19 spread that occurred in 54 countries in the
European continent and 47 countries in the African continent from the beginning of the
outbreak to 15 June 2020. To do this, we used the available data on the website of the
World Health Organization (WHO) [1, 2]. Although mathematicians cannot provide vac-
cine or cure the disease in an infected person, they can use their mathematical tools to
foresee what could possibly happen in the near future with some limitations [3–14]. With
the new trend of spread, it is possible that the world will face a second wave of COVID-19
spread, this will be the aim of our work. The paper is organized as follows. In Sect. 2, we present the definitions of differential
and integral operators where singular and nonsingular kernels are used. In Sect. 3, the pa-
rameter estimations are presented for the infected and deaths in Africa and Europe using
the Box–Jenkins model. In Sect. 1 Introduction 4, the simulations for smoothing method for the infected
and deaths in Africa and Europe are presented. In Sect. 5, the predictions about the cases
of infections and deaths in Africa and Europe are provided. In Sect. 6, we give an analy-
sis of COVID-19 spread based on fractal interpolation and fractal dimension. In Sect. 7,
existence and uniqueness for a mathematical model with stochastic component are in-
vestigated. Also the numerical simulations for such a model are depicted. In Sect. 8, we
present a modified scheme based on the Newton polynomial. In Sect. 9, we provide nu-
merical solutions for the suggested COVID-19 model with different differential operators. 2 Differential and integral operators In this section, we present some definitions of differential and integral operators with sin-
gular and nonsingular kernels. The fractional derivatives with power-law, exponential de-
cay, and Mittag-Leffler kernel are given as follows: Definition 1 Definition 1 C
0 Dα
t f (t) =
1
(1 – α)
t
0
d
dτ f (τ)(t – τ)–α dτ,
CF
0 Dα
t f (t) = M(α)
1 – α
t
0
d
dτ f (τ)exp
–
α
1 – α (t – τ)
dτ,
(1)
ABC
0
Dα
t f (t) = AB(α)
1 – α
t
0
d
dτ f (τ)Eα
–
α
1 – α (t – τ)α
dτ. (1) The fractional integrals with power-law, exponential decay, and Mittag-Leffler kernel are
given as follows: C
0 Jα
t f (t) =
1
(α)
t
0
(t – τ)α–1f (τ)dτ,
CF
0 Jα,β
t
f (t) = 1 – α
M(α)f (t) +
α
M(α)
t
0
f (τ)dτ,
(2)
AB
0 Jα,β
t
f (t) = 1 – α
AB(α)f (t) +
α
AB(α)(α)
t
0
(t – τ)α–1f (τ)dτ. (2) AB
0 Jα,β
t
f (t) = 1 – α
AB(α)f (t) +
α
AB(α)(α)
t
0
(t – τ)α–1f (τ)dτ. Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 3 of 107 ( 2021) 2021:57
Page 3 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Page 3 of 107 The fractal-fractional derivatives with power-law kernel, exponential decay, and Mittag-
Leffler kernel are given as follows: FFP
0
Dα,β
t
f (t) =
1
(1 – α)
d
dtβ
t
0
f (τ)(t – τ)–α dτ,
FFE
0
Dα,β
t
f (t) = M(α)
1 – α
d
dtβ
t
0
f (τ)exp
–
α
1 – α (t – τ)
dτ,
(3)
FFM
0
Dα,β
t
f (t) = AB(α)
1 – α
d
dtβ
t
0
f (τ)Eα
–
α
1 – α (t – τ)α
dτ, (3) where df (t)
dtβ = lim
t→t1
f (t) – f (t1)
t2–β – t2–β
1
(2 – β). Definition 1 (4) (4) The fractal-fractional integrals with power-law, exponential decay, and Mittag-Leffler ker-
nel are as follows: The fractal-fractional integrals with power-law, exponential decay, and Mittag-Leffler ker-
nel are as follows: FFP
0
Jα,β
t
f (t) =
1
(α)
t
0
(t – τ)α–1τ 1–βf (τ)dτ,
FFE
0
Jα,β
t
f (t) = 1 – α
M(α)t1–βf (t) +
α
M(α)
t
0
τ 1–βf (τ)dτ,
(5)
FFM
0
Jα,β
t
f (t) = 1 – α
AB(α)t1–βf (t) +
α
AB(α)(α)
t
0
(t – τ)α–1τ 1–βf (τ)dτ. (5) 3 Box–Jenkin’s model development Autoregressive integrated moving average (ARIMA) approach suggested by Box and Jenk-
ins is one of the most powerful techniques used in time series analysis. The ARIMA model
is composed of three parts. First, the autoregressive part is a linear regression which has a
relation between past values and future values of data series; second, the integrated part
expresses how many times the data series has to be differenced to obtain a stationary se-
ries; and the last one is the moving average part which has a relation between past forecast
errors and future values of data series [14]. These processes can be presented by the mod-
els AR(p), MA(q), ARMA(p,q), and ARIMA(p,d,q). We should decide which model we
will choose for our data series. To do this, partial autocorrelation (PACF) and the auto-
correlation (ACF) are helpful to obtain parameters for the AR model and the MA model,
respectively. Figures 1 and 2 depict graphs of autocorrelation functions for the infected and deaths
in Africa and Europe. Now we introduce these models. Let Yt be the value of the time series at time t. Time
series as a p-order autoregressive process is as follows: (6) Yt = δ + ϕ1Yt–1 + ϕ2Yt–2 + ··· + ϕpYt–p + εt,
(6) Yt = δ + ϕ1Yt–1 + ϕ2Yt–2 + ··· + ϕpYt–p + εt, which is shown as AR(p). Here, δ and εt describe constant and error terms, respectively. Time series as a qth degree of moving average process is given by Yt = μ + εt + θ1εt–1 + θ2εt–2 + ··· + θqεt–q,
(7) (7) Yt = μ + εt + θ1εt–1 + θ2εt–2 + ··· + θqεt–q, Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 4 of 107 Figure 1 Autocorrelation function for the infected and deaths in Africa
Figure 2 Autocorrelation function for the infected and deaths in Europe Figure 1 Autocorrelation function for the infected and deaths in Africa Figure 1 Autocorrelation function for the infected and deaths in Africa Figure 2 Autocorrelation function for the infected and deaths in Europe nd deaths in Europe Figure 2 Autocorrelation function for the infected and deaths in Europe which is shown as MA(q). 3 Box–Jenkin’s model development (14) AIC
1056.6482,
(14)
BIC = 1064.7768. BIC = 1064.7768. 3 Box–Jenkin’s model development The ARMA(p,q) expression is obtained as a combination of
AR(p) and MA(q) equations: Yt = δ + ϕ1Yt–1 + ϕ2Yt–2 + ··· + ϕpYt–p + εt + θ1εt–1 + θ2εt–2 + ··· + θqεt–q. (8) (8) When the time series is not stationary, we take the difference d times to make it stationary. The ARIMA(p,q) model is given by When the time series is not stationary, we take the difference d times to make it stationary. The ARIMA(p,q) model is given by
1 – ϕ1l – ϕ2l2 – ··· – ϕplp
dYt = δ + εt + θ1εt–1 + θ2εt–2 + ··· + θqεt–q. (9) (9) In the ARIMA technique, the model performance can be measured by using some cri-
teria, for instance, Akaike information criteria(AIC), Bayesian information criteria(BIC). Here, we benefit from the Akaike information criteria given as follows: AIC = –2log(l) + 2k,
(10)
BIC = –2log(l) + k lnn, AIC = –2log(l) + 2k,
BIC = –2log(l) + k lnn, (10) AIC = –2log(l) + 2k, where l states likelihood of the data, n is the number of data points, and k also defines
the intercept of the ARIMA model. The numerical simulation are depicted in Figs. 3, 4, 5
and 6. where l states likelihood of the data, n is the number of data points, and k also defines
the intercept of the ARIMA model. The numerical simulation are depicted in Figs. 3, 4, 5
and 6. Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Page 5 of 107 According to data series for the infected in Africa, we use the ARIMA(2,1,0) model
which is given by
1 – ϕ1l – ϕ2l2
(1 – l)Yt = c + εt. (11)
1 – ϕ1l – ϕ2l2
(1 – l)Yt = c + εt. (11) Here, Here, AIC = 1670.1734,
(12)
BIC = 1680.9388. AIC = 1670.1734, (12) In Table 1, we give parameter estimation for infections in Africa. In Table 1, we give parameter estimation for infections in Africa. According to data series for deaths in Africa, we use the AR(1) model which is given by (1 – ϕ1l)Yt = c + εt. (13) (1 – ϕ1l)Yt = c + εt. (13) Here, Here, AIC = 1056.6482,
(14) AIC = 1056.6482,
(14)
BIC = 1064.7768. In Table 2, we give parameter estimation for deaths in Africa. In Table 2, we give parameter estimation for deaths in Africa. Table 1 Model estimation for infections in Africa
Parameter
Value
Standard error
TStatistic
Constant
89.2032
56.6511
1.5746
AR{1}
–0.44796
0.099221
–4.5147
AR{2}
–0.17789
0.068294
–2.6047
Variance
168,446.2911
12,738.3089
13.2236
Figure 3 ARIMA model for the infected in Africa Table 1 Model estimation for infections in Africa
Parameter
Value
Standard error
TStatistic
Constant
89.2032
56.6511
1.5746
AR{1}
–0.44796
0.099221
–4.5147
AR{2}
–0.17789
0.068294
–2.6047
Variance
168,446.2911
12,738.3089
13.2236 Figure 3 ARIMA model for the infected in Africa Figure 3 ARIMA model for the infected in Africa Figure 3 ARIMA model for the infected in Africa Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 6 of
Table 2 Model estimation for deaths in Africa
Parameter
Value
Standard error
TStatistic
Constant
12.581
6.0023
2.096
AR{1}
0.75094
0.082701
9.0802
Variance
694.3043
92.168
7.533
Figure 4 AR model for deaths in Africa Atangana and ˙I˘gret Araz Advances in Difference Equations Page 6 of 107 ( 2021) 2021:57 Table 2 Model estimation for deaths in Africa
Parameter
Value
Standard error
TStatistic
Constant
12.581
6.0023
2.096
AR{1}
0.75094
0.082701
9.0802
Variance
694.3043
92.168
7.533 Figure 4 AR model for deaths in Africa Figure 4 AR model for deaths in Africa Figure 4 AR model for deaths in Africa Table 3 Model estimation for the infected in Europe
Parameter
Value
Standard error
TStatistic
Constant
83.7826
118.7108
0.70577
AR{1}
0.3216
0.59303
0.5423
AR{2}
0.035772
0.16277
0.21977
MA{1}
–0.53222
0.58359
–0.91197
Variance
7,214,609.9182
569,786.6944
12.6619 Table 3 Model estimation for the infected in Europe According to data series for the infected in Europe, we use the ARIMA(2,1,1) model
which is given by
1 – ϕ1l – ϕ2l2
(1 – l)Yt = c + (1 + θ1l)εt. (15)
1 – ϕ1l – ϕ2l2
(1 – l)Yt = c + (1 + θ1l)εt. (15) (15) Here, AIC = 2690.5358,
(16)
BIC = 2705.2796. (16) In Table 3, we give parameter estimation for the infected in Europe. In Table 3, we give parameter estimation for the infected in Europe. According to data series for deaths in Europe, we use the AR(1) model which is given by According to data series for deaths in Europe, we use the AR(1) model which is given by (1 – ϕ1l)Yt = c + εt. 4 Brown’s exponential smoothing method Brown’s linear exponential smoothing is one type of double exponential smoothing based
on two different smoothed series. The formula is composed of an extrapolation of a line
through the two centers. The Brown exponential smoothing method is helpful to model
the time series having trend but no seasonality. For non-adaptive Brown exponential smoothing, the procedure can be described as fol-
lows. Firstly, we start with the following initialization: Firstly, we start with the following initialization: Firstly, we start with the following initialization: 1) S0 = u0,
2) T0 = u0, 3) a0 = 2S0 – T0, 4) F1 = a0 + b0. 4) F1 = a0 + b0. Then we have the following calculations: Then we have the following calculations: Then we have the following calculations: 1) St = αut + (1 – α)St–1,
2) Tt = αSt + (1 – α)Tt–1,
3)
2S
T 63 1) St = αut + (1 – α)St–1,
2) Tt = αSt + (1 – α)Tt–1,
3) at = 2St + Tt63,
4) α(St – Tt) = (1 – α)bt,
5) Ft+1 = at + bt, 4) α(St – Tt) = (1 – α)bt, 5) Ft+1 = at + bt, where 0 < α < 1 is the smoothing factor. St and Tt are the simply smoothed value and
doubly smoothed value for the (t + 1)th time period, respectively. Also at and bt describe
the intercept and the slope, respectively. In Figs. 7, 8, 9, and 10, we present the simulation for smoothing method for the infected
and deaths in Africa and Europe where the smoothing factor was chosen as α = 0.99. In Table 2, we give parameter estimation for deaths in Africa. (17) (1 – ϕ1l)Yt = c + εt. (17) (17) (1 – ϕ1l)Yt = c + εt. Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 7 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 7 of 107 Figure 5 ARIMA model for the infected in Europe
Table 4 Model estimation for deaths in Europe
Parameter
Value
Standard error
TStatistic
Constant
151.4852
163.967
0.92388
AR{1}
0.8865
0.041096
21.5714
Variance
460,062.22
27,485.093
16.7386
Figure 6 AR model for deaths in Europe
Here, Figure 5 ARIMA model for the infected in Europe Figure 5 ARIMA model for the infected in Europe Figure 6 AR model for deaths in Europe Figure 6 AR model for deaths in Europe Here, Here, (18) In Table 4, we give parameter estimation for deaths in Europe. In Table 4, we give parameter estimation for deaths in Europe. Page 8 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations 5 Future prediction of daily new numbers of the infected and deaths: Africa With the collected data using some statistical formula, it is possible to predict what will
possibly happen in the near future. Having in mind what could possibly happen, several
measures could be taken to avoid the worst case scenario. In this section, with the data
collected for 101 countries from Africa (47) and Europe (54), we aim at presenting possible Figure 7 Exponential smoothing for the infected in Africa Figure 7 Exponential smoothing for the infected in Africa ( 2021) 2021:57
Page 9 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 9 of 107 Figure 8 Exponential smoothing for deaths in Africa
Figure 9 Exponential smoothing for the infected in Europe
Figure 10 Exponential smoothing for deaths in Europe
scenarios or events that could be observed in the near future, the daily numbers of deaths
and infections. Numerical simulation are presented in Figs. 11, 12, 13 and 14. In Figs. 15, 16, 17, and 18, we present fitting with smoothing spline for the infected and
deaths in Africa and Europe. Figure 8 Exponential smoothing for deaths in Africa Figure 8 Exponential smoothing for deaths in Africa
Figure 9 Exponential smoothing for the infected in Europe Figure 8 Exponential smoothing for deaths in Africa Figure 9 Exponential smoothing for the infected in Europe Figure 9 Exponential smoothing for the infected in Europe Figure 9 Exponential smoothing for the infected in Europe Figure 10 Exponential smoothing for deaths in Europe Figure 10 Exponential smoothing for deaths in Europe scenarios or events that could be observed in the near future, the daily numbers of deaths
and infections. Numerical simulation are presented in Figs. 11, 12, 13 and 14. scenarios or events that could be observed in the near future, the daily numbers of deaths
and infections. Numerical simulation are presented in Figs. 11, 12, 13 and 14. In Figs. 15, 16, 17, and 18, we present fitting with smoothing spline for the infected and
deaths in Africa and Europe. scenarios or events that could be observed in the near future, the daily numbers of deaths
and infections. Numerical simulation are presented in Figs. 11, 12, 13 and 14. In Figs. 15, 16, 17, and 18, we present fitting with smoothing spline for the infected and
deaths in Africa and Europe. 6 An analysis of COVID-19 spread based on fractal interpolation and fractal
dimension In this section, we present some information about fractal dimension, interpolation, and
blancmange curve. 5 Future prediction of daily new numbers of the infected and deaths: Africa scenarios or events that could be observed in the near future, the daily numbers of deaths
and infections. Numerical simulation are presented in Figs. 11, 12, 13 and 14. In Figs. 15, 16, 17, and 18, we present fitting with smoothing spline for the infected and
deaths in Africa and Europe. p
g
,
,
In Figs. 15, 16, 17, and 18, we present fitting with smoothing spline for the infected and
deaths in Africa and Europe. Atangana and ˙I˘gret Araz Advances in Difference Equations Page 10 of 107 ( 2021) 2021:57 Figure 11 Prediction for the infected in Africa using Forecast Sheet
Figure 12 Prediction for deaths in Africa using Forecast Sheet Figure 11 Prediction for the infected in Africa using Forecast Sheet Figure 11 Prediction for the infected in Africa using Forecast Sheet Figure 12 Prediction for deaths in Africa using Forecast Sheet 6.1 Fractal dimension Fractal dimensions enable us to compare fractals. Fractal dimensions are important be-
cause they can be defined in connection with real-world data, and they can be measured
approximately by means of experiments. These numbers allow us to compare sets in the
real world with the laboratory fractals. Atangana and ˙I˘gret Araz Advances in Difference Equations Page 11 of 107 ( 2021) 2021:57 Figure 13 Prediction for the infected in Europe using Forecast Sheet
Figure 14 Prediction for deaths in Europe using Forecast Sheet
Theorem (The box counting theorem) Let Nn(A) be the number of boxes of side length
(1/2n). Then the fractal dimension D of A is given as [15]
l [N (A)] Figure 13 Prediction for the infected in Europe using Forecast Sheet Figure 13 Prediction for the infected in Europe using Forecast Sheet Figure 14 Prediction for deaths in Europe using Forecast Sheet Theorem (The box counting theorem) Let Nn(A) be the number of boxes of side length
(1/2n). Then the fractal dimension D of A is given as [15] Theorem (The box counting theorem) Let Nn(A) be the number of boxes of side length
(1/2n). Then the fractal dimension D of A is given as [15] D = lim
n→∞
ln[Nn(A)]
ln(2n)
. (19) D = lim
n→∞
ln[Nn(A)]
ln(2n)
. (19) 6.2 Fractal interpolation 6.2 Fractal interpolation (22) (22) When solving this system for tn, un, vn, and wn in terms of the data and yn, we obtain the
following: When solving this system for tn, un, vn, and wn in terms of the data and yn, we obtain the
following: tn = xn – xn–1
xN – x0
,
un = Fn – Fn–1
xN – x0
– yn
Fn – F0
xN – x0
,
(23)
vn = xNxn–1 – x0xn
xN – x0
,
wn = xNFn–1 – x0Fn
xN – x0
– yn
xNF0 – x0Fn
xN – x0
, tn = xn – xn–1
xN – x0
,
un = Fn – Fn–1
xN – x0
– yn
Fn – F0
xN – x0
,
vn = xNxn–1 – x0xn
xN – x0
,
wn = xNFn–1 – x0Fn
xN – x0
– yn
xNF0 – x0Fn
xN – x0
, (23) where 0 ≤yn < 1 is called the scaling factor [15]. where 0 ≤yn < 1 is called the scaling factor [15]. 6.2 Fractal interpolation Euclidean geometry and calculus enable us to model using some lines and curves, the
shapes that we encounter in the nature [15, 16]. In this section, we present an interpolation
function which interpolates the data. Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 12 of 107 ( 2021) 2021:57
Page 12 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 12 of 107 Figure 15 Fitting for the infected in Africa
Figure 16 Fitting for deaths in Africa
Figure 17 Fitting for the infected in Europe
Figure 18 Fitting for deaths in Europe Figure 15 Fitting for the infected in Africa Figure 15 Fitting for the infected in Africa Figure 16 Fitting for deaths in Africa Figure 16 Fitting for deaths in Africa Figure 16 Fitting for deaths in Africa Figure 17 Fitting for the infected in Europe Figure 17 Fitting for the infected in Europe Figure 17 Fitting for the infected in Europe Figure 18 Fitting for deaths in Europe ( 2021) 2021:57
Page 13 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations Page 13 of 107 Definition 2 An interpolation function f : [x0,xN] →R corresponding to the set of data
{(xi,Fi) ∈R2 : i = 0,1,2,...,N} [15] f (xi) = Fi
for i = 1,2,...,N,
(20) f (xi) = Fi
for i = 1,2,...,N,
(20)
where x0 < x1 < x2 ··· < xN f (xi) = Fi
for i = 1,2,...,N, f (xi) = Fi
for i = 1,2,...,N, (20) where x0 < x1 < x2 ··· < xN. where x0 < x1 < x2 ··· < xN. where x0 < x1 < x2 ··· < xN. where x0 < x1 < x2 ··· < xN. Let f : [x0,xN] →R denote the unique continuous function which is called a piece-
wise linear interpolation function. Also this function is linear on each of the subintervals
[xi–1,xi], and it is represented by f (x) = Fi–1 + (x – xi–1)
(xi – xi–1)(Fi – Fi–1)
for x ∈[xi–1,xi],i = 1,2,...,N. (21) (21) We have the following transformation, which is iterated: We have the following transformation, which is iterated: have the following transformation, which is iterated: fn
x
y
=
tn
0
un
yn
x
y
+
vn
wn
. 6.3 Blancmange curve The blancmange function can be given as an example of fractal interpolation function,
and this function is defined by ∞
n=0
S(2nx)
2n
,
x ∈[0,1],
(24) ∞
n=0
S(2nx)
2n
,
x ∈[0,1], (24) where S(x) = minm∈Z |x – m|, x ∈R. where S(x) = minm∈Z |x – m|, x ∈R. However, many problems cannot be depicted when c = 2 [16]. Then we discuss the lim-
itations of this blancmange; for example, t can only go from 0 to 1, the periodic parameter
is 2. Therefore, we change 2 to c, where c is a real number from 1 to a. Therefore, in this
section, we extend the blancmange function to a large interval also with any given periodic
parameter. So, we have the following formula: ∞
n=0
S(cnx)
cn
,
x ∈[0,a],
(25) ∞
n=0
S(cnx)
cn
,
x ∈[0,a], (25) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 14 of 107 Figure 19 Blancmange function c = 2
Figure 20 Blancmange function c = 3.7
Figure 21 Blancmange function c = 1.3 Figure 19 Blancmange function c = 2 Figure 19 Blancmange function c = 2 Figure 19 Blancmange function c = 2 Figure 20 Blancmange function c = 3.7 Figure 20 Blancmange function c = 3.7 Figure 21 Blancmange function c = 1.3 Figure 21 Blancmange function c = 1.3 Figure 21 Blancmange function c = 1.3 Figure 21 Blancmange function c = 1.3 where c is the real number. We now present the extended blancmange function for differ-
ent periodic parameters and different w. where c is the real number. We now present the extended blancmange function for differ-
ent periodic parameters and different w. The simulation are presented in Figs. 19, 20, 21, and 18. 7 Mathematical model for COVID-19 outbreak For the class S(t) :
–γ1 →–γ1 + (11S + 12)
·
B1(t),
For the class I(t) :
–λ →–λ + (21I + 22)
·
B2(t),
For the class IA(t) :
–θ →–θ + (31IA + 32)
·
B3(t),
For the class ID(t) :
–η →–η + (41ID + 42)
·
B4(t),
For the class IR(t) :
–v →–v + (51IR + 52)
·
B5(t),
For the class IT(t) :
–σ →–σ + (61IT + 62)
·
B6(t),
For the class R(t) :
– →– + (71R + 72)
·
B7(t),
For the class D(t) :
τ →τ no change,
For the class V(t) :
–μ1 →–μ1 + (81V + 82)
·
B8(t). For the class S(t) :
–γ1 →–γ1 + (11S + 12)
·
B1(t),
For the class I(t) :
–λ →–λ + (21I + 22)
·
B2(t),
For the class IA(t) :
–θ →–θ + (31IA + 32)
·
B3(t),
For the class ID(t) :
–η →–η + (41ID + 42)
·
B4(t),
For the class IR(t) :
–v →–v + (51IR + 52)
·
B5(t),
For the class IT(t) :
–σ →–σ + (61IT + 62)
·
B6(t),
For the class R(t) :
– →– + (71R + 72)
·
B7(t),
For the class D(t) :
τ →τ no change,
For the class V(t) :
–μ1 →–μ1 + (81V + 82)
·
B8(t). Therefore, the associated stochastic model is given as follows: 7 Mathematical model for COVID-19 outbreak We consider the following mathematical model of COVID-19 spread: ·
S = –
δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT) + γ1 + μ1
S, Page 15 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ·
I = δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT)
S – (ε + ξ + λ + μ1)I,
·
IA = ξI – (θ + μ + χ + μ1)IA,
·
ID = εI – (η + ϕ + μ1)ID,
·
IR = ηID + θIA – (v + ξ + μ1)IR,
·
IT = μIA + vIR – (σ + τ + μ1)IT,
·
R = λI + ϕID + χIA + ξIR + σIT – ( + μ1)R, ·
I = δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT)
S – (ε + ξ + λ + μ1)I,
·
IA = ξI – (θ + μ + χ + μ1)IA,
·
ID = εI – (η + ϕ + μ1)ID,
·
IR = ηID + θIA – (v + ξ + μ1)IR,
·
IT = μIA + vIR – (σ + τ + μ1)IT,
·
R = λI + ϕID + χIA + ξIR + σIT – ( + μ1)R, (26) ·
IR = ηID + θIA – (v + ξ + μ1)IR, IT = μIA + vIR – (σ + τ + μ1)IT, ·
R = λI + ϕID + χIA + ξIR + σIT – ( + μ1)R, ·
D = τIT, ·
D = τIT, ·
D = τIT, ·
V = γ1S + R – μ1V. The above model was suggested by Atangana and Seda, the model has a deterministic
character. In this section, we convert the model to a stochastic one by introducing the ef-
fect of environmental white noise. To achieve this, we reformulate the model by adding
the nonlinear perturbation into each equation of the system. The perturbation may de-
pend on square of the classes S, I, IA, ID, IR, IT, R, D, and V respectively. Here, we perturb
only the rate of each class. However, for the vaccine class, it will be perturbed by a natural
death rate. Therefore, the associated stochastic model is given as follows: (27) In this conversion, the function Bi(t) represents the standard Brownian motions valid
within the set of probability (,A,{At}t≥0,P), where {At}t≥0 is filtration valid under the
condition described in [17]. Here, i,j∈[1,2,3,4,5,6,7,8] are positive and are the intensities of
the environmental random disturbance. In this conversion, the function Bi(t) represents the standard Brownian motions valid
within the set of probability (,A,{At}t≥0,P), where {At}t≥0 is filtration valid under the
condition described in [17]. Here, i,j∈[1,2,3,4,5,6,7,8] are positive and are the intensities of
the environmental random disturbance. 7.1 Existence and uniqueness In this subsection, we present the existence and uniqueness of the system solutions of the
stochastic model. To achieve the existence and uniqueness, we convert the system into
Volterra type. But first we do the following for simplicity: dS = F1(t,S,I,IA,ID,IR,IT,R,V)dt + G1(t,S)dB1(t),
dI = F2(t,S,I,IA,ID,IR,IT,R,V)dt + G2(t,I)dB2(t),
dIA = F3(t,I,IA)dt + G3(t,IA)dB3(t),
dID = F4(t,I,ID,)dt + G4(t,ID)dB4(t),
(28)
dIR = F5(t,IA,ID,IR)dt + G5(t,IR)dB5(t),
dIT = F6(t,IA,IR,IT)dt + G6(t,IT)dB6(t),
dR = F7(t,I,IA,ID,IR,IT,R)dt + G7(t,R)dB7(t),
dV = F8(t,S,R,V)dt + G8(t,V)dB8(t). dS = F1(t,S,I,IA,ID,IR,IT,R,V)dt + G1(t,S)dB1(t),
dI = F2(t,S,I,IA,ID,IR,IT,R,V)dt + G2(t,I)dB2(t), dS = F1(t,S,I,IA,ID,IR,IT,R,V)dt + G1(t,S)dB1(t),
dI = F2(t,S,I,IA,ID,IR,IT,R,V)dt + G2(t,I)dB2(t),
dIA = F3(t,I,IA)dt + G3(t,IA)dB3(t),
dID = F4(t,I,ID,)dt + G4(t,ID)dB4(t),
(28)
dIR = F5(t,IA,ID,IR)dt + G5(t,IR)dB5(t),
dIT = F6(t,IA,IR,IT)dt + G6(t,IT)dB6(t),
dR = F7(t,I,IA,ID,IR,IT,R)dt + G7(t,R)dB7(t),
dV = F8(t,S,R,V)dt + G8(t,V)dB8(t). dIA = F3(t,I,IA)dt + G3(t,IA)dB3(t), dID = F4(t,I,ID,)dt + G4(t,ID)dB4(t),
(28) dID = F4(t,I,ID,)dt + G4(t,ID)dB4(t),
(28) (28) dIR = F5(t,IA,ID,IR)dt + G5(t,IR)dB5(t), dIT = F6(t,IA,IR,IT)dt + G6(t,IT)dB6(t), dV = F8(t,S,R,V)dt + G8(t,V)dB8(t). Therefore, converting to Volterra, we get Therefore, the associated stochastic model is given as follows: Therefore, the associated stochastic model is given as follows: dS =
–
δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT) + γ1 + μ1
S
dt
+ (11S + 12)S dB1(t), dI =
δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT)
S – (ε + ξ + λ + μ1)I
dt
+ (21I + 22)I dB2(t), dI =
δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT)
S – (ε + ξ + λ + μ1)I
dt
+ (21I + 22)I dB2(t), dIA =
ξI – (θ + μ + χ + μ1)IA
dt + (31IA + 32)IA dB3(t), dIA =
ξI – (θ + μ + χ + μ1)IA
dt + (31IA + 32)IA dB3(t), dIA =
ξI – (θ + μ + χ + μ1)IA
dt + (31IA + 32)IA dB3(t), Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations Page 16 of 107 ( 2021) 2021:57 dID =
εI – (η + ϕ + μ1)ID
dt + (41ID + 42)ID dB4(t),
(27)
dIR =
ηID + θIA – (v + ξ + μ1)IR
dt + (51IR + 52)ID dB5(t),
dIT =
μIA + vIR – (σ + τ + μ1)IT
dt + (61IT + 62)IT dB6(t),
dR =
λI + ϕID + χIA + ξIR + σIT – ( + μ1)R
dt + (71R + 72)RdB7(t),
dV = [γ1S + R – μ1V]dt + +(71V + 72)V dB8(t). dID =
εI – (η + ϕ + μ1)ID
dt + (41ID + 42)ID dB4(t),
(27)
dIR =
ηID + θIA – (v + ξ + μ1)IR
dt + (51IR + 52)ID dB5(t),
dIT =
μIA + vIR – (σ + τ + μ1)IT
dt + (61IT + 62)IT dB6(t),
dR =
λI + ϕID + χIA + ξIR + σIT – ( + μ1)R
dt + (71R + 72)RdB7(t),
dV = [γ1S + R – μ1V]dt + +(71V + 72)V dB8(t). Therefore, converting to Volterra, we get S(t) = S(0) +
t
0
F1(τ,S,I,IA,ID,IR,IT,R,V)dτ +
t
0
G1(τ,S)dB1(τ),
I(t) = I(0) +
t
0
F2(τ,S,I,IA,ID,IR,IT,R,V)dτ +
t
0
G2(τ,I)dB2(τ),
IA(t) = IA(0) +
t
0
F3(τ,I,IA)dτ +
t
0
G3(τ,IA)dB3(τ),
ID(t) = ID(0) +
t
0
F4(τ,I,ID)dτ +
t
0
G4(τ,ID)dB4(τ),
IR(t) = IR(0) +
t
0
F5(τ,IA,ID,IR)dτ +
t
0
G5(τ,IR)dB5(τ),
(29
IT(t) = IT(0) +
t
0
F6(τ,IA,IR,IT)dτ +
t
0
G6(τ,IT)dB6(τ), (29) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Page 17 of 107 R(t) = R(0) +
t
0
F7(τ,I,IA,ID,IR,IT,R)dτ +
t
0
G7(τ,R)dB7(τ),
V(t) = V(0) +
t
0
F8(τ,S,R,V)dτ +
t
0
G8(τ,S)dB8(τ). We present the existence and uniqueness of the stochastic system of COVID-19 model. This will be achieved via the following theorem. We present the existence and uniqueness of the stochastic system of COVID-19 model. This will be achieved via the following theorem. Theorem Assume that there exist positive constants Ki, Ki such that Theorem Assume that there exist positive constants Ki, Ki such that Theorem Assume that there exist positive constants Ki, Ki such that (i) Fi(x,t) – Fi(xi,t)
2 < Ki|x – xi|2,
(30)
Gi(x,t) – Gi(xi,t)
2 < Ki|x – xi|2 (30) (ii) ∀(x,t) ∈R8 × [0,T] (ii) ∀(x,t) ∈R8 × [0,T]
Fi(x,t)
2,
Gi(x,t)
2 < K
1 + |x|2
. (31) (ii) ∀(x,t) ∈R8 × [0,T] ( )
( , )
[ ,
]
Fi(x,t)
2,
Gi(x,t)
2 < K
1 + |x|2
. (31) Fi(x,t)
2,
Gi(x,t)
2 < K
1 + |x|2
. (31) (31) Then there exists a unique solution X(t) ∈R8 for our model and it belongs to M2([0,T],
R8). The proof can be found in [17], but we have to verify (i) and (ii) for our system. Without
loss of generality, we start our investigation with functions F1(t,S,I,IA,ID,IR,IT,R,V) and
G1(t,S). For the function F, the proof will be performed for (t,S). Thus F1(t,S) – F1(t,S1)
2 =
δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT) + γ1 + μ1
(S – S1)
2. Theorem Assume that there exist positive constants Ki, Ki such that (32) (32) We define the following norm: We define the following norm: ∥ϕ∥∞= sup
t∈[0,T]
|ϕ|2,
(33) ∥ϕ∥∞= sup
t∈[0,T]
|ϕ|2, ∥ϕ∥∞= sup
t∈[0,T]
|ϕ|2, (33) then then F1(S,t) – F1(S1,t)
2 ≤sup
t∈[0,T]
δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT)
(S – S1)
2
≤
δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT)
2
∞|S – S1|2
(34)
≤K1|S – S1|2 ≤
δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT)
2
∞|S – S1|2
(34)
≤K1|S – S1|2 (34) and and G1(S,t) – G1(S1,t)
2 =
(11S + 12)S – (11S1 + 12)S1
2
=
11
S2 – S2
1
– 12(S – S1)
2
=
11(S + S1) + 12
2|S – S1|2
=
2
11(S + S1)2 + 21112(S + S1) + 2
12
|S – S1|2
=
2
11
S2 + 2SS1 + S2
1
+ 21112(S + S1) + 2
12
|S – S1|2
(35) ions ( 2021) 2021:57
Page 18 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 18 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Page 18 of 107 ≤
⎧
⎪⎪⎨
⎪⎪⎩
2
11
supt∈[0,T] |S2(t)| + 2supt∈[0,T] |S(t)|supt∈[0,T] |S1(t)|
+ supt∈[0,T] |S2
1(t)|
+ 21112
supt∈[0,T]
S(t)
+ supt∈[0,T]
S1(t)
+ 2
12
⎫
⎪⎪⎬
⎪⎪⎭
× |S – S1|2
≤
2
11(∥S2∥∞+ 2∥S∥∞∥S1∥∞+ ∥S2
1∥∞)
+ 21112∥S∥∞∥S1∥∞+ 2
12
|S – S1|2
≤K1|S – S1|2, where K1 = 2
11
S2
∞+ 2∥S∥∞∥S1∥∞+
S2
1
∞
+ 21112∥S∥∞∥S1∥∞+ 2
12
(36)
= 2
11
∥S∥∞+ ∥S1∥∞
2 + 21112∥S∥∞∥S1∥∞+ 2
12. (36) imilarly, Similarly, Similarly, K2 = 2
21
∥I∥∞+ ∥I1∥∞
2 + 22122∥I∥∞∥I1∥∞+ 2
22,
K3 = 2
31
∥IA∥∞+ ∥IA1∥∞
2 + 23132∥IA∥∞∥IA1∥∞+ 2
32,
K4 = 2
41
∥ID∥∞+ ∥ID1∥∞
2 + 24142∥ID∥∞∥ID1∥∞+ 2
42,
K5 = 2
51
∥IR∥∞+ ∥IR1∥∞
2 + 25152∥IR∥∞∥IR1∥∞+ 2
52,
(37)
K6 = 2
61
∥IT∥∞+ ∥IT1∥∞
2 + 26162∥IT∥∞∥IT1∥∞+ 2
62,
K7 = 2
71
∥R∥∞+ ∥R1∥∞
2 + 27172∥R∥∞∥R1∥∞+ 2
72,
K8 = 2
81
∥V∥∞+ ∥V1∥∞
2 + 28182∥V∥∞∥V1∥∞+ 2
82. Theorem Assume that there exist positive constants Ki, Ki such that Similarly, we evaluate Similarly, we evaluate F4(ID,t) – F4(ID1,t)
2 = |η + ϕ + μ1|2|ID – ID1|2
≤K4|ID – ID1|2,
(42)
F5(IR,t) – F5(IR1,t)
2 = |v + ξ + μ1|2|IR – IR1|2
≤K5|IR – IR1|2,
F6(IT,t) – F6(IT1,t)
2 = |σ + τ + μ1|2|IT – IT1|2
≤K6|IT – IT1|,
F7(R,t) – F7(R1,t)
2 = | + μ1|2|R – R1|2
≤K7|R – R1|,
F8(V,t) – F8(V1,t)
2 = |μ1|2|V – V1|2
≤K8|V – V1|2. F4(ID,t) – F4(ID1,t)
2 = |η + ϕ + μ1|2|ID – ID1|2
≤K4|ID – ID1|2,
F5(IR,t) – F5(IR1,t)
2 = |v + ξ + μ1|2|IR – IR1|2
≤K5|IR – IR1|2,
F6(IT,t) – F6(IT1,t)
2 = |σ + τ + μ1|2|IT – IT1|2
≤K6|IT – IT1|,
F7(R,t) – F7(R1,t)
2 = | + μ1|2|R – R1|2
≤K7|R – R1|,
F8(V,t) – F8(V1,t)
2 = |μ1|2|V – V1|2
≤K8|V – V1|2. F4(ID,t) – F4(ID1,t)
2 = |η + ϕ + μ1|2|ID – ID1|2
≤K4|ID – ID1|2,
F5(IR,t) – F5(IR1,t)
2 = |v + ξ + μ1|2|IR – IR1|2
≤K5|IR – IR1|2, (42) ≤K5|IR – IR1|2, F6(IT,t) – F6(IT1,t)
2 = |σ + τ + μ1|2|IT – IT1|2
≤K6|IT – IT1|,
F7(R,t) – F7(R1,t)
2 = | + μ1|2|R – R1|2
≤K7|R – R1|,
F8(V,t) – F8(V1,t)
2 = |μ1|2|V – V1|2
≤K8|V – V1|2. For both classes Gi and Fi, we have verified condition (i). Now we verify the second con-
dition. For both classes Gi and Fi, we have verified condition (i). Now we verify the second con-
dition. Theorem Assume that there exist positive constants Ki, Ki such that K2 = 2
21
∥I∥∞+ ∥I1∥∞
2 + 22122∥I∥∞∥I1∥∞+ 2
22,
K3 = 2
31
∥IA∥∞+ ∥IA1∥∞
2 + 23132∥IA∥∞∥IA1∥∞+ 2
32,
K4 = 2
41
∥ID∥∞+ ∥ID1∥∞
2 + 24142∥ID∥∞∥ID1∥∞+ 2
42,
K5 = 2
51
∥IR∥∞+ ∥IR1∥∞
2 + 25152∥IR∥∞∥IR1∥∞+ 2
52,
(37)
K6 = 2
61
∥IT∥∞+ ∥IT1∥∞
2 + 26162∥IT∥∞∥IT1∥∞+ 2
62,
K7 = 2
71
∥R∥∞+ ∥R1∥∞
2 + 27172∥R∥∞∥R1∥∞+ 2
72,
K8 = 2
81
∥V∥∞+ ∥V1∥∞
2 + 28182∥V∥∞∥V1∥∞+ 2
82. (37) Also F2(I,t) – F2(I1,t)
2 =
δ(t)α(I – I1) – (ε + ξ + λ + μ1)(I – I1)
2
=
δ(t)α – (ε + ξ + λ + μ1)
(I – I1)
2
≤sup
t∈[0,T]
δ(t)α – (ε + ξ + λ + μ1)
2|I – I1|2
(38)
≤
δ(t)
∞
α – (ε + ξ + λ + μ1)
2|I – I1|2
≤K2|I – I1|2, F2(I,t) – F2(I1,t)
2 =
δ(t)α(I – I1) – (ε + ξ + λ + μ1)(I – I1)
2
=
δ(t)α – (ε + ξ + λ + μ1)
(I – I1)
2
≤sup
t∈[0,T]
δ(t)α – (ε + ξ + λ + μ1)
2|I – I1|2
(38)
≤
δ(t)
∞
α – (ε + ξ + λ + μ1)
2|I – I1|2
≤K2|I – I1|2, (38) where where K2 =
δ(t)
∞
α – (ε + ξ + λ + μ1)
2. (39) K2 =
δ(t)
∞
α – (ε + ξ + λ + μ1)
2. (39) Also Also F3(IA,t) – F3(IA1,t)
2 =
–(θ + μ + χ + μ1)(IA – IA1)
2
≤2
(θ + μ + χ + μ1)
2|IA – IA1|2
(40)
≤K3|IA – IA1|2, (40) Page 19 of 107 ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations where where (41) K3 = 2
(θ + μ + χ + μ1)
2. Theorem Assume that there exist positive constants Ki, Ki such that F1(S,t)
2 =
– δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT) + γ1 + μ1
S
2
≤
S – δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT) + γ1 + μ1
S
2
≤|S|2 – δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT) + γ1 + μ1
2
<
|S|2 + 1
– δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT) + γ1 + μ1
2
(43)
<
|S|2 + 1
– δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT) + γ1 + μ1
2
<
|S|2 + 1
sup
t∈[0,T]
– δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT) + γ1 + μ1
2 F1(S,t)
2 =
– δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT) + γ1 + μ1
S
2
≤
S – δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT) + γ1 + μ1
S
2
≤|S|2 – δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT) + γ1 + μ1
2
<
|S|2 + 1
– δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT) + γ1 + μ1
2
(43)
<
|S|2 + 1
– δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT) + γ1 + μ1
2
<
|S|2 + 1
sup
t∈[0,T]
– δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT) + γ1 + μ1
2 (43) < K1
|S|2 + 1
, < K1
|S|2 + 1
, where K1 = sup
t∈[0,T]
– δ(t)
αI + w(βID + γ IA + δ1IR + δ2IT) + γ1 + μ1
2. Theorem Assume that there exist positive constants Ki, Ki such that (44) (44) hen Then Then G1(S,t) – G1(S1,t)
2 =
(11S + 12)S
2
≤
11S2 + 12S22
≤(11 + 12)2S22
(45)
≤(11 + 12)2 sup
t∈[0,T]
S2|S|2 (45) Page 20 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ≤(11 + 12)2S2
∞
|S|2 + 1
≤K
1
|S|2 + 1
, where K
1 = (11 + 12)2S2
∞. (46) Similarly, Similarly, K
2 = (21 + 22)2I2
∞,
K
3 = (31 + 32)2I2
A
∞,
K
4 = (41 + 42)2I2
D
∞,
K
5 = (51 + 52)2I2
R
∞,
K
6 = (61 + 62)2I2
T
∞,
K
7 = (71 + 72)2R2
∞,
K
8 = (81 + 82)2V 2
∞. K
2 = (21 + 22)2I2
∞,
K
3 = (31 + 32)2I2
A
∞,
K
4 = (41 + 42)2I2
D
∞,
K
5 = (51 + 52)2I2
R
∞,
K
6 = (61 + 62)2I2
T
∞,
K
7 = (71 + 72)2R2
∞,
K
8 = (81 + 82)2V 2
∞. Theorem Assume that there exist positive constants Ki, Ki such that (47) Also, we have Also, we have F2(I,t)
2 =
δ(t)
w(βID + γ IA + δ1IR + δ2IT)
S + δ(t)αIS – (ε + ξ + λ + μ1)I
2
≤
δ(t)
w(βID + γ IA + δ1IR + δ2IT)
S + δ(t)αS – (ε + ξ + λ + μ1)
|I|2
<
|S|2 + 1
sup
t∈[0,T]
δ(t)
w(βID + γ IA + δ1IR + δ2IT)
S + δ(t)αS F2(I,t)
2 =
δ(t)
w(βID + γ IA + δ1IR + δ2IT)
S + δ(t)αIS – (ε + ξ + λ + μ1)I
2
≤
δ(t)
w(βID + γ IA + δ1IR + δ2IT)
S + δ(t)αS – (ε + ξ + λ + μ1)
|I|2
<
|S|2 + 1
sup
t∈[0,T]
δ(t)
w(βID + γ IA + δ1IR + δ2IT)
S + δ(t)αS (48) – (ε + ξ + λ + μ1)
2
< K2
|I|2 + 1
, F3(IA,t)
2 =
ξI – (θ + μ + χ + μ1)IA
2
≤
ξI – (θ + μ + χ + μ1)
2|IA|2
≤
|IA|2 + 1
sup
t∈[0,T]
ξI – (θ + μ + χ + μ1)
2 F5(IR,t)
2 ≤
|IR|2 + 1
sup
t∈[0,T]
ηID + θIA – (v + ξ + μ1)
2 ≤K5
|IR|2 + 1
, Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 21 of 107 ( 2021) 2021:57 F6(IT,t)
2 ≤
|IT|2 + 1
sup
t∈[0,T]
μIA + vIR – (σ + τ + μ1)
2
≤K6
|IT|2 + 1
,
F6(IT,t)
2 ≤
|IT|2 + 1
sup
t∈[0,T]
μIA + vIR – (σ + τ + μ1)
2
≤K6
|IT|2 + 1
,
F7(R,t)
2 ≤
|R|2 + 1
sup
t∈[0,T]
λI + ϕID + χIA + ξIR + σIT – ( + μ1)
2
≤K7
|R|2 + 1
. Finally, we have Finally, we have F8(V,t)
2 ≤
|V|2 + 1
sup
t∈[0,T]
|γ1S + R – μ1|2
≤K8
|V|2 + 1
. Both Gi and Fi verify the second condition. Therefore, according to the above theorem,
the system has a unique system solution. 7.2 Numerical simulation for the stochastic model Numerical solutions of the suggested stochastic model are presented in Figs. 22–25. The
numerical solution depicts the future stochastic behavior of the susceptible class, five sub-
classes of the infected population, the recovered class, the death class, and the vaccination
class. These are depicted in figures below. 8 Atangana–Seda modified scheme The mathematical model considered in this work has the ability to depict two to three
waves of COVID-19 spread. The model is subjected to a system of initial conditions. Ad-
ditionally, the model is nonlinear, thus it is impossible to obtain exact solutions to the
system, thus numerical schemes are needed. We present a numerical scheme based on
the Newton polynomial [18]. However, one needs the initial condition and two additional
components for the scheme to be implemented. In this section, we present a modified ver-
sion that will not need the two additional components, and then the scheme will be used
later to provide numerical solutions for the suggested COVID-19 model with different
differential operators. We start with the classical case, the following is considered: dy(t)
dt
= f
t,y(t)
. dy(t)
dt
= f
t,y(t)
. (49) (49) Then Then yn+1 = yn +
5
12f
tn–2,yn–2
– 4
3f
tn–1,yn–1
+ 5
12f
tn,yn
t. (50) (50) To reduce these requirements, we proceed as follows: To reduce these requirements, we proceed as follows: yn – yn–1
t
= f
tn,yn
⇒
yn–1 = yn – f
tn,yn
t. (51) (51) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 22 of 107 ( 2021) 2021:57
Page 22 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 22 of 107 Figure 22 Stochastic behavior of S(t) and I(t) classes Figure 22 Stochastic behavior of S(t) and I(t) classes Figure 22 Stochastic behavior of S(t) and I(t) classes On the other hand, On the other hand, On the other hand, yn–1 – yn–2
t
= f
tn–1,yn–1
(52) yn–1 – yn–2
t
= f
tn–1,yn–1 (52) y
– y
t
= f
tn–1,yn–1
(52) t
r or yn–2 = yn–1 – f
tn–1,yn–1
t
(53)
= yn –
tf
tn,yn
–
tf
tn–1,yn – f
tn,yn
t
. (53) Replacing yn–2 and yn–1 with their values, we obtain Replacing yn–2 and yn–1 with their values, we obtain yn+1 = yn + 5
12
tf
tn–2,yn –
tf
tn,yn
–
tf
tn–1,yn – f
tn,yn
t
(54)
– 4
3f
tn–1,yn – f
tn,yn
t
+ 23
12f
tn,yn
t. 8 Atangana–Seda modified scheme (54) The above does not need y1 and y2, only the initial condition. With the Caputo–Fabrizio
derivative, we consider the following: CF
0 Dα
t y(t) = f
t,y(t)
. (55) CF
0 Dα
t y(t) = f
t,y(t)
. (55) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 23 of 107 ( 2021) 2021:57
Page 23 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 23 of 107 Figure 23 Stochastic behavior of IA(t) and ID(t) classes Figure 23 Stochastic behavior of IA(t) and ID(t) classes From the definition of the Caputo–Fabrizio integral, we can reformulate the above equa-
tion as follows: From the definition of the Caputo–Fabrizio integral, we can reformulate the above equa-
tion as follows: y(t) – y(0) = 1 – α
M(α)f
t,y(t)
+
α
M(α)
t
0
f
τ,y(τ)
dτ. (56) (56) have, at the point tn+1 = (n + 1)
t, We have, at the point tn+1 = (n + 1)
t, We have, at the point tn+1 = (n + 1)
t, y(tn+1) – y(0) = 1 – α
M(α)f
tn+1,y(tn+1)
+
α
M(α)
tn+1
0
f
τ,y(τ)
dτ,
(57) (57) and at the point tn = n
t, y(tn) – y(0) = 1 – α
M(α)f
tn,y(tn)
+
α
M(α)
tn
0
f
τ,y(τ)
dτ. (58) (58) Taking the difference of these equations, we can write the following: y(tn+1) – y(tn) = 1 – α
M(α)
f
tn+1,y(tn+1)
– f
tn,y(tn)
(59)
+
α
M(α)
tn+1
tn
f
τ,y(τ)
dτ (59) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 24 of 107 ( 2021) 2021:57
Page 24 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:5 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 24 of 107 Figure 24 Stochastic behavior of IR(t) and IT(t) classes Figure 24 Stochastic behavior of IR(t) and IT(t) classes Figure 24 Stochastic behavior of IR(t) and IT(t) classes With the Caputo derivative, we write With the Caputo derivative, we write ⎧
⎨
⎩
C
0 Dα
t y(t) = f (t,y(t)),
y(0) = y0. 8 Atangana–Seda modified scheme After putting the Newton polynomial into the above equation, the above equation can be
written as follows: After putting the Newton polynomial into the above equation, the above equation can be
written as follows: yn+1 = y0 +
(
t)α
(α + 1)
n
j=2
f
tj–2,yj–2
(n – j + 1)α – (n – j)α
+
(
t)α
(α + 2)
n
j=2
f
tj–1,yj–1
– f
tj–2,yj–2
×
(n – j + 1)α(n – j + 3 + 2α)
– (n – j)α(n – j + 3 + 3α)
(62)
+
α(
t)α
2(α + 3)
n
j=2
f (tj,yj) – 2f (tj–1,yj–1)
+ f (tj–2,yj–2)
×
⎡
⎢⎢⎢⎢⎣
(n – j + 1)α
2(n – j)2 + (3α + 10)(n – j)
+ 2α2 + 9α + 12
– (n – j)α
2(n – j)2 + (5α + 10)(n – j)
+ 6α2 + 18α + 12
⎤
⎥⎥⎥⎥⎦
, (62) ( 2021) 2021:57
Page 26 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Page 26 of 107 where where f
tj–1,yj–1
= f
tj–1,yj – f
tj,yj
t
,
(63)
f
tj–2,yj–2
= f
tj–2,yj –
tf
tj,yj
–
tf
tj–1,yj – f
tj,yj
t
. (63) With Atangana–Baleanu, we have With Atangana–Baleanu, we have ⎧
⎨
⎩
ABC
0
Dα
t y(t) = f (t,y(t)),
y(0) = y0. (64) (64) ⎨
⎩
0
Dt y(t) = f (t,y(t)),
y(0) = y0. (64) transform the above equation into We transform the above equation into We transform the above equation into We transform the above equation into y(t) – y(0) = 1 – α
AB(α)f
t,y(t)
+
α
AB(α)(α)
t
0
f
τ,y(τ)
(t – τ)α–1 dτ. 8 Atangana–Seda modified scheme (60) (60) We convert the above into We convert the above into y(t) – y(0) =
1
(α)
t
0
f
τ,y(τ)
(t – τ)α–1 dτ. (61) (61) At the point tn+1 = (n + 1)
t, we have the following: At the point tn+1 = (n + 1)
t, we have the following: At the point tn+1 = (n + 1)
t, we have the following: At the point tn+1 = (n + 1)
t, we have the following: y(tn+1) – y(0) =
1
(α)
tn+1
0
f
τ,y(τ)
(tn+1 – τ)α–1 dτ, Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 25 of 107 ( 2021) 2021:57
Page 25 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 20 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 25 of 107 Figure 25 Stochastic behavior of R(t) and V(t) classes
d
it Figure 25 Stochastic behavior of R(t) and V(t) classes Figure 25 Stochastic behavior of R(t) and V(t) classes and we write and we write y(tn+1) = y(0) +
1
(α)
n
j=2
tj+1
tj
f
τ,y(τ)
(tn+1 – τ)α–1 dτ. 8 Atangana–Seda modified scheme (67) After putting the Newton polynomial into the above equation, the above equation can be
written as follows: After putting the Newton polynomial into the above equation, the above equation can be
written as follows: yn+1 = y0 + 1 – α
AB(α)f
tn+1,y(tn) –
tf
tn,y(tn)
(68) (68) +
α(
t)α
AB(α)(α + 1)
n
j=2
f
tj–2,yj –
tf (tj,yj)
–
tf (tj–1,yj – f (tj,yj)
t)
×
(n – j + 1)α – (n – j)α
+
α(
t)α
AB(α)(α + 2)
n
j=2
f (tj–1,yj – f (tj,yj)
t)
– f (tj–2,yj –
tf (tj,yj) –
tf (tj–1,yj – f (tj,yj)
t))
×
(n – j + 1)α(n – j + 3 + 2α)
– (n – j)α(n – j + 3 + 3α) +
α(
t)α
AB(α)(α + 2)
n
j=2
f (tj–1,yj – f (tj,yj)
t)
– f (tj–2,yj –
tf (tj,yj) –
tf (tj–1,yj – f (tj,yj)
t))
×
(n – j + 1)α(n – j + 3 + 2α)
– (n – j)α(n – j + 3 + 3α) +
α(
t)α
2AB(α)(α + 3)
n
j=2
⎡
⎢⎣
f (tj,yj) – 2f (tj–1,yj–1)
+ f
tj–2,yj –
tf (tj,yj)
–
tf (tj–1,yj – f (tj,yj)
t)
⎤
⎥⎦ nce Equations ( 2021) 2021:57
Page 27 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 27 of 107 angana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Page 27 of 107 ×
⎡
⎢⎢⎢⎢⎣
(n – j + 1)α
2(n – j)2 + (3α + 10)(n – j)
+ 2α2 + 9α + 12
– (n – j)α
2(n – j)2 + (5α + 10)(n – j)
+ 6α2 + 18α + 12
⎤
⎥⎥⎥⎥⎦
. With the Caputo–Fabrizio fractal-fractional derivative, we consider With the Caputo–Fabrizio fractal-fractional derivative, we consider FFE
0
Dα,β
t
y(t) = f
t,y(t)
,
y(0) = y0. (69) FFE
0
Dα,β
t
y(t) = f
t,y(t)
,
(69) (69) y(0) = y0. 8 Atangana–Seda modified scheme (65) (65) At the point tn+1 = (n + 1)
t, we have the following: At the point tn+1 = (n + 1)
t, we have the following: y(tn+1) – y(0) = 1 – α
AB(α)f
t,y(t)
(66) y(tn+1) – y(0) = 1 – α
AB(α)f
t,y(t)
(66) +
α
AB(α)(α)
tn+1
0
f
τ,y(τ)
(tn+1 – τ)α–1 dτ, and we write y(tn+1) = y(0) + 1 – α
AB(α)f
tn+1,yn+1
(67)
+
α
AB(α)(α)
n
j=2
tj+1
tj
f
τ,y(τ)
(tn+1 – τ)α–1 dτ. 8 Atangana–Seda modified scheme (75) We can rearrange the above scheme as follows: We can rearrange the above scheme as follows: yn+1 – yn = 1 – α
M(α)
F(tn+1,y(tn) –
tf (tn,y(tn)))
– F(tn,y(tn))
(76) yn+1 – yn = 1 – α
M(α)
F(tn+1,y(tn) –
tf (tn,y(tn)))
– F(tn,y(tn)) (76) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 28 of 107 Equations ( 2021) 2021:57
Page 28 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 28 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Page 28 of 107 +
α
M(α)
⎧
⎪⎨
⎪⎩
5
12F(tn–2,yn –
tf (tn,yn) –
tF(tn–1,yn – f (tn,yn)
t))
t
– 4
3F(tn–1,yn – f (tn,yn)
t)
t
+ 23
12F(tn,yn)
t
⎫
⎪⎬
⎪⎭
. If we replace F(t,y(t)) with its value, we can solve our equation numerically with the fol-
lowing scheme: yn+1 – yn = 1 – α
M(α)
t1–β
n+1 f (tn+1,y(tn) –
tf (tn,y(tn)))
– t1–β
n
f (tn,y(tn))
(77)
+
α
M(α)
⎧
⎪⎨
⎪⎩
t1–β
n–2
5
12F(tn–2,yn –
tf (tn,yn) –
tf (tn–1,yn – f (tn,yn)
t))
t
– 4
3t1–β
n–1 f (tn–1,yn – f (tn,yn)
t)
t
+ 23
12t1–β
n
f (tn,yn)
t
⎫
⎪⎬
⎪⎭
. (77) With the Atangana–Baleanu fractal-fractional derivative, we write With the Atangana–Baleanu fractal-fractional derivative, we write FFM
0
Dα,β
t
y(t) = f
t,y(t)
,
(78) FFM
0
Dα,β
t
y(t) = f
t,y(t)
,
(78)
y(0) = y0. (78) 0
Dt
y(t) = f
t,y(t)
,
(78)
y(0) = y0. Applying the new fractional integral with Mittag-Leffler kernel, we transform the above
equation into Applying the new fractional integral with Mittag-Leffler kernel, we transform the above
equation into y(t) = y(0) + 1 – α
AB(α)t1–βf
t,y(t)
(79)
+
α
AB(α)(α)
t
0
f
τ,y(τ)
(t – τ)α–1τ 1–β dτ. 8 Atangana–Seda modified scheme Applying the associated integral operator with exponential kernel, we can reformulate
equation (69) as follows: Applying the associated integral operator with exponential kernel, we can reformulate
equation (69) as follows: y(t) = 1 – α
M(α)t1–βf
t,y(t)
+
α
M(α)
t
0
f
τ,y(τ)
τ 1–β dτ. (70) (70) At the point tn+1 = (n + 1)
t, At the point tn+1 = (n + 1)
t, y(tn+1) = 1 – α
M(α)t1–β
n+1 f
tn+1,y(tn+1)
+
α
M(α)
tn+1
0
f
τ,y(τ)
τ 1–β dτ,
(71) (71) and at the point tn = n
t, we have and at the point tn = n
t, we have and at the point tn = n
t, we have y(tn) = 1 – α
M(α)t1–β
n
f
tn,y(tn)
+
α
M(α)
tn
0
f
τ,y(τ)
τ 1–β dτ. (72) (72) If we take the difference of these equations, we obtain the following equation: y(tn+1) – y(tn) = 1 – α
M(α)
t1–β
n+1 f (tn+1,y(tn+1))
– t1–β
n
f (tn,y(tn))
(73)
+
α
M(α)
tn+1
tn
f
τ,y(τ)
τ 1–β dτ. y(tn+1) – y(tn) = 1 – α
M(α)
t1–β
n+1 f (tn+1,y(tn+1))
– t1–β
n
f (tn,y(tn))
(73) (73) or brevity, we consider For brevity, we consider For brevity, we consider y(tn+1) – y(tn) = 1 – α
M(α)
F
tn+1,y(tn+1)
– F
tn,y(tn)
(74)
+
α
M(α)
tn+1
tn
F
τ,y(τ)
dτ, (74) where F
t,y(t)
= f
t,y(t)
t1–β. (75) F
t,y(t)
= f
t,y(t)
t1–β. 8 Atangana–Seda modified scheme y(t) = y(0) + 1 – α
AB(α)t1–βf
t,y(t)
(79) At the point tn+1 = (n + 1)
t, we obtain the following: At the point tn+1 = (n + 1)
t, we obtain the following: y(tn+1) = y(0) + 1 – α
AB(α)t1–β
n+1 f
tn+1,y(tn+1)
(80)
+
α
AB(α)(α)
tn+1
0
f
τ,y(τ)
(tn+1 – τ)α–1τ 1–β dτ. (80) +
α
AB(α)(α)
tn+1
0
f
τ,y(τ)
(tn+1 – τ)α–1τ 1–β dτ. For simplicity, we shall take F
t,y(t)
= f
t,y(t)
t1–β. (81) F
t,y(t)
= f
t,y(t)
t1–β. (81) We also have y(tn+1) = y(0) + 1 – α
AB(α)F
tn+1,y(tn+1)
(82)
+
α
AB(α)(α)
n
j=2
tj+1
tj
F
τ,y(τ)
(tn+1 – τ)α–1 dτ. (82) Replacing them into the above equation and substituting F(t,y(t)) = f (t,y(t))t1–β, we can
get the following numerical scheme: Replacing them into the above equation and substituting F(t,y(t)) = f (t,y(t))t1–β, we can
get the following numerical scheme: yn+1 = 1 – α
AB(α)t1–β
n+1 f
tn+1,y(tn+1)
(83) (83) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 29 of 107 ( 2021) 2021:57 +
α(
t)α
AB(α)(α + 1)
n
j=2
t1–β
j–2 f
tj–2,yj –
tf (tj,yj)
–
tf (tj–1,yj – f (tj,yj)
t)
×
(n – j + 1)α – (n – j)α
+
α(
t)α
AB(α)(α + 2)
n
j=2
⎡
⎢⎣
t1–β
j–1 f (tj–1,yj – f (tj,yj)
t)
– t1–β
j–2 f
tj–2,yj –
tf (tj,yj)
–
tf (tj–1,yj – f (tj,yj)
t)
⎤
⎥⎦
×
(n – j + 1)α(n – j + 3 + 2α)
– (n – j)α(n – j + 3 + 3α)
+
α(
t)α
2AB(α)(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎣
t1–β
j
g(tj,yj)
– 2t1–β
j–1 f (tj–1,yj – f (tj,yj)
t)
+ t1–β
j–2 f
tj–2,yj –
tf (tj,yj)
–
tf (tj–1,yj – f (tj,yj)
t)
⎤
⎥⎥⎥⎥⎦
×
⎡
⎢⎢⎢⎢⎣
(n – j + 1)α
2(n – j)2 + (3α + 10)(n – j)
+ 2α2 + 9α + 12
– (n – j)α
2(n – j)2 + (5α + 10)(n – j)
+ 6α2 + 18α + 12
⎤
⎥⎥⎥⎥⎦
. 8 Atangana–Seda modified scheme (88) (88) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 30 of 107 ( 2021) 2021:57
Page 30 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Page 30 of 107 Replacing them into the above equation and substituting F(t,y(t)) = f (t,y(t))t1–β, we can
get the following numerical scheme: yn+1 =
(
t)α
(α + 1)
n
j=2
t1–β
j–2 f
tj–2,yj –
tf (tj,yj)
–
tf (tj–1,yj – f (tj,yj)
t)
(89)
×
(n – j + 1)α – (n – j)α
+
(
t)α
(α + 2)
n
j 2
⎡
⎢⎣
t1–β
j–1 f (tj–1,yj – f (tj,yj)
t)
– t1–β
j 2 f
tj–2,yj –
tf (tj,yj)
j
j
⎤
⎥⎦ yn+1 =
(
t)α
(α + 1)
n
j=2
t1–β
j–2 f
tj–2,yj –
tf (tj,yj)
–
tf (tj–1,yj – f (tj,yj)
t)
(89)
×
(n – j + 1)α – (n – j)α yn+1 =
(
t)α
(α + 1)
n
j=2
t1–β
j–2 f
tj–2,yj –
tf (tj,yj)
–
tf (tj–1,yj – f (tj,yj)
t)
(89)
×
(n – j + 1)α – (n – j)α
+
(
t)α
(α + 2)
n
j=2
⎡
⎢⎣
t1–β
j–1 f (tj–1,yj – f (tj,yj)
t)
– t1–β
j–2 f
tj–2,yj –
tf (tj,yj)
–
tf (tj–1,yj – f (tj,yj)
t)
⎤
⎥⎦
×
(n – j + 1)α(n – j + 3 + 2α)
– (n – j)α(n – j + 3 + 3α)
+
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎣
t1–β
j
g(tj,yj)
– 2t1–β
j–1 f (tj–1,yj – f (tj,yj)
t)
+ t1–β
j–2 f
tj–2,yj –
tf (tj,yj)
–
tf (tj–1,yj – f (tj,yj)
t)
⎤
⎥⎥⎥⎥⎦
×
⎡
⎢⎢⎢⎢⎣
(n – j + 1)α
2(n – j)2 + (3α + 10)(n – j)
+ 2α2 + 9α + 12
– (n – j)α
2(n – j)2 + (5α + 10)(n – j)
+ 6α2 + 18α + 12
⎤
⎥⎥⎥⎥⎦
. 8 Atangana–Seda modified scheme +
α(
t)α
2AB(α)(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎣
t1–β
j
g(tj,yj)
– 2t1–β
j–1 f (tj–1,yj – f (tj,yj)
t)
+ t1–β
j–2 f
tj–2,yj –
tf (tj,yj)
–
tf (tj–1,yj – f (tj,yj)
t)
⎤
⎥⎥⎥⎥⎦
×
⎡
⎢⎢⎢⎢⎣
(n – j + 1)α
2(n – j)2 + (3α + 10)(n – j)
+ 2α2 + 9α + 12
– (n – j)α
2(n – j)2 + (5α + 10)(n – j)
+ 6α2 + 18α + 12
⎤
⎥⎥⎥⎥⎦
. With the Caputo fractal-fractional derivative, we consider the following: FFP
0
Dα,β
t
y(t) = f
t,y(t)
,
(84)
y(0) = y0. FFP
0
Dα,β
t
y(t) = f
t,y(t)
,
(84) (84) y(0) = y0. Applying the new fractional integral with power-law kernel, we transform the above equa-
tion into y(t) = y(0) +
1
(α)
t
0
f
τ,y(τ)
(t – τ)α–1τ 1–β dτ. (85) (85) At the point tn+1 = (n + 1)
t, we obtain the following: At the point tn+1 = (n + 1)
t, we obtain the following: At the point tn+1 = (n + 1)
t, we obtain the following: y(tn+1) = y(0) +
1
(α)
tn+1
0
f
τ,y(τ)
(tn+1 – τ)α–1τ 1–β dτ. (86) (86) For simplicity, we shall take For simplicity, we shall take F
t,y(t)
= f
t,y(t)
t1–β. (87)
also have F
t,y(t)
= f
t,y(t)
t1–β. F
t,y(t)
= f
t,y(t)
t1–β. (87) We also have y(tn+1) = y(0) +
1
(α)
n
j=2
tj+1
tj
F
τ,y(τ)
(tn+1 – τ)α–1 dτ. 8 Atangana–Seda modified scheme For simplicity, we take For simplicity, we take For simplicity, we take For simplicity, we take F
t,y(t)
= f
t,y(t)
–β′(t)ln(t) + 2 – β(t)
t
t2–β(t),
(93) (93) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Page 31 of 107 and we have and we have y(tn+1) – y(tn) = 1 – α
M(α)
F
tn+1,y(tn+1)
– F
tn,y(tn)
(94)
+
α
M(α)
tn+1
tn
F
τ,y(τ)
dτ. (94) If we do the same routine and replace F(t,y(t)) with its value, we have the following nu-
merical approximation: If we do the same routine and replace F(t,y(t)) with its value, we have the following nu-
merical approximation: yn+1 = yn + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎢⎣
t2–β(tn+1)
n+1
(– β(tn+2)–β(tn+1)
t
lntn+1 + 2–β(tn+1)
tn+1
)
× f (tn+1,y(tn) –
tf (tn,y(tn)))
– t2–β(tn)
n
(– β(tn+1)–β(tn)
t
lntn + 2–β(tn)
tn
)
× f (tn,y(tn))
⎤
⎥⎥⎥⎥⎥⎦
(95)
+
α
M(α)
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12t2–β(tn)
n
(– β(tn+1)–β(tn)
t
lntn + 2–β(tn)
tn
)
× 23
12f (tn,yn)
t
– 4
3t2–β(tn–1)
n–1
(– β(tn)–β(tn–1)
t
lntn–1 + 2–β(tn–1)
tn–1
)
× f (tn–1,yn – f (tn,yn)
t)
t
+ 5
12t2–β(tn–2)
n–2
(– β(tn–1)–β(tn–2)
t
lntn–2 + 2–β(tn–2)
tn–2
)
× 5
12f
tn–2,yn –
tf (tn,yn)
–
tf (tn–1,yn – f (tn,yn)
t)
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
. (95) We deal with our problem involving the new constant fractional order and variable fractal
dimension We deal with our problem involving the new constant fractional order and variable fractal
dimension FFM
0
Dα,β(t)
t
y(t) = f
t,y(t)
,
(96) FFM
0
Dα,β(t)
t
y(t) = f
t,y(t)
,
(96)
y(0) = y0, (96) FFM
0
Dα,β(t)
t
y(t) = f
t,y(t)
,
(96)
y(0) = y0, y(0) = y0, where the kernel is the Mittag-Leffler kernel. If we integrate the above equation with the
new integral operator including the Mittag-Leffler kernel, the above equation can be con-
verted to where the kernel is the Mittag-Leffler kernel. 8 Atangana–Seda modified scheme (89) +
(
t)α
(α + 2)
n
j=2
⎡
⎢⎣
t1–β
j–1 f (tj–1,yj – f (tj,yj)
t)
– t1–β
j–2 f
tj–2,yj –
tf (tj,yj)
–
tf (tj–1,yj – f (tj,yj)
t)
⎤
⎥⎦ +
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎣
t1–β
j
g(tj,yj)
– 2t1–β
j–1 f (tj–1,yj – f (tj,yj)
t)
+ t1–β
j–2 f
tj–2,yj –
tf (tj,yj)
–
tf (tj–1,yj – f (tj,yj)
t)
⎤
⎥⎥⎥⎥⎦
×
⎡
⎢⎢⎢⎢⎣
(n – j + 1)α
2(n – j)2 + (3α + 10)(n – j)
+ 2α2 + 9α + 12
– (n – j)α
2(n – j)2 + (5α + 10)(n – j)
+ 6α2 + 18α + 12
⎤
⎥⎥⎥⎥⎦
. Finally, we present the numerical scheme with fractal-fractional derivative with variable
order. We start with the Caputo–Fabrizio case: FFE
0
Dα,β(t)
t
y(t) = f
t,y(t)
,
(90)
y(0) = y0. FFE
0
Dα,β(t)
t
y(t) = f
t,y(t)
,
(90) (90) 0
Dt
y(t) = f
t,y(t)
,
(90)
y(0) = y0. y(0) = y0. The above equation can be reformulated as follows: y(t) = 1 – α
M(α)t2–β(t)
–β′(t)ln(t) + 2 – β(t)
t
f
t,y(t)
(91)
+
α
M(α)
t
0
f
τ,y(τ)
β′(τ)ln(τ) + 2 – β(τ)
τ
τ 2–β(τ) dτ. (91) We write the above equation as follows: We write the above equation as follows: We write the above equation as follows: y(tn+1) – y(tn) = 1 – α
M(α)
t2–β(tn+1)
n+1
(– β(tn+2)–β(tn+1)
t
lntn+1 + 2–β(tn+1)
tn+1
)f (tn+1,y(tn+1))
– t2–β(tn)
n
(– β(tn+1)–β(tn)
t
lntn + 2–β(tn)
tn
)f (tn,y(tn))
(92)
+
α
M(α)
tn+1
tn
f
τ,y(τ)
β′(τ)ln(τ) + 2 – β(τ)
τ
τ 2–β(τ) dτ. 8 Atangana–Seda modified scheme If we integrate the above equation with the
new integral operator including the Mittag-Leffler kernel, the above equation can be con-
verted to y(t) = 1 – α
AB(α)t2–β(t)
–β′(t)ln(t) + 2 – β(t)
t
f
t,y(t)
+
α
AB(α)(α)
t
0
f
τ,y(τ)
(t – τ)α–1
(97)
×
–β′(τ)ln(τ) + 2 – β(τ)
τ
τ 2–β(τ) dτ. (97) At the point tn+1 = (n + 1)
t, we have the following: At the point tn+1 = (n + 1)
t, we have the following: y(tn+1) = 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
&
× f
tn+1,y(tn+1)
(98) y(tn+1) = 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
& ( )
%
n+1
&
× f
tn+1,y(tn+1)
(98) (98) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 32 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Page 32 of 107 +
α
AB(α)(α)
tn+1
0
f
τ,y(τ)
(tn+1 – s)α–1
×
–β′(τ)ln(τ) + 2 – β(τ)
τ
τ 2–β(τ) dτ. For brevity, we consider For brevity, we consider For brevity, we consider For brevity, we consider F
τ,y(τ)
= f
τ,y(τ)
–β′(τ)ln(τ) + 2 – β(τ)
τ
τ 2–β(τ),
(99) (99) and we can write the following: and we can write the following: y(tn+1) = 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
&
(100)
× f
tn+1,y(tn+1)
+
α
AB(α)(α)
n
j=2
tj+1
tj
F
τ,y(τ)
(tn+1 – τ)α–1 dτ. (100) One can replace the Newton polynomial in the above equation as follows. 8 Atangana–Seda modified scheme Replacing the function G(t,y(t)) with its value, we can present the following scheme for
numerical solution of our equation: yn+1 = 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
&
× f
tn+1,yn + f
tn,yn
t
(102) yn+1 = 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
& × f
tn+1,yn + f
tn,yn
t
(102) (102) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 33 of Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Page 33 of 107 +
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
× f
tj–2,yj –
tf (tj,yj)
–
tf (tj–1,yj – f (tj,yj)
t)
(n – j + 1)α – (n – j)α
+
α(
t)α
AB(α)(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
[–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
]
× f (tj–1,yj – f (tj,yj)
t)
– t
2–β(tj–2)
j–2
[–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
]
× f
tj–2,yj –
tf (tj,yj)
–
tf (tj–1,yj – f (tj,yj)
t)
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
(n – j + 1)α(n – j + 3 + 2α)
– (n – j)α(n – j + 3 + 3α)
+
α(
t)α
2AB(α)(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
[–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
]
× f (tj,yj)
t
– 2t
2–β(tj–1)
j–1
[–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
]
× f (tj–1,yj – f (tj,yj)
t)
+ t
2–β(tj–2)
j–2
[–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
]
× f
tj–2,yj –
tf (tj,yj)
–
tf (tj–1,yj – f (tj,yj)
t)
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
⎡
⎢⎢⎢⎢⎣
(n – j + 1)α
2(n – j)2 + (3α + 10)(n – j)
+ 2α2 + 9α + 12
– (n – j)α
2(n – j)2 + (5α + 10)(n – j)
+ 6α2 + 18α + 12
⎤
⎥⎥⎥⎥⎦
. 8 Atangana–Seda modified scheme Thus, we have
the following scheme: yn+1 = 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
&
× f
tn+1,y(tn+1)
+
α(
t)α
AB(α)(α + 1)
n
j=2
F
tj–2,yj–2
(n – j + 1)α – (n – j)α
+
α(
t)α
AB(α)(α + 2)
n
j=2
F
tj–1,yj–1
– F
tj–2,yj–2
×
(n – j + 1)α(n – j + 3 + 2α)
– (n – j)α(n – j + 3 + 3α) yn+1 = 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
&
(101)
× f
tn+1,y(tn+1)
(101) +
α(
t)α
AB(α)(α + 1)
n
j=2
F
tj–2,yj–2
(n – j + 1)α – (n – j)α
+
α(
t)α
AB(α)(α + 2)
n
j=2
F
tj–1,yj–1
– F
tj–2,yj–2
×
(n – j + 1)α(n – j + 3 + 2α)
– (n – j)α(n – j + 3 + 3α) ×
(n – j + 1)α(n – j + 3 + 2α)
– (n – j)α(n – j + 3 + 3α) +
α(
t)α
2AB(α)(α + 3)
n
j=2
F
tj,yj
– 2F
tj–1,yj–1
+ F
tj–2,yj–2
×
⎡
⎢⎢⎢⎢⎣
(n – j + 1)α
2(n – j)2 + (3α + 10)(n – j)
+ 2α2 + 9α + 12
– (n – j)α
2(n – j)2 + (5α + 10)(n – j)
+ 6α2 + 18α + 12
⎤
⎥⎥⎥⎥⎦
. 8 Atangana–Seda modified scheme ×
⎡
⎢⎢⎢⎢⎣
(n – j + 1)α
2(n – j)2 + (3α + 10)(n – j)
+ 2α2 + 9α + 12
– (n – j)α
2(n – j)2 + (5α + 10)(n – j)
+ 6α2 + 18α + 12
⎤
⎥⎥⎥⎥⎦
. We deal with our problem involving the new constant fractional order and variable fractal
dimension We deal with our problem involving the new constant fractional order and variable fractal
dimension FFP
0
Dα,β(t)
t
y(t) = f
t,y(t)
,
(103) FFP
0
Dα,β(t)
t
y(t) = f
t,y(t)
,
y(0) = y0,
(103) (103) y(0) = y0, where the kernel is the power-law kernel. If we integrate equation (103) with the new
integral operator including the power-law kernel, the above equation can be converted to y(t) =
1
(α)
t
0
f
τ,y(τ)
(t – τ)α–1
–β′(τ)ln(τ) + 2 – β(τ)
τ
τ 2–β(τ) dτ. (104) (104) At the point tn+1 = (n + 1)
t, we have the following: At the point tn+1 = (n + 1)
t, we have the following: y(tn+1) =
1
(α)
tn+1
0
f
τ,y(τ)
(tn+1 – s)α–1
(105)
×
–β′(τ)ln(τ) + 2 – β(τ)
τ
τ 2–β(τ) dτ. (105) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 34 of 107 ( 2021) 2021:57 For brevity, we consider For brevity, we consider For brevity, we consider F
τ,y(τ)
= f
τ,y(τ)
–β′(τ)ln(τ) + 2 – β(τ)
τ
τ 2–β(τ), F
τ,y(τ)
= f
τ,y(τ)
–β′(τ)ln(τ) + 2 – β(τ)
τ
τ 2–β(τ),
(106) (106) and we can write the following: and we can write the following: and we can write the following: y(tn+1) =
1
(α)
n
j=2
tj+1
tj
F
τ,y(τ)
(tn+1 – τ)α–1 dτ. y(tn+1) =
1
(α)
n
j=2
tj+1
tj
F
τ,y(τ)
(tn+1 – τ)α–1 dτ. 9 Application to COVID-19 model In this section, using the suggested numerical scheme, we present its application to solve
the mathematical model of COVID-19 with possibility of waves. The numerical scheme
will be applied for all cases where the differential operators are with classical differential
operators, modern fractional differential operators, and variable orders, although only few
examples will be used for numerical simulations. Firstly, we shall use the Caputo–Fabrizio
fractional derivative CF
0 Dα
t S = –
δ(t)
αI∗+ wβI∗
D + γ wI∗
A + wδ1I∗
R + wδ2I∗
T
+ γ1 + μ1
S,
CF
0 Dα
t I =
δ(t)
αI∗+ wβI∗
D + γ wI∗
A + wδ1I∗
R + wδ2I∗
T
S – (ε + ξ + λ + μ1)I,
CF
0 Dα
t IA = ξI – (θ + μ + χ + μ1)IA,
CF
0 Dα
t ID = εI – (η + ϕ + μ1)ID,
CF
0 Dα
t IR = ηID + θIA – (v + ξ + μ1)IR,
(110)
CF
0 Dα
t IT = μIA + vIR – (σ + τ + μ1)IT,
CF
0 Dα
t R = λI + ϕID + χIA + ξIR + σIT – ( + μ1)R,
CF
0 Dα
t D = τIT,
CF
0 Dα
t V = γ1S + R – μ1V. CF
0 Dα
t S = –
δ(t)
αI∗+ wβI∗
D + γ wI∗
A + wδ1I∗
R + wδ2I∗
T
+ γ1 + μ1
S,
CF
0 Dα
t I =
δ(t)
αI∗+ wβI∗
D + γ wI∗
A + wδ1I∗
R + wδ2I∗
T
S – (ε + ξ + λ + μ1)I,
CF
0 Dα
t IA = ξI – (θ + μ + χ + μ1)IA,
CF
0 Dα
t ID = εI – (η + ϕ + μ1)ID,
CF
0 Dα
t IR = ηID + θIA – (v + ξ + μ1)IR,
(110)
CF
0 Dα
t IT = μIA + vIR – (σ + τ + μ1)IT,
CF
0 Dα
t R = λI + ϕID + χIA + ξIR + σIT – ( + μ1)R,
CF
0 Dα
t D = τIT,
CF
0 Dα
t V = γ1S + R – μ1V. (110) CF
0 Dα
t V = γ1S + R – μ1V. 8 Atangana–Seda modified scheme Replacing the function G(t,y(t)) with its value, we can present the following scheme for
numerical solution of our equation: yn+1 =
(
t)α
(α + 1)
n
j=2
t
2–β(tj–2)
j–2
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
× f
tj–2,yj –
tf (tj,yj)
–
tf (tj–1,yj – f (tj,yj)
t)
(n – j + 1)α – (n – j)α
+
(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
[–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
]
× f (tj–1,yj – f (tj,yj)
t)
– t
2–β(tj–2)
j–2
[–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
]
× f
tj–2,yj –
tf (tj,yj)
–
tf (tj–1,yj – f (tj,yj)
t)
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎦
(109) (109) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 35 of 1 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Page 35 of 107 +
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
[–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
]
× f (tj,yj)
t
– 2t
2–β(tj–1)
j–1
[–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
]
× f (tj–1,yj – f (tj,yj)
t)
+ t
2–β(tj–2)
j–2
[–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
]
× f
tj–2,yj –
tf (tj,yj)
–
tf (tj–1,yj – f (tj,yj)
t)
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
⎡
⎢⎢⎢⎢⎣
(n – j + 1)α
2(n – j)2 + (3α + 10)(n – j)
+ 2α2 + 9α + 12
– (n – j)α
2(n – j)2 + (5α + 10)(n – j)
+ 6α2 + 18α + 12
⎤
⎥⎥⎥⎥⎦
. 8 Atangana–Seda modified scheme (107) (107) Thus, we have the following scheme: Thus, we have the following scheme: yn+1 =
(
t)α
(α + 1)
n
j=2
F
tj–2,yj–2
(n – j + 1)α – (n – j)α
(108)
+
(
t)α
(α + 2)
n
j=2
F
tj–1,yj–1
– F
tj–2,yj–2
×
(n – j + 1)α(n – j + 3 + 2α)
– (n – j)α(n – j + 3 + 3α)
+
(
t)α
2(α + 3)
n
j=2
F
tj,yj
– 2F
tj–1,yj–1
+ F
tj–2,yj–2
×
⎡
⎢⎢⎢⎢⎣
(n – j + 1)α
2(n – j)2 + (3α + 10)(n – j)
+ 2α2 + 9α + 12
– (n – j)α
2(n – j)2 + (5α + 10)(n – j)
+ 6α2 + 18α + 12
⎤
⎥⎥⎥⎥⎦
. yn+1 =
(
t)α
(α + 1)
n
j=2
F
tj–2,yj–2
(n – j + 1)α – (n – j)α
(108)
+
(
t)α
(α + 2)
n
j=2
F
tj–1,yj–1
– F
tj–2,yj–2
×
(n – j + 1)α(n – j + 3 + 2α)
– (n – j)α(n – j + 3 + 3α) (108) +
(
t)α
2(α + 3)
n
j=2
F
tj,yj
– 2F
tj–1,yj–1
+ F
tj–2,yj–2
×
⎡
⎢⎢⎢⎢⎣
(n – j + 1)α
2(n – j)2 + (3α + 10)(n – j)
+ 2α2 + 9α + 12
– (n – j)α
2(n – j)2 + (5α + 10)(n – j)
+ 6α2 + 18α + 12
⎤
⎥⎥⎥⎥⎦
. 9 Application to COVID-19 model For simplicity, we rearrange the above equation as follows: CF
0 Dα
t S = S∗(t,S,I,IA,ID,IR,IT,R,D,V),
CF
0 Dα
t I = I∗(t,S,I,IA,ID,IR,IT,R,D,V),
CF
0 Dα
t IA = I∗
A(t,S,I,IA,ID,IR,IT,R,D,V),
CF
0 Dα
t ID = I∗
D(t,S,I,IA,ID,IR,IT,R,D,V),
CF
0 Dα
t IR = I∗
R(t,S,I,IA,ID,IR,IT,R,D,V),
(111)
CF
0 Dα
t IT = I∗
T(t,S,I,IA,ID,IR,IT,R,D,V), CF
0 Dα
t S = S∗(t,S,I,IA,ID,IR,IT,R,D,V),
CF
0 Dα
t I = I∗(t,S,I,IA,ID,IR,IT,R,D,V),
CF
0 Dα
t IA = I∗
A(t,S,I,IA,ID,IR,IT,R,D,V),
CF
0 Dα
t ID = I∗
D(t,S,I,IA,ID,IR,IT,R,D,V),
CF
0 Dα
t IR = I∗
R(t,S,I,IA,ID,IR,IT,R,D,V),
CF
0 Dα
t IT = I∗
T(t,S,I,IA,ID,IR,IT,R,D,V), (111) Page 36 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) Thus, we can have the following scheme for our model: Thus, we can have the following scheme for our model: Sn+1 = Sn + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎣
S∗
⎛
⎜⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎟⎠
– S∗
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎦
(112) Sn+1 = Sn + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎣
S∗
⎛
⎜⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎟⎠
– S∗
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎦
(112)
+
α
M(α)
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12S∗(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3S∗
⎛
⎜⎜⎝
tn,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎟⎠
t
+ 5
12S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
, Sn+1 = Sn + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎣
S∗
⎛
⎜⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎟⎠
– S∗
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎦
(112)
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪
23
12S∗(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
⎛
tn,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
⎞
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪ +
α
M(α)
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12S∗(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3S∗
⎛
⎜⎜⎝
tn,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎟⎠
t
+ 5
12S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
, + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎣
I∗
⎛
⎜⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎟⎠
– I∗
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎦
(113) +
α
M(α)
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12I∗(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3I∗
⎛
⎜⎜⎝
tn,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎟⎠
t
+ 5
12I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
, In+1
A
= In
A + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎣
I∗
A
⎛
⎜⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎟⎠
– I∗
A
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎦
(114) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 37 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:5 Atangana and ˙I˘gret Araz Advances in Difference Equations Page 37 of 107 ( 2021) 2021:57 +
α
M(α)
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12I∗
A(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3I∗
A
⎛
⎜⎜⎝
tn,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎟⎠
t
+ 5
12I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
, In+1
D
= In
D + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎣
I∗
D
⎛
⎜⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎟⎠
– I∗
D
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎦
(115) +
α
M(α)
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12I∗
D(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3I∗
D
⎛
⎜⎜⎝
tn,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎟⎠
t
+ 5
12I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
, 1 – α
M(α)
⎡
⎢⎢⎢⎢⎣
I∗
R
⎛
⎜⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎟⎠
– I∗
R
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎦
(116) +
α
M(α)
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12I∗
R(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3I∗
R
⎛
⎜⎜⎝
tn,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎟⎠
t
+ 5
12I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
, In+1
T
= In
T + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎣
I∗
T
⎛
⎜⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎟⎠
– I∗
T
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎦
(117) In+1
T
= In
T + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎣
I∗
T
⎛
⎜⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎟⎠
– I∗
T
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎦
(117) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 38 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:5 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 38 of 107 +
α
M(α)
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12I∗
T(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3I∗
T
⎛
⎜⎜⎝
tn,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎟⎠
t
+ 5
12I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
, Rn+1 = Rn + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎣
R∗
⎛
⎜⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎟⎠
– R∗
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎦
(118) +
α
M(α)
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12R∗(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3R∗
⎛
⎜⎜⎝
tn,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎟⎠
t
+ 5
12R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
, + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎣
D∗
⎛
⎜⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎟⎠
– D∗
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎦
(119) +
α
M(α)
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12D∗(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3D∗
⎛
⎜⎜⎝
tn,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎟⎠
t
+ 5
12D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
, V n+1 = V n + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎣
V ∗
⎛
⎜⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎟⎠
– V ∗
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎦
(120) V n+1 = V n + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎣
V ∗
⎛
⎜⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎟⎠
– V ∗
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎦
(120) Equations ( 2021) 2021:57
Page 39 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 39 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 39 of 107 +
α
M(α)
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12V ∗(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3V ∗
⎛
⎜⎝
tn,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎠
t
+ 5
12V ∗
⎛
⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
. 9 Application to COVID-19 model With the Atangana–Baleanu fractional derivative, we can solve numerically our model as
follows: With the Atangana–Baleanu fractional derivative, we can solve numerically our model as
follows: Sn+1 = 1 – α
AB(α)S∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
(121) (121) +
α(
t)α
AB(α)(α + 1) +
α(
t)
AB(α)(α + 1)
×
n
j=2
S∗
⎛
⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
S∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– S∗
⎛
⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
S∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2S∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,Rj –
tRj∗,
Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ S∗
⎛
⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, ×
n
j=2
S∗
⎛
⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
× +
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
S∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– S∗
⎛
⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× 2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
S∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2S∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,Rj –
tRj∗,
Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ S∗
⎛
⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, Araz Advances in Difference Equations ( 2021) 2021:57
In+1 = 1 – α
AB(α)I∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
×
n
j=2
I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2I∗
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,Ij
D –
tIj∗
D ,
Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
+ I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
In+1
A
= 1 – α
AB(α)I∗
A
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 40 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Page 40 of 107 In+1 = 1 – α
AB(α)I∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ ×
n
j=2
I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× +
α(
t)
2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2I∗
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,Ij
D –
tIj∗
D ,
Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
+ I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ + 2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2I∗
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,Ij
D –
tIj∗
D ,
Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
+ I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
, In+1
A
= 1 – α
AB(α)I∗
A
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ In+1
A
= 1 – α
AB(α)I∗
A
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 41 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 41 of 107 ( 2021) 2021:57 ×
n
j=2
I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
A
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
A(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2I∗
A
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
In+1
D
= 1 – α
AB(α)I∗
D
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
×
n
j=2
I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× ( ) (
)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
A(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2I∗
A
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, In+1
D
= 1 – α
AB(α)I∗
D
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ +
α(
t)α
AB(α)(α + 1)
×
n
j=2
I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 42 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations Page 42 of 107 ( 2021) 2021:57 ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
D
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× + 2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
D(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2I∗
D
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× In+1
R
= 1 – α
AB(α)I∗
R
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ +
α(
t)α
AB(α)(α + 1)
×
n
j=2
I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
R
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3) ×
n
j=2
I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
R
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 43 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021 Atangana and ˙I˘gret Araz Advances in Difference Equations Page 43 of 107 ( 2021) 2021:57 ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
R(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2I∗
R
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, In+1
T
= 1 – α
AB(α)I∗
T
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ In+1
T
= 1 – α
AB(α)I∗
T
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ +
α(
t)α
AB(α)(α + 1)
×
n
j=2
I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
T
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× ×
n
j=2
I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
T
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× +
α(
t)α
2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
T(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2I∗
T
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, et Araz Advances in Difference Equations ( 2021) 2021:57
Rn+1 = 1 – α
AB(α)R∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
×
n
j=2
R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
R∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
R∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2R∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
Dn+1 = 1 – α
AB(α)D∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 44 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 44 of 107 ( 2021) 2021:57 Rn+1 = 1 – α
AB(α)R∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ Dn+1 = 1 – α
AB(α)D∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ Dn+1 = 1 – α
AB(α)D∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ +
α(
t)α
AB(α)(α + 1) +
α(
t)α
AB(α)(α + 1) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 45 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Page 45 of 107 ×
n
j=2
D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
D∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
D∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2D∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
V n+1 = 1 – α
AB(α)V ∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
×
n
j=2
V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× ( ) (
)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
D∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2D∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, V n+1 = 1 – α
AB(α)V ∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ +
α(
t)α
AB(α)(α + 1)
×
n
j=2
V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2) AB(α)(α + 1)
×
n
j=2
V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 46 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 46 of 107 ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
V ∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
V ∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2V ∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, =
⎡
⎢⎢⎢⎢⎣
(n – j + 1)α
2(n – j)2 + (3α + 10)(n – j)
+ 2α2 + 9α + 12
– (n – j)α
2(n – j)2 + (5α + 10)(n – j)
+ 6α2 + 18α + 12
⎤
⎥⎥⎥⎥⎦
,
(122)
=
(n – j + 1)α(n – j + 3 + 2α)
– (n – j)α(n – j + 3 + 3α)
,
=
(n – j + 1)α – (n – j)α
. 9 Application to COVID-19 model =
⎡
⎢⎢⎢⎢⎣
(n – j + 1)α
2(n – j)2 + (3α + 10)(n – j)
+ 2α2 + 9α + 12
– (n – j)α
2(n – j)2 + (5α + 10)(n – j)
+ 6α2 + 18α + 12
⎤
⎥⎥⎥⎥⎦
,
(122) (122) =
(n – j + 1)α(n – j + 3 + 2α)
– (n – j)α(n – j + 3 + 3α)
,
=
(n – j + 1)α – (n – j)α
. 9 Application to COVID-19 model ( 2021) 2021:57
Page 47 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations Page 47 of 107 ( 2021) 2021:57 With the Caputo fractional derivative, we can obtain the following: Sn+1 =
(
t)α
(α + 1)
n
j=2
S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
S∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
(123) × ×
+
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
S∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2S∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
, ×
,
In+1 =
(
t)α
(α + 1)
n
j=2
I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× 2021) 2021:57
Page 48 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 48 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:5 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 48 of 107 +
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2I∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
, ×
,
In+1
A
=
(
t)α
(α + 1)
n
j=2
I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
A
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
+
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
A(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2I∗
A
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ × ×
,
In+1
D
=
(
t)α
(α + 1)
n
j=2
I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 49 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Page 49 of 107 +
(
t)α
(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
D
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
(
t)α
2(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
D(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2I∗
D
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,Ij
D –
tIj∗
D ,
Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
+ I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
In+1
R
=
(
t)α
(α + 1)
n
j=2
I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
R
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× +
(
t)α
(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
D
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
D(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2I∗
D
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,Ij
D –
tIj∗
D ,
Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
+ I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ In+1
R
=
(
t)α
(α + 1)
n
j=2
I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
R
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× 2021) 2021:57
Page 50 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
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(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
R(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2I∗
R
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ⎢⎢⎢⎢⎣
+ I∗
R
⎜⎜⎜⎜⎝
Ij
R –
tIj
R –
tI(j
)
R
,Ij
T –
tIj
T –
tI(j
)
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎟⎟⎟⎟⎠
⎥⎥⎥⎥⎦
×
,
In+1
T
=
(
t)α
(α + 1)
n
j=2
I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+ α(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
T
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
I∗
T(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2I∗
T
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
, ×
, ×
, Rn+1 =
(
t)α
(α + 1)
n
j=2
R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× ( 2021) 2021:57
Page 51 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
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(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
R∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
R∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2R∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
Dn+1 =
(
t)α
(α + 1)
n
j=2
D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
D∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ +
(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
R∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
+
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
R∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2R∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ × ×
, Dn+1 =
(
t)α
(α + 1)
n
j=2
D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
D∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ × × ( 2021) 2021:57
Page 52 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 52 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 52 of 107 +
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
D∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2D∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
, ×
, V n+1 =
(
t)α
(α + 1)
n
j=2
V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
V ∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
+
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
V ∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2V ∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
. 9 Application to COVID-19 model × × ×
. We now do the same routine for fractal-fractional derivatives. We start with the Caputo–
Fabrizio fractal-fractional derivative FFE
0
Dα
t S = S∗(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα
t I = I∗(t,S,I,IA,ID,IR,IT,R,D,V), Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 53 of 1 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations Page 53 of 107 FFE
0
Dα
t IA = I∗
A(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα
t ID = I∗
D(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα
t IR = I∗
R(t,S,I,IA,ID,IR,IT,R,D,V),
(124)
FFE
0
Dα
t IT = I∗
T(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα
t R = R∗(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα
t D = D∗(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα
t V = V ∗(t,S,I,IA,ID,IR,IT,R,D,V). 9 Application to COVID-19 model (124) After applying the fractional integral with exponential kernel and putting the Newton
polynomial into these equations, we can solve our model as follows: After applying the fractional integral with exponential kernel and putting the Newton
polynomial into these equations, we can solve our model as follows: Sn+1 = Sn + 1 – α
M(α)
⎡
⎢⎢⎢⎣
t1–β
n+1 S∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
– t1–β
n
S∗
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎦
+
α
M(α)
(125) (125) ×
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12t1–β
n
S∗(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3t1–β
n–1 S∗
⎛
⎜⎝
tn–1,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎠
t
+ 5
12t1–β
n–2 S∗
⎛
⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
,
In+1 = Sn + 1 – α
(126) ×
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12t1–β
n
I∗(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3t1–β
n–1 I∗
⎛
⎜⎝
tn–1,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎠
t
+ 5
12t1–β
n–2I∗
⎛
⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
, ons ( 2021) 2021:57
Page 54 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 54 of 107 ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations Page 54 of 107 In+1
A
= In
A + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎣
t1–β
n+1 I∗
A
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
– t1–β
n
I∗
A
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎦
+
α
M(α)
(127) (127) ×
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12t1–β
n
I∗
A(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3t1–β
n–1I∗
A
⎛
⎜⎝
tn–1,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎠
t
+ 5
12t1–β
n–2 I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
, In+1
D
= In
D + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎣
t1–β
n+1 I∗
D
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
– t1–β
n
I∗
D
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎦
+
α
( )
(128) In+1
D
= In
D + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎣
t1–β
n+1 I∗
D
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
– t1–β
n
I∗
D
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎦
α
(128) (128) ×
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12t1–β
n
I∗
D(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3t1–β
n–1I∗
D
⎛
⎜⎝
tn–1,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎠
t
+ 5
12t1–β
n–2 I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
, (129) ons ( 2021) 2021:57
Page 55 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 55 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 55 of 107 ×
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12t1–β
n
I∗
R(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3t1–β
n–1 I∗
R
⎛
⎜⎝
tn–1,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎠
t
+ 5
12t1–β
n–2I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
, In+1
T
= In
T + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎣
t1–β
n+1 I∗
T
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
– t1–β
n
I∗
T
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎦
(130) (130) ×
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12t1–β
n
I∗
T(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3t1–β
n–1I∗
T
⎛
⎜⎝
tn–1,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎠
t
+ 5
12t1–β
n–2 I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
, ×
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12t1–β
n
R∗(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3t1–β
n–1 R∗
⎛
⎜⎝
tn–1,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎠
t
+ 5
12t1–β
n–2 R∗
⎛
⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
, Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 56 of 107 ( 2021) 2021:57
Page 56 of 107 ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations Page 56 of 107 Dn+1 = Dn + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎣
t1–β
n+1 D∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
– t1–β
n
D∗
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎦
+
α
M(α)
(132) ×
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12t1–β
n
D∗(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3t1–β
n–1 D∗
⎛
⎜⎝
tn–1,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎠
t
+ 5
12t1–β
n–2 D∗
⎛
⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
, V n+1 = V n + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎣
t1–β
n+1 V ∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
– t1–β
n
V ∗
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎦
+
α
M(α)
(133) V n+1 = V n + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎣
t1–β
n+1 V ∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
– t1–β
n
V ∗
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎦
+
α
M(α)
(133) V n+1 = V n + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎣
t1–β
n+1 V ∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
– t1–β
n
V ∗
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎦
+
α
M(α)
(133) ×
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12t1–β
n
V ∗(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3t1–β
n–1 V ∗
⎛
⎜⎝
tn–1,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎠
t
+ 5
12t1–β
n–2V ∗
⎛
⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
. 9 Application to COVID-19 model ×
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12t1–β
n
V ∗(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3t1–β
n–1 V ∗
⎛
⎜⎝
tn–1,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎠
t
+ 5
12t1–β
n–2V ∗
⎛
⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
. 9 Application to COVID-19 model For the Atangana–Baleanu fractal-fractional derivative, we can have the following numer-
ical scheme: Sn+1 = 1 – α
AB(α)t1–β
n+1 S∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
(134) (134) +
α(
t)α
AB(α)(α + 1) +
α(
t)α
AB(α)(α + 1) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 57 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Araz Advances in Difference Equations ( 2021) 2021:57
×
n
j=2
t1–β
j–2 S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 S∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
S∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 S∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
In+1
1 – α t1–βI∗
⎛
⎜
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
tIn∗In
tIn∗In
tIn∗
⎞
⎟ ( 2021) 2021:57 Page 57 of 107 ×
n
j=2
t1–β
j–2 S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 S∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
S∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 S∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
In+1 = 1 – α
AB(α)t1–β
n+1 I∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
×
n
j=2
t1–β
j–2 I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
α(
t)α ×
n
j=2
t1–β
j–2 S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 S∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
S∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 S∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, In+1 = 1 – α
AB(α)t1–β
n+1 I∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ In+1 = 1 – α
AB(α)t1–β
n+1 I∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ +
α(
t)α
AB(α)(α + 1)
×
n
j=2
t1–β
j–2 I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 58 of 107 ( 2021) 2021:57
Page 58 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 58 of 107 ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 I∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
I∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 I∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
I∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 I∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, In+1
A
= 1 – α
AB(α)t1–β
n+1 I∗
A
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ ⎝
,
,
⎠
+
α(
t)α
AB(α)(α + 1)
×
n
j=2
t1–β
j–2 I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 I∗
A
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3) +
α(
t)α
AB(α)(α + 1)
×
n
j=2
t1–β
j–2 I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 I∗
A
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 59 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 59 of 107 ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
I∗
A(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 I∗
A
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, In+1
D
= 1 – α
AB(α)t1–β
n+1 I∗
D
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ In+1
D
= 1 – α
AB(α)t1–β
n+1 I∗
D
⎛
⎜⎝
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
×
n
j=2
t1–β
j–2 I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 I∗
D
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
I∗
D(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 I∗
D
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, +
α(
t)α
AB(α)(α + 1)
×
n
j=2
t1–β
j–2 I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 I∗
D
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
I∗
D(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 I∗
D
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, Araz Advances in Difference Equations ( 2021) 2021:57
In+1
R
= 1 – α
AB(α)t1–β
n+1 I∗
R
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
×
n
j=2
t1–β
j–2 I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 I∗
R
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
I∗
R(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 I∗
R
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
In+1
T
= 1 – α
AB(α)t1–β
n+1 I∗
T
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 60 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Page 60 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations In+1
R
= 1 – α
AB(α)t1–β
n+1 I∗
R
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ ×
n
j=2
t1–β
j–2 I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 I∗
R
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
I∗
R(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 I∗
R
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, In+1
T
= 1 – α
AB(α)t1–β
n+1 I∗
T
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ In+1
T
= 1 – α
AB(α)t1–β
n+1 I∗
T
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ +
α(
t)α
AB(α)(α + 1) +
α(
t)α
AB(α)(α + 1) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 61 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Page 61 of 107 ×
n
j=2
t1–β
j–2 I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 I∗
T
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
I∗
T(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–12I∗
T
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
Rn+1 = 1 – α
AB(α)t1–β
n+1 R∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
×
n
j=2
t1–β
j–2 R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α ×
n
j=2
t1–β
j–2 I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 I∗
T
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
I∗
T(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–12I∗
T
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, Rn+1 = 1 – α
AB(α)t1–β
n+1 R∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ Rn+1 = 1 – α
AB(α)t1–β
n+1 R∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ +
α(
t)α
AB(α)(α + 1)
×
n
j=2
t1–β
j–2 R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× ( 2021) 2021:57
Page 62 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 62 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 62 of 107 ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 R∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
R∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 R∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
Dn+1 = 1 – α
AB(α)t1–β
n+1 D∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
×
n
j=2
t1–β
j–2 D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 D∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
α(
t)α ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
R∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 R∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, Dn+1 = 1 – α
AB(α)t1–β
n+1 D∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ Dn+1 = 1 – α
AB(α)t1–β
n+1 D∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ ⎝
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎠
+
α(
t)α
AB(α)(α + 1)
×
n
j=2
t1–β
j–2 D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 D∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3) +
α(
t)α
AB(α)(α + 1)
×
n
j=2
t1–β
j–2 D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 D∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 63 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 63 of 107 ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
D∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 D∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
V n+1 = 1 – α
AB(α)t1–β
n+1 V ∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
×
n
j=2
t1–β
j–2 V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 V ∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
V ∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 V ∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
. 9 Application to COVID-19 model ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
D∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 D∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, V n+1 = 1 – α
AB(α)t1–β
n+1 V ∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ AB(α)
⎝
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎠
+
α(
t)α
AB(α)(α + 1)
×
n
j=2
t1–β
j–2 V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 V ∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
V ∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 V ∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
. 9 Application to COVID-19 model +
α(
t)α
AB(α)(α + 1)
×
n
j=2
t1–β
j–2 V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 V ∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
V ∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 V ∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
. 9 Application to COVID-19 model Now we apply ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
V ∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 V ∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 V ∗
⎛
⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
. Now we apply FFE
0
Dα,β(t)
t
S = S∗(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα,β(t)
t
I = I∗(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα,β(t)
t
IA = I∗
A(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα,β(t)
t
ID = I∗
D(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα,β(t)
t
IR = I∗
R(t,S,I,IA,ID,IR,IT,R,D,V),
(136)
FFE
0
Dα,β(t)
t
IT = I∗
T(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα,β(t)
t
R = R∗(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα,β(t)
t
D = D∗(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα,β(t)
t
V = V ∗(t,S,I,IA,ID,IR,IT,R,D,V). FFE
0
Dα,β(t)
t
S = S∗(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα,β(t)
t
I = I∗(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα,β(t)
t
IA = I∗
A(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα,β(t)
t
ID = I∗
D(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα,β(t)
t
IR = I∗
R(t,S,I,IA,ID,IR,IT,R,D,V),
(136)
FFE
0
Dα,β(t)
t
IT = I∗
T(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα,β(t)
t
R = R∗(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα,β(t)
t
D = D∗(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα,β(t)
t
V = V ∗(t,S,I,IA,ID,IR,IT,R,D,V). FFE
0
Dα,β(t)
t
S = S∗(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα,β(t)
t
I = I∗(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα,β(t)
t
IA = I∗
A(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα,β(t)
t
ID = I∗
D(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα,β(t)
t
IR = I∗
R(t,S,I,IA,ID,IR,IT,R,D,V),
(136)
FFE
0
Dα,β(t)
t
IT = I∗
T(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα,β(t)
t
R = R∗(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα,β(t)
t
D = D∗(t,S,I,IA,ID,IR,IT,R,D,V),
FFE
0
Dα,β(t)
t
V = V ∗(t,S,I,IA,ID,IR,IT,R,D,V). 9 Application to COVID-19 model Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations Page 64 of 107 ( 2021) 2021:57 For the power-law kernel, we can have the following: For the power-law kernel, we can have the following: For the power-law kernel, we can have the following: Sn+1 =
(
t)α
(α + 1)
×
n
j=2
t1–β
j–2 S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
(
t)α
(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 S∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
– t1–β
j–2 S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
(
t)α
2(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
S∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 S∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t1–β
j–2 S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
(135)
In+1 =
(
t)α
(α + 1)
n
j=2
t1–β
j–2 I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
(
t)α
(α + 2) (135) ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 S∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
– t1–β
j–2 S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
S∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 S∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t1–β
j–2 S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, In+1 =
(
t)α
(α + 1)
n
j=2
t1–β
j–2 I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× et Araz Advances in Difference Equations ( 2021) 2021:57
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 I∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
– t1–β
j–2 I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
(
t)α
2(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
I∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 I∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t1–β
j–2 I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
In+1
A
=
(
t)α
(α + 1)
n
j=2
t1–β
j–2 I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
(
t)α
(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 I∗
A
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
– t1–β
j–2 I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
(
t)α ( 2021) 2021:57
Page 65 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 65 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 65 of 107 ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 I∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
– t1–β
j–2 I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
I∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 I∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t1–β
j–2 I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, In+1
A
=
(
t)α
(α + 1)
n
j=2
t1–β
j–2 I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× In+1
A
=
(
t)α
(α + 1)
n
j=2
t1–β
j–2 I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 I∗
A
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
– t1–β
j–2 I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 66 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 66 of 107 ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
I∗
A(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 I∗
A
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t1–β
j–2 I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, In+1
D
=
(
t)α
(α + 1)
n
j=2
t1–β
j–2 I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× + (α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 I∗
D
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
– t1–β
j–2 I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
(
) 2(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
I∗
D(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 I∗
D
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t1–β
j–2 I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 67 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Page 67 of 107 In+1
R
=
(
t)α
(α + 1)
n
j=2
t1–β
j–2 I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
(
t)α
(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–12I∗
R
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,Ij
D –
tIj∗
D ,
Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
– t1–β
j–2 I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
(
t)α
2(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
I∗
R(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 I∗
R
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
In+1
T
=
(
t)α
(α + 1)
n
j=2
t1–β
j–2 I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+ α(
t)α
(α + 2) In+1
R
=
(
t)α
(α + 1)
n
j=2
t1–β
j–2 I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× (
)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–12I∗
R
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,Ij
D –
tIj∗
D ,
Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
– t1–β
j–2 I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
+
(
t)α
2(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
I∗
R(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 I∗
R
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
In+1
T
=
(
t)α
(α + 1)
n
j=2
t1–β
j–2 I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+ α(
t)α
(α + 2) In+1
T
=
(
t)α
(α + 1)
n
j=2
t1–β
j–2 I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 68 of 107 ( 2021) 2021:57
Page 68 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 68 of 107 ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 I∗
T
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
(
t)α
2(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
I∗
T(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 I∗
T
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
Rn+1 =
(
t)α
(α + 1)
n
j=2
t1–β
j–2 R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
(
t)α
(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 R∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
(
t)α ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 I∗
T
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× Rn+1 =
(
t)α
(α + 1)
n
j=2
t1–β
j–2 R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 R∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 69 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 69 of 107 ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
R∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 R∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, ×
n
j=2
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
– 2t1–β
j–1 R∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
Dn+1 =
(
t)α
(α + 1)
n
j=2
t1–β
j–2 D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
(
t)α
(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 D∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
(
t)α
2(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
D∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 D∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
V n+1 =
(
t)α
(α + 1)
n
j=2
t1–β
j–2 V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× Dn+1 =
(
t)α
(α + 1)
n
j=2
t1–β
j–2 D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 D∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 70 of 107 uations ( 2021) 2021:57
Page 70 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Page 70 of 107 +
(
t)α
(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j–1 V ∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t1–β
j–2 V ∗
⎛
⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
(
t)α
2(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t1–β
j
V ∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t1–β
j–1 V ∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t1–β
j–2 V ∗
⎛
⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
. 9 Application to COVID-19 model (136) After applying the fractional integral with exponential kernel and putting the Newton
polynomial into these equations, we can solve our model as follows: Sn+1 = Sn + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t2–β(tn+1)
n+1
(– β(tn+2)–β(tn+1)
t
lntn+1 + 2–β(tn+1)
tn+1
)
× S∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
– t2–β(tn)
n
(– β(tn+1)–β(tn)
t
lntn + 2–β(tn)
tn
)
S∗
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
(137) (137) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 20 Atangana and ˙I˘gret Araz Advances in Difference Equations Page 71 of 107 ( 2021) 2021:57 +
α
M(α)
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12t2–β(tn)
n
(– β(tn+1)–β(tn)
t
lntn + 2–β(tn)
tn
)
× S∗(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3t2–β(tn–1)
n–1
(– β(tn)–β(tn–1)
t
lntn–1 + 2–β(tn–1)
tn–1
)
× S∗
⎛
⎜⎝
tn–1,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎠
t
+ 5
12t2–β(tn–2)
n–2
(– β(tn–1)–β(tn–2)
t
lntn–2 + 2–β(tn–2)
tn–2
)
× S∗
⎛
⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
,
In+1 = In + 1 – α
M(α) In+1
A
= In
A + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t2–β(tn+1)
n+1
(– β(tn+2)–β(tn+1)
t
lntn+1 + 2–β(tn+1)
tn+1
)
× I∗
A
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
– t2–β(tn)
n
(– β(tn+1)–β(tn)
t
lntn + 2–β(tn)
tn
)
× I∗
A
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:5 Atangana and ˙I˘gret Araz Advances in Difference Equations Page 72 of 107 ( 2021) 2021:57 +
α
M(α)
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12t2–β(tn)
n
(– β(tn+1)–β(tn)
t
lntn + 2–β(tn)
tn
)
× I∗
A(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3t2–β(tn–1)
n–1
(– β(tn)–β(tn–1)
t
lntn–1 + 2–β(tn–1)
tn–1
)
× I∗
A
⎛
⎜⎝
tn–1,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎠
t
+ 5
12t2–β(tn–2)
n–2
(– β(tn–1)–β(tn–2)
t
lntn–2 + 2–β(tn–2)
tn–2
)
× I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
,
In+1
D
= In
D + 1 – α
M(α) In+1
R
= In
R + 1 – α
M(α)
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t2–β(tn+1)
n+1
(– β(tn+2)–β(tn+1)
t
lntn+1 + 2–β(tn+1)
tn+1
)
× I∗
R
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
– t2–β(tn)
n
(– β(tn+1)–β(tn)
t
lntn + 2–β(tn)
tn
)
× I∗
R
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 20 Atangana and ˙I˘gret Araz Advances in Difference Equations Page 73 of 107 ( 2021) 2021:57 +
α
M(α)
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12t2–β(tn)
n
(– β(tn+1)–β(tn)
t
lntn + 2–β(tn)
tn
)
× I∗
D(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3t2–β(tn–1)
n–1
(– β(tn)–β(tn–1)
t
lntn–1 + 2–β(tn–1)
tn–1
)
× I∗
R
⎛
⎜⎝
tn–1,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎠
t
+ 5
12t2–β(tn–2)
n–2
(– β(tn–1)–β(tn–2)
t
lntn–2 + 2–β(tn–2)
tn–2
)
× I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
,
In+1
T
= In
T + 1 – α
M(α) T
T
M(α)
×
⎡
⎢⎢⎢⎢⎢⎢⎣
t2–β(tn+1)
n+1
(– β(tn+2)–β(tn+1)
t
lntn+1 + 2–β(tn+1)
tn+1
)
× I∗
T
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,In
D +
tIn∗
D ,
In
R +
tIn∗
R ,In
T +
tIn∗
T ,Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
– t2–β(tn)
n
(– β(tn+1)–β(tn)
t
lntn + 2–β(tn)
tn
)
× I∗
T
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎥⎥⎦
+
α
M(α)
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12t2–β(tn)
n
(– β(tn+1)–β(tn)
t
lntn + 2–β(tn)
tn
)
× I∗
T(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3t2–β(tn–1)
n–1
(– β(tn)–β(tn–1)
t
lntn–1 + 2–β(tn–1)
tn–1
)
× I∗
T
⎛
⎜⎝
tn–1,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎠
t
+ 5
12t2–β(tn–2)
n–2
(– β(tn–1)–β(tn–2)
t
lntn–2 + 2–β(tn–2)
tn–2
)
× I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
,
Rn+1 = Rn + 1 – α
M(α)
×
⎡
⎢⎢⎢⎢⎢⎢⎣
t2–β(tn+1)
n+1
(– β(tn+2)–β(tn+1)
t
lntn+1 + 2–β(tn+1)
tn+1
)
× R∗
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,In
D +
tIn∗
D ,
In
R +
tIn∗
R ,In
T +
tIn∗
T ,Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
– t2–β(tn)
n
(– β(tn+1)–β(tn)
t
lntn + 2–β(tn)
tn
)
R∗
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎥⎥⎦ ×
⎡
⎢⎢⎢⎢⎢⎢⎣
t2–β(tn+1)
n+1
(– β(tn+2)–β(tn+1)
t
lntn+1 + 2–β(tn+1)
tn+1
)
× R∗
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,In
D +
tIn∗
D ,
In
R +
tIn∗
R ,In
T +
tIn∗
T ,Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
– t2–β(tn)
n
(– β(tn+1)–β(tn)
t
lntn + 2–β(tn)
tn
)
R∗
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎥⎥⎦ Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 74 of 107 +
α
M(α)
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12t2–β(tn)
n
(– β(tn+1)–β(tn)
t
lntn + 2–β(tn)
tn
)
× R∗(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3t2–β(tn–1)
n–1
(– β(tn)–β(tn–1)
t
lntn–1 + 2–β(tn–1)
tn–1
)
× R∗
⎛
⎜⎝
tn–1,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎠
t
+ 5
12t2–β(tn–2)
n–2
(– β(tn–1)–β(tn–2)
t
lntn–2 + 2–β(tn–2)
tn–2
)
× R∗
⎛
⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
,
Dn+1 = Dn + 1 – α
M(α) (138) ×
⎡
⎢⎢⎢⎢⎢⎢⎣
t2–β(tn+1)
n+1
(– β(tn+2)–β(tn+1)
t
lntn+1 + 2–β(tn+1)
tn+1
)
× V ∗
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,In
D +
tIn∗
D ,
In
R +
tIn∗
R ,In
T +
tIn∗
T ,Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
– t2–β(tn)
n
(– β(tn+1)–β(tn)
t
lntn + 2–β(tn)
tn
)
× V ∗
tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n
⎤
⎥⎥⎥⎥⎥⎥⎦ Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 75 of 107 +
α
M(α)
⎧
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎩
23
12t2–β(tn)
n
(– β(tn+1)–β(tn)
t
lntn + 2–β(tn)
tn
)
× R∗(tn,Sn,In,In
A,In
D,In
R,In
T,Rn,Dn,V n)
t
– 4
3t2–β(tn–1)
n–1
(– β(tn)–β(tn–1)
t
lntn–1 + 2–β(tn–1)
tn–1
)
× V ∗
⎛
⎜⎝
tn–1,Sn –
tSn∗,In –
tIn∗,In
A –
tIn∗
A ,
In
D –
tIn∗
D ,In
R –
tIn∗
R ,In
T –
tIn∗
T ,
Rn –
tRn∗,Dn –
tDn∗,V n –
tV n∗
⎞
⎟⎠
t
+ 5
12t2–β(tn–2)
n–2
(– β(tn–1)–β(tn–2)
t
lntn–2 + 2–β(tn–2)
tn–2
)
× V ∗
⎛
⎜⎜⎜⎜⎜⎜⎝
tn–2,Sn –
tSn∗–
tS(n–1)∗,In –
tIn∗–
tI(n–1)∗,
In
A –
tIn∗
A –
tI(n–1)∗
A
,In
D –
tIn∗
D –
tI(n–1)∗
D
,
In
R –
tIn∗
R –
tI(n–1)∗
R
,In
T –
tIn∗
T –
tI(n–1)∗
T
,
Rn –
tRn∗–
tR(n–1)∗,Dn –
tDn∗–
tD(n–1)∗,
V n –
tV n∗–
tV (n–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎠
t
⎫
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎪⎭
. 9 Application to COVID-19 model For the Atangana–Baleanu fractal-fractional derivative, we can have the following numer-
ical scheme: Sn+1 = 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
&
(139) (139) × S∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ × S∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ +
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& × S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× ⎜⎜⎜⎜⎝
IR
tIR
tIR
,IT
tIT
tIT
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎟⎟⎟⎟⎠
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× S∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t
2–β(tj–2)
j–2
%
–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
&
× S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
× Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 76 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 76 of 107 ( 2021) 2021:57 +
α(
t)α
2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× S∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× S∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
In+1 = 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
&
× I∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
&
× I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3) +
α(
t)α
2AB(α)(α + 3)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× S∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× S∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
In+1 = 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
& In+1 = 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
& × I∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ +
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& +
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& +
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& × I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 77 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Araz Advances in Difference Equations ( 2021) 2021:57
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× I∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
In+1
A
= 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
&
× I∗
A
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
&
× I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
A
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3) ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× I∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
%
& In+1
A
= 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
& × I∗
A
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& × I∗
A
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ × I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 78 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Araz Advances in Difference Equations ( 2021) 2021:57
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× I∗
A(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
A
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
In+1
D
= 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
&
× I∗
D
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
&
× I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
D
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB( )(
3) ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× I∗
A(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
A
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, In+1
D
= 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
& × I∗
D
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& × I∗
D
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ +
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& × I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 79 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Araz Advances in Difference Equations ( 2021) 2021:57
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× I∗
D(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
D
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
In+1
R
= 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
&
× I∗
R
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
&
× I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
R
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3) ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× I∗
D(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
D
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, In+1
R
= 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
& × I∗
R
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& × I∗
R
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ × I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 8 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 20 Atangana and ˙I˘gret Araz Advances in Difference Equations Page 80 of 107 ( 2021) 2021:57 Araz Advances in Difference Equations ( 2021) 2021:57
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× I∗
R(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
R
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
In+1
T
= 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
&
× I∗
T
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
&
× I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
T
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB( )(
3) ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× I∗
R(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
R
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
1
α
% β(t
)
β(t
)
2
β(t
)& × I∗
T
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& × I∗
T
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ +
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& × I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 81 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Araz Advances in Difference Equations ( 2021) 2021:57
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× I∗
T(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
T
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
Rn+1 = 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
&
× R∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
&
× R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× R∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3) ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× I∗
T(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
T
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
1
α
% β(t
)
β(t
)
2
β(t
)& Rn+1 = 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
& × R∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ +
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& × R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations Page 82 of 107 ( 2021) 2021:57 Araz Advances in Difference Equations ( 2021) 2021:57
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× R∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× R∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
Dn+1 = 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
&
× D∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
&
× D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× D∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3) ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× R∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× R∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
, × D∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ × D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021: Page 83 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 raz Advances in Difference Equations ( 2021) 2021:57
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× D∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× D∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
,
V n+1 = 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
&
× V ∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
&
× V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
α(
t)α
AB(α)(α + 2)
×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× V ∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
– t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
+
α(
t)α
2AB(α)(α + 3) ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× D∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× D∗
⎛
⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
V n+1 = 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
& V n+1 = 1 – α
AB(α)t2–β(tn+1)
n+1
%
–β(tn+2) – β(tn+1)
t
lntn+1 + 2 – β(tn+1)
tn+1
& × V ∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠
+
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& × V ∗
⎛
⎜⎝
tn+1,Sn +
tSn∗,In +
tIn∗,In
A +
tIn∗
A ,
In
D +
tIn∗
D ,In
R +
tIn∗
R ,In
T +
tIn∗
T ,
Rn +
tRn∗,Dn +
tDn∗,V n +
tV n∗
⎞
⎟⎠ +
α(
t)α
AB(α)(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& × V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations Page 84 of 107 ( 2021) 2021:57 ×
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× V ∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× V ∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦
×
. 9 Application to COVID-19 model ×
. 9 Application to COVID-19 model For the power-law kernel, we can have the following: For the power-law kernel, we can have the following: For the power-law kernel, we can have the following: Sn+1 =
(
t)α
(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
&
(140) (140) ⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× +
(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× S∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
– t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ × × Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 85 of 107 +
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× S∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× S∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ +
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× S∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× S∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× S∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
,
In+1 =
(
t)α
(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
&
× I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
– t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
, In+1 =
(
t)α
(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& × I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× +
(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
– t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ × Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 86 of 107 +
α(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× I∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ +
α(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× I∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
, ×
, In+1
A
=
(
t)α
(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& × I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× × Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Pa Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations Page 87 of 107 ( 2021) 2021:57 +
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× I∗
A(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
A
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ +
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× I∗
A(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
A
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
A
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
,
In+1
D
=
(
t)α
(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
&
× I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
D
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
– t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
,
In+1
D
=
(
t)α
(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
&
× I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
D
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
– t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
, ×
, In+1
D
=
(
t)α
(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& × I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× × Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 88 o Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 88 of 107 +
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× I∗
D(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
D
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ +
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× I∗
D(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
D
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
D
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
,
In+1
R
=
(
t)α
(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
&
× I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
R
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
– t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
, ×
, In+1
R
=
(
t)α
(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& × I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× × Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 202 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 89 of 107 +
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× I∗
R(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
R
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ +
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× I∗
R(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
R
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
R
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
,
In+1
T
=
(
t)α
(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
&
× I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
T
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
– t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
,
In+1
T
=
(
t)α
(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
&
× I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
T
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
– t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
, ×
, In+1
T
=
(
t)α
(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& × I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× × Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 90 o Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 90 of 107 +
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× I∗
T(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
T
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ +
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× I∗
T(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× I∗
T
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× I∗
T
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
, Rn+1 =
(
t)α
(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& × R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× +
(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× R∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
– t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ × Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 91 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:5 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 91 of 107 +
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× R∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× R∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ +
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× R∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× R∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× R∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
,
Dn+1 =
(
t)α
(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
&
× D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
×
+
(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× D∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
– t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
, ×
,
Dn+1 =
(
t)α
(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& ×
, Dn+1 =
(
t)α
(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& × D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× × Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 92 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 92 of 107 +
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× D∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× D∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ +
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× D∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× D∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× D∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
, ×
, V n+1 =
(
t)α
(α + 1)
n
j=2
t
2–β(tj–2)
j–2
%
–β(tj–1) – β(tj–2)
t
lntj–2 + 2 – β(tj–2)
tj–2
& × V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
× +
(
t)α
(α + 2)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× V ∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
– t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ × Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:5 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 93 of 107 +
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× V ∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× V ∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ +
(
t)α
2(α + 3)
n
j=2
⎡
⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎢⎣
t
2–β(tj)
j
(–
β(tj+1)–β(tj)
t
lntj +
2–β(tj)
tj
)
× V ∗(tj,Sj,Ij,Ij
A,Ij
D,Ij
R,Ij
T,Rj,Dj,V j)
– 2t
2–β(tj–1)
j–1
(–
β(tj)–β(tj–1)
t
lntj–1 +
2–β(tj–1)
tj–1
)
× V ∗
⎛
⎜⎜⎝
tj–1,Sj –
tSj∗,Ij –
tIj∗,Ij
A –
tIj∗
A ,
Ij
D –
tIj∗
D ,Ij
R –
tIj∗
R ,Ij
T –
tIj∗
T ,
Rj –
tRj∗,Dj –
tDj∗,V j –
tV j∗
⎞
⎟⎟⎠
+ t
2–β(tj–2)
j–2
(–
β(tj–1)–β(tj–2)
t
lntj–2 +
2–β(tj–2)
tj–2
)
× V ∗
⎛
⎜⎜⎜⎜⎜⎜⎜⎝
tj–2,Sj –
tSj∗–
tS(j–1)∗,Ij –
tIj∗–
tI(j–1)∗,
Ij
A –
tIj∗
A –
tI(j–1)∗
A
,Ij
D –
tIj∗
D –
tI(j–1)∗
D
,
Ij
R –
tIj∗
R –
tI(j–1)∗
R
,Ij
T –
tIj∗
T –
tI(j–1)∗
T
,
Rj –
tRj∗–
tR(j–1)∗,Dj –
tDj∗–
tD(j–1)∗,
V j –
tV j∗–
tV (j–1)∗
⎞
⎟⎟⎟⎟⎟⎟⎟⎠
⎤
⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎥⎦ ×
. 10 Numerical simulation In this section, using the numerical solutions obtained in the previous section, we present
a numerical method for all cases. The numerical simulations are depicted for different
values of fractional order and fractal dimension as presented in Figs. 26–37. 10 Numerical simulation FFM
0
Dα,β
t
S = –
δ(t)
αI∗+ wβI∗
D + γ wI∗
A + wδ1I∗
R + wδ2I∗
T
+ γ1 + μ1
S,
FFM
0
Dα,β
t
I =
δ(t)
αI∗+ wβI∗
D + γ wI∗
A + wδ1I∗
R + wδ2I∗
T
S – (ε + ξ + λ + μ1)I,
FFM
0
Dα,β
t
IA = ξI – (θ + μ + χ + μ1)IA,
FFM
0
Dα,β
t
ID = εI – (η + ϕ + μ1)ID, FFM
0
Dα,β
t
S = –
δ(t)
αI∗+ wβI∗
D + γ wI∗
A + wδ1I∗
R + wδ2I∗
T
+ γ1 + μ1
S,
FFM
0
Dα,β
t
I =
δ(t)
αI∗+ wβI∗
D + γ wI∗
A + wδ1I∗
R + wδ2I∗
T
S – (ε + ξ + λ + μ1)I,
FFM
0
Dα,β
t
IA = ξI – (θ + μ + χ + μ1)IA, FFM
0
Dα,β
t
S = –
δ(t)
αI∗+ wβI∗
D + γ wI∗
A + wδ1I∗
R + wδ2I∗
T
+ γ1 + μ1
S,
FFM
0
Dα,β
t
I =
δ(t)
αI∗+ wβI∗
D + γ wI∗
A + wδ1I∗
R + wδ2I∗
T
S – (ε + ξ + λ + μ1)I,
FFM
0
Dα,β
t
IA = ξI – (θ + μ + χ + μ1)IA, FFM
0
Dα,β
t
IA = ξI – (θ + μ + χ + μ1)IA, FFM
0
Dα,β
t
ID = εI – (η + ϕ + μ1)ID, Figure 26 Numerical visualization of COVID-19 model for α = 0.76 Figure 26 Numerical visualization of COVID-19 model for α = 0.76 ( 2021) 2021:57
Page 94 of 107 ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations Page 94 of 107 Figure 27 Numerical visualization of COVID-19 model for α = 0.85
Figure 28 Numerical visualization of COVID-19 model for α = 0.91
FFM
0
Dα,β
t
IR = ηID + θIA – (v + ξ + μ1)IR,
(141)
FFM
0
Dα,τ
t IT = μIA + vIR – (σ + τ + μ1)IT,
FFM
0
Dα,τ
t R = λI + ϕID + χIA + ξIR + σIT – ( + μ1)R,
FFM
0
Dα,τ
t D = τIT, Figure 27 Numerical visualization of COVID-19 model for α = 0.85 Figure 27 Numerical visualization of COVID-19 model for α = 0.85 Figure 28 Numerical visualization of COVID-19 model for α = 0.91 Figure 28 Numerical visualization of COVID-19 model for α = 0.91 Figure 28 Numerical visualization of COVID-19 model for α = 0.91 FFM
0
Dα,β
t
IR = ηID + θIA – (v + ξ + μ1)IR,
(141)
FFM
0
Dα,τ
t IT = μIA + vIR – (σ + τ + μ1)IT,
FFM
0
Dα,τ
t R = λI + ϕID + χIA + ξIR + σIT – ( + μ1)R,
FFM
0
Dα,τ
t D = τIT,
FFM
0
Dα,τ
t V = γ1S + R – μ1V, FFM
0
Dα,β
t
IR = ηID + θIA – (v + ξ + μ1)IR,
(141)
FFM
0
Dα,τ
t IT = μIA + vIR – (σ + τ + μ1)IT, (141) FFM
0
Dα,τ
t V = γ1S + R – μ1V, Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 95 of 107 ( 2021) 2021:57
Page 95 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 95 of 107 Figure 29 Numerical visualization of COVID-19 model for α = 0.76
Figure 30 Numerical visualization of COVID-19 model for α = 0.90, β = 0.85
where Figure 29 Numerical visualization of COVID-19 model for α = 0.76 Figure 29 Numerical visualization of COVID-19 model for α = 0.76 Figure 30 Numerical visualization of COVID-19 model for α = 0.90, β = 0.85 Figure 30 Numerical visualization of COVID-19 model for α = 0.90, β = 0.85 Figure 30 Numerical visualization of COVID-19 model for α = 0.90, β = 0.85 where where
δ(t) =
⎧
⎪⎪⎪⎪⎪⎪⎨
⎪⎪⎪⎪⎪⎪⎩
d0(1 – an)cos(–b t–t0
T ),0 < t < t0
d0,t0 < t < t1
d1(1 – ar)cos(–b t–t1
T ),t1 < t < t2
d1,t2 < t < t3
d2(1 – at)cos(–b t–t2
T ),t > t3
⎫
⎪⎪⎪⎪⎪⎪⎬
⎪⎪⎪⎪⎪⎪⎭
. 10 Numerical simulation (142) (142) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 96 of 107 ( 2021) 2021:57
Page 96 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations Page 96 of 107 Figure 31 Numerical visualization of COVID-19 model for α = 0.95, β = 0.95
Figure 32 Numerical visualization of COVID-19 model for α = 0.72
Also, the initial conditions are
S(0) = 800,000,
I(0) = 3,
IA(0) = 0,
ID(0) = 0,
IR(0) = 0,
(143)
IT(0) = 0,
R(0) = 0,
D(0) = 0,
V(0) = 0. Also, the parameters are chosen as follows: Figure 31 Numerical visualization of COVID-19 model for α = 0.95, β = 0.95 Figure 31 Numerical visualization of COVID-19 model for α = 0.95, β = 0.95 Figure 32 Numerical visualization of COVID-19 model for α = 0.72 Figure 32 Numerical visualization of COVID-19 model for α = 0.72 Figure 32 Numerical visualization of COVID-19 model for α = 0.72 Also, the initial conditions are Also, the initial conditions are Also, the initial conditions are S(0) = 800,000,
I(0) = 3,
IA(0) = 0,
ID(0) = 0,
IR(0) = 0,
(143)
IT(0) = 0,
R(0) = 0,
D(0) = 0,
V(0) = 0. (143) Also, the parameters are chosen as follows: Also, the parameters are chosen as follows: o, the parameters are chosen as follows: = 810,000,
η = 0.12,
χ = 0.15,
v = 0.4,
γ = 0.09,
(144)
β = 0.75,
γ1 = 0.4,
μ1 = 0.3,
ε = 0.161,
τ = 0.0199,
= 0.015,
λ = 0.0345,
ϕ = 0.0345,
δ1 = 0.5,
ξ = 0.015, = 810,000,
η = 0.12,
χ = 0.15,
v = 0.4,
γ = 0.09,
(144)
β = 0.75,
γ1 = 0.4,
μ1 = 0.3,
ε = 0.161,
τ = 0.0199, (144) = 0.015,
λ = 0.0345,
ϕ = 0.0345,
δ1 = 0.5,
ξ = 0.015, = 0.015,
λ = 0.0345,
ϕ = 0.0345,
δ1 = 0.5,
ξ = 0.015, ( 2021) 2021:57
Page 97 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations Page 97 of 107 Figure 33 Numerical visualization of COVID-19 model for α = 0.82
Figure 34 Numerical visualization of COVID-19 model for α = 0.90
σ = 0.015,
δ0 = 0.99,
t = 900,
t0 = 30,
δ2 = 0.4,
w = 0.4,
b = 0.2,
an = 0.1,
ar = 0.2,
at = 0.3,
d0 = 0.02,
d1 = 0.2,
d2 = 0.15. 11 Likelihood with hyper-Poisson distribution
Using the suggested numerical model, we obtain the approximate solution (S∗(t),I∗(t), Figure 33 Numerical visualization of COVID-19 model for α = 0.82 Figure 33 Numerical visualization of COVID-19 model for α = 0.82 Figure 34 Numerical visualization of COVID-19 model for α = 0.90 Figure 34 Numerical visualization of COVID-19 model for α = 0.90 Figure 34 Numerical visualization of COVID-19 model for α = 0.90 Figure 34 Numerical visualization of COVID-19 model for α = 0.90 σ = 0.015,
δ0 = 0.99,
t = 900,
t0 = 30,
δ2 = 0.4,
w = 0.4,
b = 0.2,
an = 0.1,
ar = 0.2,
at = 0.3,
d0 = 0.02,
d1 = 0.2,
d2 = 0.15. 11 Likelihood with hyper-Poisson distribution Using the suggested numerical model, we obtain the approximate solution (S∗(t),I∗(t),
I∗
A(t),I∗
D(t),I∗
R(t),I∗
T(t),R∗(t),D∗(t),V ∗(t)). We are more interested in I∗(t), R∗(t), and D∗(t)
and the approximate solution I, R, D because we have the collected data zt
I, zt
R, zt
D which
represent the number of infections, recovered, and deaths daily. We assume that such fol-
low hyper-Poisson distribution with parameters. The hyper-Poisson distribution is given Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 98 of 107 ( 2021) 2021:57
Page 98 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 98 of 107 Figure 35 Numerical visualization of COVID-19 model for α = 1
Figure 36 Numerical visualization of COVID-19 model for α = 0.89, β = 0.85
as follows:
P(X
k)
(β)
λ
0 k
0 1 2
(145) Figure 35 Numerical visualization of COVID-19 model for α = 1 Figure 35 Numerical visualization of COVID-19 model for α = 1 Figure 35 Numerical visualization of COVID-19 model for α = 1 Figure 36 Numerical visualization of COVID-19 model for α = 0.89, β = 0.85 Figure 36 Numerical visualization of COVID-19 model for α = 0.89, β = 0.85 Figure 36 Numerical visualization of COVID-19 model for α = 0.89, β = 0.85 Figure 36 Numerical visualization of COVID-19 model for α = 0.89, β = 0.85 as follows: as follows: P(X = k) =
(β)
(k + β)(1,β,λ),
λ > 0,k = 0,1,2,...,n,
(145) (145) where (1,β,λ) =
∞
k=0
(1)kλk
(β)kk! ,
(β)k = β(β + 1)···(β + k)
(146) (1,β,λ) =
∞
k=0
(1)kλk
(β)kk! 11 Likelihood with hyper-Poisson distribution ,
(β)k = β(β + 1)···(β + k (146) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 99 of 107 ( 2021) 2021:57
Page 99 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 99 of 107 Figure 37 Numerical visualization of COVID-19 model for α = 0.95, β = 0.97 Figure 37 Numerical visualization of COVID-19 model for α = 0.95, β = 0.97 Figure 37 Numerical visualization of COVID-19 model for α = 0.95, β = 0.97 ′′ with parameters k1, k2, k3 ′′ with parameters k1, k2, k3 k1 = 1I∗(t),
k2 = 2R∗(t),
k3 = 3D∗(t) k1 = 1I∗(t),
k2 = 2R∗(t),
(147)
k3 = 3D∗(t) (147) and zt
I ∼HP
k1 = 1I∗(t)
,
zt
R ∼HP
k2 = 2R∗(t)
,
(148)
zt
D ∼HP
k3 = 3D∗(t)
. (148) Here, the parameters 1, 2, and 3 are a combination of collection accuracy and de-
tectability of infected, recovered, and dead. Thus the likelihood function is defined as fol-
lows: L(k1) =
n
-
t=0
g
zt
I/k1
,
L(k2) =
n
-
t=0
g
zt
R/k2
,
L(k3) =
n
-
t=0
g
zt
D/k3
. L(k1) =
n
-
t=0
g
zt
I/k1
,
L(k2) =
n
-
t=0
g
zt
R/k2
,
(149)
L(k3) =
n
-
t=0
g
zt
D/k3
. (149) Thus L(k1) =
n
-
t=0
(β)λzt
I
(zt
I + β)(1,β,λ), L(k1) =
n
-
t=0
(β)λzt
I
(zt
I + β)(1,β,λ), Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 100 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 100 of 107 ( 2021) 2021:57 L(k2) =
n
-
t=0
(β)λzt
R
(zt
R + β)(1,β,λ),
(150)
L(k3) =
n
-
t=0
(β)λzt
D
(zt
D + β)(1,β,λ). L(k2) =
n
-
t=0
(β)λzt
R
(zt
R + β)(1,β,λ),
L(k3) =
n
-
t=0
(β)λzt
D
(zt
D + β)(1,β,λ). 11 Likelihood with hyper-Poisson distribution (162) (
β
)
L(k3) =
n
t=0
log
(β)λzt
D
(zt
D + β)(1,β,λ)
=
n
t=0
log(β) + zt
D logλ – log
zt
D + β
– log(1,β,λ)
,
(159)
∂logL(k3)
∂zt
D
=
n
t=0
log(3) +
n
t=0
log
D∗
–
n
t=0
((zt
D + β))′
(zt
D + β) =
n
t=0
log
(β)λzt
D
(zt
D + β)(1,β,λ)
(159) L(k3) =
n
t=0
log
(β)λzt
D
(zt
D + β)(1,β,λ)
n
( )
t
t
(
)
(159) (159) ∂logL(k3)
∂zt
D
=
n
t=0
log(3) +
n
t=0
log
D∗
–
n
t=0
((zt
D + β))′
(zt
D + β)
= n
log(3) + log
D∗
–
n
t=0
((zt
D + β))′
(zt
D + β) = nlog
3D∗
–
n
t=0
((zt
D + β))′
(zt
D + β) , ∂logL(k3)
∂R∗
= nzt
D
D∗′
D∗–
n
t=0
(1,β,3D∗)′
(1,β,3D∗)
= nzt
D
D∗′
D∗– n(1,β,3D∗)′
(1,β,3D∗) ,
∂logL(k3)
∂3
= nzt
D
3′
3
– n(1,β,3D∗)′
(1,β,3D∗)
= –n(1,β,3D∗)′
(1,β,3D∗) . ∂logL(k3)
∂R∗
= nzt
D
D∗′
D∗–
n
t=0
(1,β,3D∗)′
(1,β,3D∗)
= nzt
D
D∗′
D∗– n(1,β,3D∗)′
(1,β,3D∗) ,
(161)
∂logL(k3)
∂3
= nzt
D
3′
3
– n(1,β,3D∗)′
(1,β,3D∗)
= –n(1,β,3D∗)′
(1,β,3D∗) . (162) (161) (162) 11 Likelihood with hyper-Poisson distribution (150) Without loss of generality, we consider L(k1): Without loss of generality, we consider L(k1): logL(k1) =
n
t=0
log
(β)λzt
I
(zt
I + β)(1,β,λ)
=
n
t=0
log(β) + zt
I log
1I∗
– log
zt
I + β
– log
1,β,1I∗
(151) logL(k1) =
n
t=0
log
(β)λzt
I
(zt
I + β)(1,β,λ)
(151) (151) and and ∂logL(k1)
∂zt
I
=
n
t=0
log(1) +
n
t=0
log
I∗
–
n
t=0
((zt
I + β))′
(zt
I + β)
= n
log(1) + log
I∗
–
n
t=0
((zt
I + β))′
(zt
I + β)
= nlog
1I∗
–
n
t=0
((zt
I + β))′
(zt
I + β) ,
(152)
∂logL(k1)
∂I∗
= nzt
I
I∗′
I∗–
n
t=0
(1,β,1I∗)′
(1,β,1I∗)
= nzt
I
I∗′
I∗– n(1,β,1I∗)′
(1,β,1I∗) ,
(153)
∂logL(k1)
∂1
= nzt
I
1′
1
– n(1,β,1I∗)′
(1,β,1I∗)
= –n(1,β,1I∗)′
(1,β,1I∗) ,
(154)
L(k2) =
n
t=0
log
(β)λzt
R
(zt
R + β)(1,β,λ)
=
n
t=0
log(β) + zt
R logλ – log
zt
R + β
– log(1,β,λ)
,
(155)
∂logL(k2)
∂zt
R
=
n
t=0
log(2) +
n
t=0
log
R∗
–
n
t=0
((zt
R + β))′
(zt
R + β)
n
((zt
R + β))′ ∂logL(k1)
∂zt
I
=
n
t=0
log(1) +
n
t=0
log
I∗
–
n
t=0
((zt
I + β))′
(zt
I + β)
= n
log(1) + log
I∗
–
n
t=0
((zt
I + β))′
(zt
I + β)
= nlog
1I∗
–
n
t=0
((zt
I + β))′
(zt
I + β) ,
(152) (152) = nlog
1I∗
–
n
t=0
((zt
I + β))′
(zt
I + β) , t=0
(zI + β)
∂logL(k1)
∂I∗
= nzt
I
I∗′
I∗–
n
t=0
(1,β,1I∗)′
(1,β,1I∗)
= nzt
I
I∗′
I∗– n(1,β,1I∗)′
(1,β,1I∗) ,
(153)
∂logL(k1)
∂1
= nzt
I
1′
1
– n(1,β,1I∗)′
(1,β,1I∗)
= –n(1,β,1I∗)′
(1,β,1I∗) ,
(154)
n
(β)λzt
R ∂logL(k1)
∂I∗
= nzt
I
I∗′
I∗–
n
t=0
(1,β,1I∗)′
(1,β,1I∗)
= nzt
I
I∗′
I∗– n(1,β,1I∗)′
(1,β,1I∗) ,
(153)
∂logL(k1)
∂1
= nzt
I
1′
1
– n(1,β,1I∗)′
(1,β,1I∗)
= –n(1,β,1I∗)′
(1,β,1I∗) ,
(154)
L(k2) =
n
t=0
log
(β)λzt
R
(zt
R + β)(1,β,λ)
=
n
t=0
log(β) + zt
R logλ – log
zt
R + β
– log(1,β,λ)
,
(155)
∂logL(k2)
∂zt
=
n
log(2) +
n
log
R∗
–
n
((zt
R + β))′
(zt + β) ∂logL(k1)
∂I∗
= nzt
I
I∗′
I∗–
n
t=0
(1,β,1I∗)′
(1,β,1I∗)
= nzt
I
I∗′
I∗– n(1,β,1I∗)′
(1,β,1I∗) ,
∂logL(k1)
∂1
= nzt
I
1′
1
– n(1,β,1I∗)′
(1,β,1I∗) (153) (154) L(k2) =
n
t=0
log
(β)λzt
R
(zt
R + β)(1,β,λ)
=
n
t=0
log(β) + zt
R logλ – log
zt
R + β
– log(1,β,λ)
,
(155)
∂logL(k2)
∂zt
R
=
n
t=0
log(2) +
n
t=0
log
R∗
–
n
t=0
((zt
R + β))′
(zt
R + β)
= n
log(2) + log
R∗
–
n
t=0
((zt
R + β))′
(zt
R + β)
= nlog
2R∗
–
n
t=0
((zt
R + β))′
(zt
R + β) ,
(156) (155) (156) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 101 of 107 ( 2021) 2021:57
Page 101 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 101 of 107 ( 2021) 2021:57 raz Advances in Difference Equations ( 2021) 2021:57
P
∂logL(k2)
∂R∗
= nzt
R
R∗′
R∗–
n
t=0
(1,β,2R∗)′
(1,β,2R∗)
= nzt
R
R∗′
R∗– n(1,β,2R∗)′
(1,β,2R∗) ,
(157)
∂logL(k2)
∂2
= nzt
R
2′
2
– n(1,β,2R∗)′
(1,β,2R∗)
= –n(1,β,2R∗)′
(1,β,2R∗) ,
(158)
L(k3) =
n
t=0
log
(β)λzt
D
(zt
D + β)(1,β,λ)
=
n
t=0
log(β) + zt
D logλ – log
zt
D + β
– log(1,β,λ)
,
(159)
∂logL(k3)
∂zt
D
=
n
t=0
log(3) +
n
t=0
log
D∗
–
n
t=0
((zt
D + β))′
(zt
D + β)
= n
log(3) + log
D∗
–
n
t=0
((zt
D + β))′
(zt
D + β)
= nlog
3D∗
–
n
t=0
((zt
D + β))′
(zt
D + β) ,
(160)
∂logL(k3)
∂R∗
= nzt
D
D∗′
D∗–
n
t=0
(1,β,3D∗)′
(1,β,3D∗)
= nzt
D
D∗′
D∗– n(1,β,3D∗)′
(1,β,3D∗) ,
(161)
∂logL(k3)
∂3
= nzt
D
3′
3
– n(1,β,3D∗)′
(1,β,3D∗)
= –n(1,β,3D∗)′
(1,β,3D∗) . 11 Likelihood with hyper-Poisson distribution (162) ∂logL(k2)
∂R∗
= nzt
R
R∗′
R∗–
n
t=0
(1,β,2R∗)′
(1,β,2R∗)
= nzt
R
R∗′
R∗– n(1,β,2R∗)′
(1,β,2R∗) ,
(157)
∂logL(k2)
∂2
= nzt
R
2′
2
– n(1,β,2R∗)′
(1,β,2R∗)
= –n(1,β,2R∗)′
(1,β,2R∗) ,
(158)
L(k3) =
n
t=0
log
(β)λzt
D
(zt
D + β)(1,β,λ)
=
n
t=0
log(β) + zt
D logλ – log
zt
D + β
– log(1,β,λ)
,
(159)
∂logL(k3)
∂zt
D
=
n
t=0
log(3) +
n
t=0
log
D∗
–
n
t=0
((zt
D + β))′
(zt
D + β) ∂logL(k2)
∂R∗
= nzt
R
R∗′
R∗–
n
t=0
(1,β,2R∗)′
(1,β,2R∗)
= nzt
R
R∗′
R∗– n(1,β,2R∗)′
(1,β,2R∗) ,
(157)
∂logL(k2)
∂2
= nzt
R
2′
2
– n(1,β,2R∗)′
(1,β,2R∗)
= –n(1,β,2R∗)′
(1,β,2R∗) ,
(158)
L(k3) =
n
t=0
log
(β)λzt
D
(zt
D + β)(1,β,λ)
(159) ∂logL(k2)
∂R∗
= nzt
R
R∗′
R∗–
n
t=0
(1,β,2R∗)′
(1,β,2R∗)
= nzt
R
R∗′
R∗– n(1,β,2R∗)′
(1,β,2R∗) ,
(157)
∂logL(k2)
∂2
= nzt
R
2′
2
– n(1,β,2R∗)′
(1,β,2R∗)
(
(158) (157) gL(k2)
2
= nzt
R
2′
2
– n(1,β,2R∗)′
(1,β,2R∗)
(
β R∗)
(158) (158) (1,β,2R )
L(k3) =
n
t=0
log
(β)λzt
D
(zt
D + β)(1,β,λ)
=
n
t=0
log(β) + zt
D logλ – log
zt
D + β
– log(1,β,λ)
,
(159)
∂logL(k3)
∂zt
D
=
n
t=0
log(3) +
n
t=0
log
D∗
–
n
t=0
((zt
D + β))′
(zt
D + β)
= n
log(3) + log
D∗
–
n
t=0
((zt
D + β))′
(zt
D + β)
= nlog
3D∗
–
n
t=0
((zt
D + β))′
(zt
D + β) ,
(160)
∂logL(k3)
∂R∗
= nzt
D
D∗′
D∗–
n
t=0
(1,β,3D∗)′
(1,β,3D∗)
= nzt
D
D∗′
D∗– n(1,β,3D∗)′
(1,β,3D∗) ,
(161)
∂logL(k3)
∂3
= nzt
D
3′
3
– n(1,β,3D∗)′
(1,β,3D∗)
= –n(1,β,3D∗)′
(1,β,3D∗) . 12 Likelihood with Weibull distribution We will do the same routine for the Weibull distribution known as We will do the same routine for the Weibull distribution known as P(X = k) = k
α
% λ
α
&k–1
exp(–λ/α)k,
λ,α > 0,k = 0,1,2,...,n,
(163) (163) with parameters k1, k2, k3 k1 = 1I∗(t),
k2 = 2R∗(t),
(164)
k3 = 3D∗(t) k1 = 1I∗(t),
k2 = 2R∗(t),
k3 = 3D∗(t) (164) and εt
I ∼W
k1 = 1I∗(t)
, εt
I ∼W
k1 = 1I∗(t)
, Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 102 of 107 Page 102 of 107 ( 2021) 2021:57 (165) εt
R ∼W
k2 = 2R∗(t)
,
εt
D ∼W
k3 = 3D∗(t)
. εt
R ∼W
k2 = 2R∗(t)
,
εt
D ∼W
k3 = 3D∗(t)
. Thus the likelihood function is given by Thus the likelihood function is given by Thus the likelihood function is given by L(k1) =
n
-
t=0
W
εt
I/k1
,
L(k2) =
n
-
t=0
W
εt
R/k2
,
(166)
L(k3) =
n
-
t=0
W
εt
D/k3
. L(k1) =
n
-
t=0
W
εt
I/k1
,
L(k2) =
n
-
t=0
W
εt
R/k2
,
L(k3) =
n
-
t=0
W
εt
D/k3
. (166) Thus L(k1) =
n
-
t=0
εt
I
α
% λ
α
&εt
I–1
exp(–λ/α)εt
I,
L(k2) =
n
-
t=0
εt
R
α
% λ
α
&εt
R–1
exp(–λ/α)εt
R,
(167)
L(k3) =
n
-
t=0
εt
D
α
% λ
α
&εt
D–1
exp(–λ/α)εt
D. 12 Likelihood with Weibull distribution (167) Without loss of generality, we consider L(k1): Without loss of generality, we consider L(k1): Without loss of generality, we consider L(k1): logL(k1) =
n
t=0
log
εt
I
α
%1I∗
α
&εt
I–1
exp
–1I∗/α
εt
I
=
logεt
I – logα +
εt
I – 1
log
1I∗
– logα
– εt
I
–1I∗/α
(168) logL(k1) =
n
t=0
log
εt
I
α
%1I∗
α
&εt
I–1
exp
–1I∗/α
εt
I
(168) and and ∂logL(k1)
∂εt
I
=
n
t=0
εt
I′
εt
I
+
n
t=0
log
1I∗
– logα
–
n
t=0
–1I∗/α
= nεt
I′
εt
I
+ n
log
1I∗
– logα
+ n
1I∗/α
,
(169) ∂logL(k1)
∂εt
I
=
n
t=0
εt
I′
εt
I
+
n
t=0
log
1I∗
– logα
–
n
t=0
–1I∗/α
= nεt
I′
εt
I
+ n
log
1I∗
– logα
+ n
1I∗/α
,
(169) = nεt
I′
εt
I
+ n
log
1I∗
– logα
+ n
1I∗/α
,
(169) (169) ∂logL(k1)
∂I∗
= n
εt
I – 1
I∗′
I∗–
n
t=0
(–1I∗/α)′
(–1I∗/α)
= n
εt
I – 1
I∗′
I∗– n(–1I∗/α)′
(–1I∗/α) ,
(170)
∂logL(k1)
∂1
= n
εt
I – 1
1′
1
– n(–1I∗/α)′
(–1I∗/α)
= –n(–1I∗/α)′
(–1I∗/α) ,
(171) ∂logL(k1)
∂I∗
= n
εt
I – 1
I∗′
I∗–
n
t=0
(–1I∗/α)′
(–1I∗/α)
= n
εt
I – 1
I∗′
I∗– n(–1I∗/α)′
(–1I∗/α) ,
∂logL(k1)
∂1
= n
εt
I – 1
1′
1
– n(–1I∗/α)′
(–1I∗/α)
= –n(–1I∗/α)′
(–1I∗/α) , (171) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 103 of 107 nce Equations ( 2021) 2021:57
Page 103 of 107 angana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57
Page 103 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Page 103 of 107 logL(k2) =
n
t=0
log
εt
R
α
%2I∗
α
&εt
R–1
exp
–2R∗/α
εt
I
logL(k2) =
n
t=0
log
εt
R
α
%2I∗
α
&εt
R–1
exp
–2R∗/α
εt
I
=
logεt
R – logα +
εt
R – 1
log
2R∗
– logα
– εt
R
–2R∗/α
(172) =
logεt
R – logα +
εt
R – 1
log
2R∗
– logα
– εt
R
–2R∗/α
(172) and and ∂logL(k2)
∂εt
R
=
n
t=0
εt
R′
εt
R
+
n
t=0
log
2R∗
– logα
–
n
t=0
–2R∗/α
= nεt
R′
εt
R
+ n
log
2R∗
– logα
+ n
–2R∗/α
, = nεt
R′
εt
R
+ n
log
2R∗
– logα
+ n
–2R∗/α
,
(173) (173) ∂logL(k2)
∂R∗
= n
εt
R – 1
R∗′
R∗–
n
t=0
(–2R∗/α)′
(–2R∗/α)
= n
εt
R – 1
R∗′
R∗– n(–2R∗/α)′
(–2R∗/α) ,
∂logL(k2)
∂2
= n
εt
R – 1
2′
2
– n(–2R∗/α)′
(–2R∗/α)
= –n(–2R∗/α)′
(–2R∗/α) , ∂logL(k2)
∂R∗
= n
εt
R – 1
R∗′
R∗–
n
t=0
(–2R∗/α)′
(–2R∗/α)
= n
εt
R – 1
R∗′
R∗– n(–2R∗/α)′
(–2R∗/α) ,
(174)
∂logL(k2)
∂2
= n
εt
R – 1
2′
2
– n(–2R∗/α)′
(–2R∗/α)
= –n(–2R∗/α)′
(–2R∗/α) ,
(175) = n
εt
R – 1
R ′
R∗– n( 2R /α)′
(–2R∗/α) ,
∂logL(k2)
∂2
= n
εt
R – 1
2′
2
– n(–2R∗/α)′
(–2R∗/α)
= –n(–2R∗/α)′
(–2R∗/α) ,
(175) (175) logL(k3) =
n
t=0
log
εt
D
α
%3D∗
α
&εt
D–1
exp
–3D∗/α
εt
D
(176) logL(k3) =
n
t=0
log
εt
D
α
%3D∗
α
&εt
D–1
exp
–3D∗/α
εt
D
=
logεt
D – logα +
εt
D – 1
log
3D∗
– logα
– εt
D
–3D∗/α
(176) and and ∂logL(k3)
∂εt
D
=
n
t=0
εt
D′
εt
D
+
n
t=0
log
3D∗
– logα
–
n
t=0
–3D∗/α
= nεt
D′
εt
D
+ n
log
3R∗
– logα
+ n
–3D∗/α
,
(177) (177) ∂logL(k3)
∂D∗
= n
εt
D – 1
D∗′
D∗–
n
t=0
(–3D∗/α)′
(–3D∗/α)
= n
εt
I – 1
D∗′
D∗– n(–3D∗/α)′
(–3D∗/α) ,
(178)
∂logL(k1)
∂1
= n
εt
D – 1
3′
3
– n(–3D∗/α)′
(–3D∗/α)
= –n(–3D∗/α)′
(–3D∗/α) . 12 Likelihood with Weibull distribution (179) ∂logL(k3)
∂D∗
= n
εt
D – 1
D∗′
D∗–
n
t=0
(–3D∗/α)′
(–3D∗/α)
= n
εt
I – 1
D∗′
D∗– n(–3D∗/α)′
(–3D∗/α) ,
∂logL(k1)
∂1
= n
εt
D – 1
3′
3
– n(–3D∗/α)′
(–3D∗/α)
= –n(–3D∗/α)′
(–3D∗/α) . (179) 13 Likelihood with Mittag-Leffler distribution
Finally, we shall use the Mittag-Leffler distribution for similar processes. The Mittag-
Leffler distribution is defined by 13 Likelihood with Mittag-Leffler distribution (185) We write L(k1): We write L(k1): logL(k1) = log
λεt
I
(αεt
I + β)Eα,β(λ)
(186)
=
n
t 0
εt
I log
1I∗
– log
αεt
I + β
– logEα,β
1I∗ logL(k1) = log
λεt
I
(αεt
I + β)Eα,β(λ) logL(k1) = log
λεt
I
(αεt
I + β)Eα,β(λ)
(186) (186) =
n
t=0
εt
I log
1I∗
– log
αεt
I + β
– logEα,β
1I∗ =
n
t=0
εt
I log
1I∗
– log
αεt
I + β
– logEα,β
1I∗ ( 2021) 2021:57
Page 105 of 107 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 105 of 107 ( 2021) 2021:57 and
∂logL(k1)
∂εt
I
=
n
t=0
log(1) +
n
t=0
log
I∗
–
n
t=0
((αεt
I + β))′
(αεt
I + β)
= n
log(1) + log
I∗
–
n
t=0
((αεt
I + β))′
(αεt
I + β)
= nlog
1I∗
–
n
t=0
((αεt
I + β))′
(αεt
I + β) ,
∂logL(k1)
∂I∗
= nεt
I
I∗′
I∗–
n
t=0
Eα,β(1I∗)′
Eα,β(1I∗)
= nεt
I
I∗′
I∗– nEα,β(1I∗)′
Eα,β(1I∗) ,
∂logL(k1)
∂1
= nεt
I
1′
1
– nEα,β(1I∗)′
Eα,β(1I∗)
= –nEα,β(1I∗)′
Eα,β(1I∗) . and ∂logL(k1)
∂εt
I
=
n
t=0
log(1) +
n
t=0
log
I∗
–
n
t=0
((αεt
I + β))′
(αεt
I + β)
= n
log(1) + log
I∗
–
n
t=0
((αεt
I + β))′
(αεt
I + β)
= nlog
1I∗
–
n
t=0
((αεt
I + β))′
(αεt
I + β) , (187) ∂logL(k1)
∂I∗
= nεt
I
I∗′
I∗–
n
t=0
Eα,β(1I∗)′
Eα,β(1I∗)
= nεt
I
I∗′
I∗– nEα,β(1I∗)′
Eα,β(1I∗) ,
∂logL(k1)
∂1
= nεt
I
1′
1
– nEα,β(1I∗)′
Eα,β(1I∗)
= –nEα,β(1I∗)′
Eα,β(1I∗) . 13 Likelihood with Mittag-Leffler distribution Finally, we shall use the Mittag-Leffler distribution for similar processes. The Mittag-
Leffler distribution is defined by P(X = k) =
λk
(αk + β)Eα,β(λ),
λ > 0,k = 0,1,2,...,n,
(180) (180) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Page 104 of 107 ( 2021) 2021:57 where Eα,β(λ) =
∞
k=0
λk
(αk + β). (181) i, i = 1,2,3 with parameters k1, k2, k3 i, i = 1,2,3 with parameters k1, k2, k3 k1 = 1I∗(t),
k2 = 2R∗(t),
k3 = 3D∗(t)
and (182) and εt
I ∼ML
k1 = 1I∗(t)
,
εt
R ∼ML
k2 = 2R∗(t)
,
εt
D ∼ML
k3 = 3D∗(t)
. (183) Thus the likelihood function is written as L(k1) =
n
-
t=0
ML
εt
I/k1
,
L(k2) =
n
-
t=0
ML
εt
R/k2
,
L(k3) =
n
-
t=0
ML
εt
D/k3
. (184) Thus L(k1) =
n
-
t=0
λεt
I
(αεt
I + β)Eα,β(λ),
L(k2) =
n
-
t=0
λεt
R
(αεt
R + β)Eα,β(λ),
L(k3) =
n
-
t=0
λεt
D
(αεt
D + β)Eα,β(λ). t=0
(
I
β)
α,β( )
L(k2) =
n
-
t=0
λεt
R
(αεt
R + β)Eα,β(λ),
(185)
L(k3) =
n
-
t=0
λεt
D
(αεt
D + β)Eα,β(λ). 13 Likelihood with Mittag-Leffler distribution (197) ∂logL(k3)
∂D∗
= nεt
D
D∗′
D∗–
n
t=0
Eα,β(3D∗)′
Eα,β(3D∗)
= nεt
D
D∗′
D∗– nEα,β(3D∗)′
Eα,β(3D∗) ,
∂logL(k3)
∂3
= nεt
D
1′
1
– nEα,β(3D∗)′
Eα,β(3D∗)
= –nEα,β(3D∗)′
Eα,β(3D∗) . (196) (197) 13 Likelihood with Mittag-Leffler distribution (188) (189) With the same routine, With the same routine, logL(k2) =
n
t=0
log
λεt
R
(αεt
I + β)Eα,β(λ)
(190)
=
n
t=0
εt
R log
1R∗
– log
αεt
R + β
– logEα,β
2R∗ (190) and ∂logL(k2)
∂εt
R
=
n
t=0
log(2) +
n
t=0
log
R∗
–
n
t=0
((αεt
R + β))′
(αεt
R + β)
= n
log(2) + log
R∗
–
n
t=0
((αεt
R + β))′
(αεt
R + β)
= nlog
2R∗
–
n
t=0
((αεt
R + β))′
(αεt
R + β) ,
(191)
∂logL(k2)
∂R∗
= nεt
R
R∗′
R∗–
n
t=0
Eα,β(2R∗)′
Eα,β(2R∗)
= nεt
I
R∗′
R∗– nEα,β(2R∗)′
Eα,β(2R∗) ,
(192)
∂logL(k2)
∂2
= nεt
R
2′
2
– nEα,β(2R∗)′
Eα,β(2R∗)
= –nEα,β(2R∗)′
Eα,β(2R∗)
(193) ∂logL(k2)
∂εt
R
=
n
t=0
log(2) +
n
t=0
log
R∗
–
n
t=0
((αεt
R + β))′
(αεt
R + β)
= n
log(2) + log
R∗
–
n
t=0
((αεt
R + β))′
(αεt
R + β)
= nlog
2R∗
–
n
t=0
((αεt
R + β))′
(αεt
R + β) ,
(191) (191) = nlog
2R∗
–
n
t=0
((αεt
R + β))′
(αεt
R + β) , ∂logL(k2)
∂R∗
= nεt
R
R∗′
R∗–
n
t=0
Eα,β(2R∗)′
Eα,β(2R∗)
= nεt
I
R∗′
R∗– nEα,β(2R∗)′
Eα,β(2R∗) ,
∂logL(k2)
∂2
= nεt
R
2′
2
– nEα,β(2R∗)′
Eα,β(2R∗) (192) (193) Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 2021:57 ( 2021) 2021:57 Page 106 of 107 and
logL(k3) =
n
t=0
log
λεt
D
(αεt
D + β)Eα,β(λ)
(194)
n and and
logL(k3) =
n
t=0
log
λεt
D
(αεt
D + β)Eα,β(λ)
(194)
=
n
t=0
εt
D log
3D∗
– log
αεt
D + β
– logEα,β
3D∗ and logL(k3) =
n
t=0
log
λεt
D
(αεt
D + β)Eα,β(λ)
(194) (194) =
t=0
εt
D log
3D∗
– log
αεt
D + β
– logEα,β
3D∗ and and ∂logL(k1)
∂εt
I
=
n
t=0
log(3) +
n
t=0
log
D∗
–
n
t=0
((αεt
D + β))′
(αεt
D + β)
= n
log(3) + log
D∗
–
n
t=0
((αεt
D + β))′
(αεt
D + β)
= nlog
3D∗
–
n
t=0
((αεt
D + β))′
(αεt
D + β) ,
(195) = n
log(3) + log
D∗
–
n
t=0
((αεt
D + β))′
(αεt
D + β)
(195) (195) = nlog
3D∗
–
n
t=0
((αεt
D + β))′
(αεt
D + β) , ∂logL(k3)
∂D∗
= nεt
D
D∗′
D∗–
n
t=0
Eα,β(3D∗)′
Eα,β(3D∗)
= nεt
D
D∗′
D∗– nEα,β(3D∗)′
Eα,β(3D∗) ,
(196)
∂logL(k3)
∂3
= nεt
D
1′
1
– nEα,β(3D∗)′
Eα,β(3D∗)
= –nEα,β(3D∗)′
Eα,β(3D∗) . 14 Conclusion Up to date humans have relied on forecasting with the aim to better control their world,
or at least to have an asymptotic idea of their future. They have many ways to achieve this,
one way is to use the deterministic approach and another is stochastic one. In this work,
we presented a comprehensive analysis ranging from stochastic, fractal to differentiation
with the aim to predict the future behavior of COVID-19 with cases studied in Africa and
Europe. With stochastic approach, we were able to detect a possibility of the second wave
of COVID-19 spread in Europe and in Africa, a continuous exponential growth could be
possible. We presented an extension of the blancmange function to capture more fractal
behaviors, and some examples were presented resembling the COVID-19 spread in vari-
ous countries in Africa and Europe. A complex and nonlinear mathematical model with
wave function was considered and solved numerically with a modified scheme. Availability of data and materials
There are no data for this paper. Competing interests
The authors declare that they have no competing interests. Acknowledgements Acknowledgements
The authors of this paper would like to thank the referees for their valuable suggestions and comments. Funding
There is no funding for this paper. Author details 1Institute for Groundwater Studies, Faculty of Natural and Agricultural Sciences, University of the Free State, Bloemfontein,
South Africa. 2Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, Institute for Groundwater Studies, Faculty of Natural and Agricultural Sciences, University of the Free State, Bloemfontein,
South Africa. 2Department of Medical Research, China Medical University Hospital, China Medical University, Taichung,
Taiwan. 3Department of Mathematic Education, Faculty of Education, Siirt University, Siirt 56100, Turkey. South Africa. Department of Medical Research, China Medical University Hospital, China Medical University, Taichung,
Taiwan. 3Department of Mathematic Education, Faculty of Education, Siirt University, Siirt 56100, Turkey. Taiwan. 3Department of Mathematic Education, Faculty of Education, Siirt University, Siirt 56100, Turkey. Authors’ contributions Authors contributions
All authors have contributed equally in this work. All authors read and approved the final manuscript. Received: 5 November 2020 Accepted: 4 January 2021 Received: 5 November 2020 Accepted: 4 January 2021 Competing interests
h
h
d
l
h Page 107 of 107 ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations ( 2021) 202 ( 2021) 2021:57 Atangana and ˙I˘gret Araz Advances in Difference Equations References 1. WHO: Coronavirus disease (Covid-2019) situation reports. https //
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https://openalex.org/W4392387958
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https://link.springer.com/content/pdf/10.1007/s11467-023-1355-6.pdf
|
English
| null |
Twistronics and moiré excitonic physics in van der Waals heterostructures
|
Frontiers of Physics
| 2,024
|
cc-by
| 38,985
|
Siwei Li (李思维)1, Ke Wei (韦可)2,†, Qirui Liu (刘祺瑞)1, Yuxiang Tang (唐宇翔)2, Tian Jiang (江天)2,‡ 1 College of Advanced Interdisciplinary Studies, National University of Defense Technology, Changsha 410073, China
2 Institute for Quantum Science and Technology, National University of Defense Technology, Changsha 410073, China
Corresponding authors. E-mail: †weikeaep@163.com, ‡tjiang@nudt.edu.cn
Received August 20, 2023; accepted October 11, 2023
© Th © The Authors 2024 © The Authors 2024 T O P I C A L R E V I E W
Volume 19 / Issue 4 / 42501 / 2024 T O P I C A L R E V I E W
Volume 19 / Issue 4 / 42501 / 2024 T O P I C A L R E V I E W
Volume 19 / Issue 4 / 42501 / 2024 Twistronics and moiré excitonic physics in
van der Waals heterostructures Siwei Li (李思维)1, Ke Wei (韦可)2,†, Qirui Liu (刘祺瑞)1, Yuxiang Tang (唐宇翔)2, Ti 1 Introduction 2 Twist-angle-tunable optoelectronic properties in
van der Waals heterostructure
2.1 Exciton properties controlled by twist-angle-
dependent interlayer interactions
2.2 Electronic band structures and relative
twist in momentum space
2.3 Twist-angle-dependent exciton dynamics
3 Moiré excitonic physics
3.1 Theoretical predictions and experimental
observations of new excitons in moiré
structure
3.2 Localization and modulation of excitons by
moiré superlattice
3.3 Spin-valley configuration of moiré excitons
3.4 Moiré system under external tunning
4 Correlated electronic states in moiré superlattice
5 Conclusions and outlook
Declarations
Acknowledgements
References 2 Twist-angle-tunable optoelectronic properties in
van der Waals heterostructure
2.1 Exciton properties controlled by twist-angle-
dependent interlayer interactions
2.2 Electronic band structures and relative
twist in momentum space
2.3 Twist-angle-dependent exciton dynamics
3 Moiré excitonic physics
3.1 Theoretical predictions and experimental
observations of new excitons in moiré
structure
3.2 Localization and modulation of excitons by
moiré superlattice
3.3 Spin-valley configuration of moiré excitons
3.4 Moiré system under external tunning
4 Correlated electronic states in moiré superlattice
5 Conclusions and outlook
Declarations
Acknowledgements
References ABSTRACT FRONTIERS OF PHYSICS FRONTIERS OF PHYSICS TOPICAL REVIEW 2
3
3
6
8
12
13
17
20
22
26
30
31
31
31
2],
oron
tion
used
rties
ance
dW
ally
well
y of
late
–26],
tical
ment
ntly
vertical heterostructures or homostructures, leading to
enriched physical properties with high flexibility and
tunability beyond the simple combination of the
constituent monolayers [45, 46]. Consequently, 2D vdW
heterostructures offer an excellent platform for exploring
modern solid-state physics, such as Bose–Einstein
condensation (BEC) [47] and fractional quantum Hall
effect [48]. The high adjustability of vdW heterostructures
allows customized properties for the new generation of
electronic and optoelectronic applications including
quantum emitters [49] and excitonic devices [26, 50, 51]. The atomic alignment between the constituent layers
of vdW heterostructure changes with interlayer lattice
constant mismatch and relative orientation angle. Thus,
the twist angle allows continuous modulation of strain
distribution, interlayer coupling, electronic band structure
and excitonic dynamics due to the incommensurate
stacking [38, 52, 53], which prompts a new topic
“twistronics” [54] that emphasizes the interplay between
lattice rotational mismatch and interlayer electronic
coupling. When the incommensurate heterostructures
are stacked with a small twist angle or lattice mismatch,
the atomic registries will change spatially with large-
scale periodicity, forming so-called moiré patterns [49,
55]. Such moiré patterns can be directly observed via
nanoscale techniques [56–58] and attract abundant
research attention due to the fascinating moiré effects in
theoretical predictions. The superlattice structure of
moiré pattern can generate strongly correlated phases,
such as insulating states [59] and unconventional super-
conductivities [60] in magic-angle graphene bilayer, and
Hofstadter’s butterfly pattern in graphene/hBN twisted
heterostructure
[48,
61]. Recently,
TMD
vdW
heterostructures with moiré superlattice show novel exci-
tonic and optical physics including excitons localized by
periodic moiré potentials [62, 63], moiré exciton minibands
[64, 65] and spatially modulated optical selection rules
[49, 66, 67]. Furthermore, as a highly tunable system,
moiré superlattice in vdW heterostructures can be feasibly
combined with external factors, which will promote the
exploration of frontier physics, including new spin opto-
electronics, many-body interactions and potential appli-
cations in quantum information processing [49]. H
f
th i t
l
t i t
difi d l
t
i ABSTRACT Heterostructures composed of two-dimensional van der Waals (vdW) materials allow highly controllable stacking, where interlayer
twist angles introduce a continuous degree of freedom to alter the electronic band structures and excitonic physics. Motivated by the
discovery of Mott insulating states and superconductivity in magic-angle bilayer graphene, the emerging research fields of “twistron-
ics” and moiré physics have aroused great academic interests in the engineering of optoelectronic properties and the exploration of
new quantum phenomena, in which moiré superlattice provides a pathway for the realization of artificial excitonic crystals. Here we
systematically summarize the current achievements in twistronics and moiré excitonic physics, with emphasis on the roles of lattice
rotational mismatches and atomic registries. Firstly, we review the effects of the interlayer twist on electronic and photonic physics,
particularly on exciton properties such as dipole moment and spin-valley polarization, through interlayer interactions and electronic
band structures. We also discuss the exciton dynamics in vdW heterostructures with different twist angles, like formation, transport
and relaxation processes, whose mechanisms are complicated and still need further investigations. Subsequently, we review the
theoretical analysis and experimental observations of moiré superlattice and moiré modulated excitons. Various exotic moiré effects
are also shown, including periodic potential, moiré miniband, and varying wave function symmetry, which result in exciton localiza-
tion, emergent exciton peaks and spatially alternating optical selection rule. We further introduce the expanded properties of moiré
systems with external modulation factors such as electric field, doping and strain, showing that moiré lattice is a promising platform
with high tunability for optoelectronic applications and in-depth study on frontier physics. Lastly, we focus on the rapidly developing
field of correlated electron physics based on the moiré system, which is potentially related to the emerging quantum phenomena. Keywords moiré superlattice, twistronics, van der Waals heterostructure, moiré exciton, correlated electronic state Keywords moiré superlattice, twistronics, van der Waals heterostructure, moiré exciton *Special Topic: Photophysics in 2D Materials, Heterostructures
and Moiré Superlattices (Eds.: Qihua Xiong, Tian Jiang, Luyi
Yang & Haiming Zhu). https://doi.org/10.1007/s11467-023-1355-6 *Special Topic: Photophysics in 2D Materials, Heterostructures
and Moiré Superlattices (Eds.: Qihua Xiong, Tian Jiang, Luyi
Yang & Haiming Zhu). Contents Consequently, 2D vdW
heterostructures offer an excellent platform for exploring
modern solid-state physics, such as Bose–Einstein
condensation (BEC) [47] and fractional quantum Hall
effect [48]. The high adjustability of vdW heterostructures
allows customized properties for the new generation of
electronic and optoelectronic applications including
quantum emitters [49] and excitonic devices [26, 50, 51]. 1 Introduction Contents 2
3
3
6
8
12
13
17
20
22
26
30
31
31
31
2],
oron
tion
used
ties
ance
dW
ally
well
y of
late
26],
ical
ment
ntly
tric
rise
vertical heterostructures or homostructures, leading to
enriched physical properties with high flexibility and
tunability beyond the simple combination of the
constituent monolayers [45, 46]. Consequently, 2D vdW
heterostructures offer an excellent platform for exploring
modern solid-state physics, such as Bose–Einstein
condensation (BEC) [47] and fractional quantum Hall
effect [48]. The high adjustability of vdW heterostructures
allows customized properties for the new generation of
electronic and optoelectronic applications including
quantum emitters [49] and excitonic devices [26, 50, 51]. The atomic alignment between the constituent layers
of vdW heterostructure changes with interlayer lattice
constant mismatch and relative orientation angle. Thus,
the twist angle allows continuous modulation of strain
distribution, interlayer coupling, electronic band structure
and excitonic dynamics due to the incommensurate
stacking [38, 52, 53], which prompts a new topic
“twistronics” [54] that emphasizes the interplay between
lattice rotational mismatch and interlayer electronic
coupling. When the incommensurate heterostructures
are stacked with a small twist angle or lattice mismatch,
the atomic registries will change spatially with large-
scale periodicity, forming so-called moiré patterns [49,
55]. Such moiré patterns can be directly observed via
nanoscale techniques [56–58] and attract abundant
research attention due to the fascinating moiré effects in
theoretical predictions. The superlattice structure of
moiré pattern can generate strongly correlated phases,
such as insulating states [59] and unconventional super-
conductivities [60] in magic-angle graphene bilayer, and
Hofstadter’s butterfly pattern in graphene/hBN twisted
heterostructure
[48,
61]. Recently,
TMD
vdW
heterostructures with moiré superlattice show novel exci-
tonic and optical physics including excitons localized by
periodic moiré potentials [62, 63], moiré exciton minibands
[64, 65] and spatially modulated optical selection rules
[49, 66, 67]. Furthermore, as a highly tunable system,
moiré superlattice in vdW heterostructures can be feasibly
combined with external factors, which will promote the
exploration of frontier physics, including new spin opto-
electronics, many-body interactions and potential appli-
cations in quantum information processing [49]. Here, we focus on the interlayer-twist-modified electronic
and optical physics in semiconducting vdW heterostruc-
tures and the burgeoning area of moiré superlattice. 2
3
3
6
8
vertical heterostructures or homostructures, leading to
enriched physical properties with high flexibility and
tunability beyond the simple combination of the
constituent monolayers [45, 46]. Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 1 Introduction Two-dimensional (2D) vdW layered materials [1, 2],
including graphene [3, 4], silicene [5], hexagonal boron
nitride (hBN) [6, 7], phosphorene [8, 9], and transition
metal dichalcogenides (TMDs) [10–12], have aroused
wide attention in recent years due to their exotic properties
[11, 13] and promising applications in high-performance
devices [9, 14–20]. The relatively weak interlayer vdW
force enables vdW materials to be exfoliated into atomically
thin layers with outstanding physical properties as well
as highly adjustable 2D structures [12]. A variety of
methods [21, 22] have been shown to efficiently regulate
material properties, such as external field [23–26],
doping [26–28], strain [29–33], integration with optical
microcavity [34], and changing dielectric environment
[35–37]. In semiconducting 2D materials, the significantly
enhanced Coulomb interactions due to reduced dielectric
screening and increased quantum confinement give rise
to rich excitonic species, which are formed by tightly
bound electron and hole pairs and could persist up to
room
temperature,
showing
strong
light–matter
couplings and many-body effects [38]. Specifically,
TMDs possess fascinating optical and electronic proper-
ties, embracing widely tunable direct bandgap [39], large
oscillator strength [40], substantial exciton binding
energy [41], and strong spin–orbit coupling that endows
TMDs with spin-valley contrasting physics [42–44]. More
importantly, different layered materials can be stacked
together through vdW interactions to form various [
]
Here, we focus on the interlayer-twist-modified electronic
and optical physics in semiconducting vdW heterostruc-
tures and the burgeoning area of moiré superlattice. This review is organized as follows. In Section 2, we first
summarize the optoelectronic properties affected by
interlayer twist in vdW heterostructures, including the
exciton properties, the electronic band structures and
the exciton dynamics. In Section 3, moiré pattern and
the emergent superlattice effects in vdW heterostructures
are systematically explained. Specifically, we discuss the
theoretical analysis and experimental observations of
diverse moiré excitons, as well as how the properties of
these excitons (localization and spin-valley configuration)
are modulated by moiré superlattice. On this basis, the Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-2 42501-2 TOPICAL REVIEW FRONTIERS OF PHYSICS Fig. 1 Schematic diagrams of five typical aligned stacking patterns of bilayer MoS2. (a) Side view and (b) Top view of
stacking orders with two different kinds of labels, where A–A, A–A′, A′–B, A–B′ and A–B correspond to 3R-like, 2H, 2H-like
(Mo), 2H-like (S) and 3R, respectively. One pair of S atoms is denoted by one yellow circle as a concise representation. 1 Introduction (a) Reproduced with permission from Ref. [69]. (b) Reproduced with permission from Ref. [77]. Fig. 1 Schematic diagrams of five typical aligned stacking patterns of bilayer MoS2. (a) Side view and (b) Top view of
stacking orders with two different kinds of labels, where A–A, A–A′, A′–B, A–B′ and A–B correspond to 3R-like, 2H, 2H-like
(Mo), 2H-like (S) and 3R, respectively. One pair of S atoms is denoted by one yellow circle as a concise representation. (a) Reproduced with permission from Ref. [69]. (b) Reproduced with permission from Ref. [77]. relative positions of atoms between layers. In 2H and 3R
patterns [see Fig. 1(b)], Mo and S atoms with opposite
charges from different layers are stacked together,
resulting in the strongest interlayer coupling. On the
contrary, if atoms with the same charge in different
layers are stacked together, the Coulomb repulsion
between them increases the interlayer distance and
reduces the coupling strength, among which the 3R-like
pattern exhibits the weakest coupling due to most of the
S atoms overlapped [77]. Various theoretical calculations
have reported the effects of stacking patterns on interlayer
interactions. Early studies mainly focused on the effect
of the interlayer distance on coupling strength under
different atomic arrangements [68, 69], which can induce
tunable optical responses such as Raman [75, 78, 87] and
photoluminescence (PL) [51, 77, 88] spectroscopy. Liu et
al. [69] evaluated the geometries and stabilities of bilayer
MoS2 with five typical stacking patterns using density
functional theory (DFT) calculations based on the local
density approximation (LDA) function. They found that
A–B and A–A′ in Fig. 1(a) had shorter interlayer
distances, lower relative energies and higher binding
energies compared with the other patterns, as these two
patterns exhibited the weakest interlayer repulsive inter-
action. Using random phase approximation (RPA), He
et al. [68] studied the binding energy and interlayer
spacing of TMD bilayers under different stacking
patterns. They found that high symmetry A–A′ (bulklike
2H symmetry) was the most stable type with short
interlayer distance, while the A–B stacking (3R type)
followed closely behind, ~5 meV/formula unit less stable. The stacking order also affects the valence band splitting
and the relative energy of the K point to Г point, where
the exciton binding energy and corresponding optical
response are influenced. In the case of incommensurate influence of external conditions such as electric field,
doping, and strain on the moiré system is further
discussed. 1 Introduction In Section 4, we introduce the recently emerging
field of correlated electronic states reported in TMDs-
based moiré superlattices that possess several advantages
compared with graphene-based twisted bilayers. The
conclusions and future outlooks are summarized in
Section 5. 2 Twist-angle-tunable optoelectronic
properties in van der Waals
heterostructure Early experimental observations on vdW heterostructures
have revealed that changing relative orientation angle or
lattice mismatch could extend the optical and electronic
properties of vdW bilayers. Most of these works focused
on bilayers stacked in high-symmetry patterns (Fig. 1),
including A–A (point group D3h), A–A′ (point group
D3d), A′ –B (point group D3d), A–B (point group C3v)
and A–B′ (point group D 3d) [68]. Then the interlayer
twist of bilayer structures is employed to realize continuous
tunning, of which excitonic physics is the most
concerned [22, 41, 65]. It is theoretically predicted that
relative twist between constituent layers can affect the
excitonic phenomena and exciton dynamics through
interlayer couplings [52] and electronic band structures
[46, 68, 69], which has been confirmed and further
explored in subsequent experiments [45, 51, 70–86]. FRONTIERS OF PHYSICS [70] found that the states at valence band
edges were affected by the twist angle, which exhibited a
long tail stretching over another layer in coherently
stacked MoSe2/WSe2, but localized in an individual
layer in incoherent case, as shown in Fig. 2(g). The
lattice mismatch controlled by twist angle would also
significantly affect the properties of devices based on
vdW heterostructures. Choi et al. [71] fabricated
p-WSe2/n-MoSe2 junctions with θ = 0°, 15° and 30°, and
measured their I–V curves under illumination with
different wavelengths. In the aligned case (θ = 0°) where
strong coupling is acquired, the sample showed a diodelike
behavior under dark condition, functioning as a well-
established p–n junction and displaying higher sensitivity
under IR illumination. Whereas in the cases of lattice
mismatch (θ = 15°, 30°), interlayer potential barrier was
built due to the vdW gap under weak coupling, and
such barrier could be overcome under ultraviolet (UV)
illumination, allowing photocurrent to flow in both
forward and reverse directions. Apart from rotation
mismatch, lattice relaxation and strain distribution of interlayer distance determined by the stacking pattern. The stacking-dependent interlayer coupling is usually
probed by Raman and PL spectra, which indicate the
lattice vibration modes and optical transitions of the
bilayer structures, respectively. Homojunctions of bilayer
TMD with varying twist angles show remarkable modu-
lations of optical transition energies and Raman modes
[75]. In stacked MoS2 bilayer with CVD-grown monolay-
ers, van der Zande and collaborators revealed the twist-
angle-controlled strong tunning of the indirect optical
transition energy and second-harmonic generation
(SHG), as well as the weak tuning of direct optical tran-
sition energy and Raman frequency [87], as shown in
Fig. 2(a). The shift of indirect optical transition energies
was attributed to the angle-dependent interlayer separa-
tion due to repulsion between S atoms [see Fig. 2(b)]. The results suggested that the effective interlayer inter-
action was maximized in the aligned (θ = 0°) or anti-
aligned (θ = 60°) conformation, and minimized at θ =
30°. Zheng et al. [74] reported the modulations of
bandgap structures and phonon vibrations in a CVD-as-
grown WS2 bilayer. These bilayer WS2 with random
twist angles formed at high growth temperatures would
exhibit a much stronger PL intensity of peak A and an
additional peak A1 compared with aligned stacking cases
(θ = 0° or 60°), accompanied with the absence of indirect
transition peak I (interlayer exciton) [see Fig. FRONTIERS OF PHYSICS 2(e)]. Such modulations were attributed to two mechanisms. On one hand, a large interlayer distance weakened the
coupling strength, which was proved by the broadening
and redshift of the out-of-plane A1g mode in Raman
spectra. On the other hand, symmetry breaking in the
random-twisted bilayers led to a higher possibility of
interlayer hoping, which would be suppressed by spin-
layer locking in aligned case. Zhang et al. [76] also
observed an extra peak in PL spectra at 77 K in as-
grown MoS2/WS2 heterostructure with type II band
alignment, which was identified as the recombination of
interlayer carriers [see Figs. 2(c) and (d)]. Twisted
heterostructures also show angle-tunable Raman modes. Wang et al. [78] reported a redshift of A1g by ~2 cm–1 in
WSe2/WS2 bilayer compared with the case of monolayer
WSe2, along with the emergence of a new peak at 309.4
cm–1 that was regarded as a general feature for strong
coupling in bilayer system. The peak separations
between A1g(Г) and E2g(Г) as well as the linewidths of
A1g(Г) in WS2/MoS2 heterostructures were periodically g
[
]
A−
A
Since the optical responses of 2D semiconducting
materials are mostly determined by their excitonic
physics, lots of researches on modulations by stacking
and twist focus on the excitonic properties [88] and
dynamics [78, 79]. Huang et al. [88] concentrated on the
trions and excitons under varying twist angles between 0°
and 120° in the stacked bilayer of CVD-grown MoS2. In
PL spectra, both the ratio of the trion (
) to exciton
( ) and the trion binding energy showed periodic and
oscillatory features as a function of twist angle θ, as
shown in Fig. 2(f). These quantities reached their
maxima in aligned cases (θ = 0° or 60°) and minima at
θ = 30° or 90°, consistent with the angle-dependent
interlayer coupling strength. Using micro-absorption
spectroscopy, Wang et al. [78] showed that in WSe2/
WS2 bilayer after annealing, the intralayer WSe2 A exciton
presented significant PL quenching and spectral broad-
ening due to angle-insensitive interlayer charge separation
under strong coupling condition, while the interlayer
excitons formed by ultrafast interlayer charge transfer
exhibit sensitively twist-angle-dependent nature. The PL
intensity of the interlayer exciton in MoSe2/WSe2
heterostructure was enhanced at coherently stacking (θ =
0° and 60°) while vanished at incoherent intermediate
angles. Utilizing vdW-corrected DFT calculations,
Nayak et al. 2.1 Exciton properties controlled by twist-angle-
dependent interlayer interactions 2.1 Exciton properties controlled by twist-angle-
dependent interlayer interactions Interlayer coupling in multilayer system depends on the Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-3 FRONTIERS OF PHYSICS TOPICAL REVIEW TOPICAL REVIEW bilayer systems (with lattice constant mismatch and/or
interlayer twist), numerical calculation for the estimation
of interlayer coupling is nontrivial due to the lack of
periodic features. For this, Wang et al. [52] adopted the
analytical effective perturbative treatment to study the
interlayer coupling represented by interlayer hopping
integral in different valleys in both commensurate and
incommensurate TMDCs bilayers. They show that the
coupling strengths in ±K valleys are sensitive to the
interlayer distance determined by the stacking pattern. modulated by twist angles, caused by the interlayer
coupling and charge transfer effects [80]. Apart from
TMD vdW heterostructures, Raman spectra of bilayer
graphene are also affected by interlayer twist [89, 90]. These twist-angle-sensitive Raman peaks demonstrate
that the interlayer interactions and lattice dynamics can
be regulated by interlayer rotation. Therefore, it is
possible to control the electron-phonon coupling strength
and
thermal
transport
properties
of
2D
vdW
heterostructures via twist angle [91]. heterostructures via twist angle [91]. A−
A
Since the optical responses of 2D semiconducting
materials are mostly determined by their excitonic
physics, lots of researches on modulations by stacking
and twist focus on the excitonic properties [88] and
dynamics [78, 79]. Huang et al. [88] concentrated on the
trions and excitons under varying twist angles between 0°
and 120° in the stacked bilayer of CVD-grown MoS2. In
PL spectra, both the ratio of the trion (
) to exciton
( ) and the trion binding energy showed periodic and
oscillatory features as a function of twist angle θ, as
shown in Fig. 2(f). These quantities reached their
maxima in aligned cases (θ = 0° or 60°) and minima at
θ = 30° or 90°, consistent with the angle-dependent
interlayer coupling strength. Using micro-absorption
spectroscopy, Wang et al. [78] showed that in WSe2/
WS2 bilayer after annealing, the intralayer WSe2 A exciton
presented significant PL quenching and spectral broad-
ening due to angle-insensitive interlayer charge separation
under strong coupling condition, while the interlayer
excitons formed by ultrafast interlayer charge transfer
exhibit sensitively twist-angle-dependent nature. The PL
intensity of the interlayer exciton in MoSe2/WSe2
heterostructure was enhanced at coherently stacking (θ =
0° and 60°) while vanished at incoherent intermediate
angles. Utilizing vdW-corrected DFT calculations,
Nayak et al. FRONTIERS OF PHYSICS [70] found that the states at valence band
edges were affected by the twist angle, which exhibited a
long tail stretching over another layer in coherently
stacked MoSe2/WSe2, but localized in an individual
layer in incoherent case, as shown in Fig. 2(g). The
lattice mismatch controlled by twist angle would also
significantly affect the properties of devices based on
vdW heterostructures. Choi et al. [71] fabricated
p-WSe2/n-MoSe2 junctions with θ = 0°, 15° and 30°, and
measured their I–V curves under illumination with
different wavelengths. In the aligned case (θ = 0°) where
strong coupling is acquired, the sample showed a diodelike
behavior under dark condition, functioning as a well-
established p–n junction and displaying higher sensitivity
under IR illumination. Whereas in the cases of lattice
mismatch (θ = 15°, 30°), interlayer potential barrier was
built due to the vdW gap under weak coupling, and
such barrier could be overcome under ultraviolet (UV)
illumination, allowing photocurrent to flow in both
forward and reverse directions. Apart from rotation
mismatch, lattice relaxation and strain distribution of Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-4 42501-4 TOPICAL REVIEW FRONTIERS OF PHYSICS Fig. 2 Experimental observations of stacking-angle modulated interlayer interactions. (a) The upper panel shows PL spec-
troscopy for the bottom (as grown, gray line) and top (transferred, green line) monolayers, as well as twisted bilayers (t-BLs)
MoS2 at various twist angles θ. The lower panel is the measured (black) and simulated (magenta) peak energies versus θ for
the indirect (circles), A (squares), and B (triangles) transitions. (b) Schematic of the interlayer separation of t-BL MoS2
depending on the stacking patterns (upper panel) and twisting angle θ (lower panel) relative to the 60° separation. (c) PL
spectra with the Lorentzian fitting at 77 K. (d) The bandgap alignment of coupled as-grown MoS2/WS2 heterostructure. The
solid black, green, and orange double-arrow lines represent the energy gap, optical gap, and exciton binding energy, respec-
tively. (e) PL spectra of the randomly twisted WS2 bilayer compared with monolayer. (f) Twist angle dependence of the
trion to exciton PL intensity ratio (left panel) and trion binding energy (right panel). (g) The intensity of the interlayer
exciton peak versus the twist angle (left panel) and the plane-averaged partial charge density of each band edge state (VBM
and CBM at K) versus the interlayer distance for θ = 60° and 27.8° (right panel) of MoSe2/WSe2 heterostructures. FRONTIERS OF PHYSICS (h‒i) Scheme of valley and spin polarizations excited by circularly polarized light in bilayers with suppressed (h) interlayer
hopping and (i) strong interlayer coupling. (a, b) Reproduced with permission from Ref. [87]. (c, d) Reproduced with permission
from Ref. [76]. (e) Reproduced with permission from Ref. [74]. (f) Reproduced with permission from Ref. [88]. (g) Reproduced
with permission from Ref. [70]. (h, i) Reproduced with permission from Ref. [77]. Fig. 2 Experimental observations of stacking-angle modulated interlayer interactions. (a) The upper panel shows PL spec-
troscopy for the bottom (as grown, gray line) and top (transferred, green line) monolayers, as well as twisted bilayers (t-BLs)
MoS2 at various twist angles θ. The lower panel is the measured (black) and simulated (magenta) peak energies versus θ for
the indirect (circles), A (squares), and B (triangles) transitions. (b) Schematic of the interlayer separation of t-BL MoS2
depending on the stacking patterns (upper panel) and twisting angle θ (lower panel) relative to the 60° separation. (c) PL
spectra with the Lorentzian fitting at 77 K. (d) The bandgap alignment of coupled as-grown MoS2/WS2 heterostructure. The
solid black, green, and orange double-arrow lines represent the energy gap, optical gap, and exciton binding energy, respec-
tively. (e) PL spectra of the randomly twisted WS2 bilayer compared with monolayer. (f) Twist angle dependence of the
trion to exciton PL intensity ratio (left panel) and trion binding energy (right panel). (g) The intensity of the interlayer
exciton peak versus the twist angle (left panel) and the plane-averaged partial charge density of each band edge state (VBM
and CBM at K) versus the interlayer distance for θ = 60° and 27.8° (right panel) of MoSe2/WSe2 heterostructures. (h‒i) Scheme of valley and spin polarizations excited by circularly polarized light in bilayers with suppressed (h) interlayer
hopping and (i) strong interlayer coupling. (a, b) Reproduced with permission from Ref. [87]. (c, d) Reproduced with permission
from Ref. [76]. (e) Reproduced with permission from Ref. [74]. (f) Reproduced with permission from Ref. [88]. (g) Reproduced
with permission from Ref. [70]. (h, i) Reproduced with permission from Ref. [77]. manipulate the valley degree of freedom in TMDCs,
where the strong spin–orbit coupling (SOC) and the
broken inversion symmetry lead to spin-valley-locked
excitons at ±K valleys, resulting in the valley contrasting
optical selection rule [42–44, 93–96]. Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 2.2 Electronic band structures and relative twist in
momentum space Electronic structure and optical properties for twist
heterostructures have also been well-explored. Jin and co-
workers [45] used cathode lens microscopy and microprobe
angle-resolved photoemission spectroscopy (μ-ARPES)
to realize a direct measurement of the electronic structure
in twisted graphene/MoS2 vdW heterostructures. The
observed band structures indicated that the electronic
structure of graphene (Gr) remained intact, while the
MoS2-derived band exhibited twist angle dependence, as
shown in Figs. 3(b) and (c). In particular, the relative
energy between Г and K decreased from ~0.2 to ~0 eV
as the twist angle varied from 5° to 28°, suggesting an
indirect bandgap character except for twist angle near
30°. These results showed a similar trend to the DFT
calculation prediction, corroborating the view that the
electron-transfer-induced strain between graphene and
MoS2 would affect the Mo–S bond and lead to the twist-
angle-tunable band structure [101]. In TMD vdW
heterostructures, the band structures exhibit direct- or
indirect-gap determined by twist angles through tuning
the relative energy levels between K and Г but have
robust type II band alignments [see Fig. 5(a)]. For
twisted MoSe2/WSe2, first-principle DFT calculations
reveal a tunable band structure shown in Fig. 3(f) [70],
where the direct K–K transitions and indirect K–I tran-
sitions are almost degenerate, and the conduction band
at Г point varying with twist angle presents a trend
similar to that in homostructure TMDs [68, 69]. However, the observed VBM is located at K instead of Г
due to the large SOC. The VBM and CBM at the K-
point mainly come from WSe2 and MoSe2, respectively. Since direct transition possesses a larger amplitude than
that of indirect transition which involves with second-
order process, the interlayer transitions observed experi-
mentally are expected to occur between band edge states
from two different layers at K point with twist-angle-
dependence [Fig. 2(g)]. For example, in twisted WSe2/
WS2 heterostructure, VBM mostly originates from
orbitals of the WSe2 layer, while CBM is mainly
contributed by the WS2 layer. Such strong type-II band
alignments are independent of the twist angle and
bandgap nature [78]. 2.2 Electronic band structures and relative twist in
momentum space The spatially indirect nature of
interlayer exciton makes its transition dipole smaller
than that of the intralayer exciton by one to two magnitudes
and consequently weaker oscillator strength [54], which The variety of stacking patterns and relative twist
angles between crystal orientations in 2D multilayer
system provides a vast realm to modify the electronic
band structures through angle- and mismatch-dependent
vdW interactions at the atomic level, flourishing novel
optoelectronic properties such as spatial- and/or momen-
tum- indirect excitons with twist-angle-tunable nature
[72]. Unlike the monolayer TMD possessing a direct
bandgap at the K point, bilayer TMD is a bulk-like indirect
gap semiconductor. Liu et al. [69] used the LDA method
to examine the band structures of bilayer MoS2 with
different stacking conformations. The indirect bandgap
was found to be determined by the energy levels of K
point in the conduction band and Г point in the valence
band. The states at the K point in the conduction band
were constitutive of the strongly localized d orbitals at
the Mo atom sites (Mo-d), whose double degeneracy
remained intact under weak interaction. Whereas, the
states at Г point in the valence band originated from the
linear combination of outer p orbitals of the S atom sites
(S-p) and internal Mo-d orbitals, so the double level
degeneracy at the Г point was lifted by the stronger
interaction between the S-p from different layers, leading
to a higher energy level at the Г point in valence band
than that at the K point. Consequently, the band structure
transformed from direct bandgap to indirect bandgap. Stronger interlayer coupling would enlarge the splitting
at the Г point in the valence band and lead to a smaller
indirect bandgap. Therefore, the direct A-exciton transi-
tion was insensitive to the interlayer interaction, while
the indirect transition was sensitively dependent on
stacking order [77]. To gain a more accurate understanding
of the stacking-induced modulation on electronic struc-
tures, excitonic effects and optical properties of bilayer
TMDs, He et al. [68] utilized a many-body framework
adopting the RPA and the GW approximation
combined with the Bethe-Salpeter equation (BSE). The
G0W0 quasiparticle band structure was represented by
three valleys at T, K and Σ of the conduction band and
two valleys at K and Г of the valence band, as shown in
Fig. 3(a). FRONTIERS OF PHYSICS TOPICAL REVIEW TOPICAL REVIEW FRONTIERS OF PHYSICS DOP of folded bilayers confirmed that the suppression
of interlayer hopping and broken inversion symmetry
generated spin-valley configuration locked to the layer
index. On the contrary, in the centrosymmetric 2H-
stacking bilayer, the interlayer hopping amplitude of
holes was estimated to be comparable with the SOC
strength [100], giving rise to the mixing of layer pseudospins
and suppressed valley- and spin-selective circular dichro-
ism, as illustrated in Figs. 2(h) and (i). and vdW interlayer distance, which is mainly determined
by the valence band splitting at K and the energy difference
between K and Г. For A excitons, although their binding
energy were sensitive to stacking patterns (about 60–
100 meV higher for A–A′ than A–B pattern) the optical
transition energies were not significantly dependent on
stacking, in accordance with the experimental results
[see Fig. 2(a)] [87]. Apart from that, the theoretically
predicted stacking-induced strong tunning on Г valley in
the valence band was also demonstrated by the observation
of twist-angle-dependent indirect exciton transition. Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) FRONTIERS OF PHYSICS The spin-valley-
locked excitons can be modulated by external electric
[23, 25, 97, 98] and magnetic fields [99], allowing desirable
potential applications [16]. In bilayer systems, the valley-
contrasting physics can also be regulated by the interlayer
coupling and structural symmetry, depending on the
stacking orders. Jiang et al. [77] fabricated MoS2 bilayers
with different stacking patterns by folding exfoliated
monolayers to break the inversion symmetry and
suppress interlayer hopping, resulting in strong valley
and spin polarizations. Circularly polarized PL spectra
showed the different degree of polarization (DOP) for
different stacking bilayers, where the DOP in folded
bilayers was comparable to that in monolayer, and much
higher than that in natural 2H bilayer. The remarkable vdW twisted bilayers can also influence the formation of
excitonic species and consequently the optical responses. Recently, Dai et al. [92] have reported a four-fold
enhancement of phonon-assisted photon up-conversion
(UPC) in 5.5° twisted WSe2 bilayer, along with PL
intensity reduced by half, which was not observed in
bilayers under other rotation angles such as 1.1° and
13.8°. Such UPC enhancement was attributed to the
significant lattice relaxation at a mediate twist angle
(5.5°) between the relaxed (1.1°) and rigid (13.8°)
regimes. This mediate angle would cause increased strain
and lattice deformation, leading to considerable interlayer
separation. As a result, the formation of indirect interlayer
exciton through non-radiative relaxation of direct
neutral exciton was suppressed, which increased the
UPC efficiency. On the other hand, the reduction of
required assisted phonons due to the spectral redshift at
the mediate angle also improved the UPC efficiency. Light with circular polarization can be utilized to Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-5 2.2 Electronic band structures and relative twist in
momentum space (d) Scheme of band alignment (left panel) and two-dimensional band
structure (right panel) of twisted MoS2/WSe2 heterostructure. is the hybrid state of both layers and moves up as
hybridization increases. (e) Measured PL peak energies and calculated transition energies of different interlayer excitons
MoS2/WSe2 heterostructure with different twist angles. (f) Geometries (upper panel) and corresponding DFT-calculated
band structures (lower panel) of MoSe2/WSe2 heterostructures under twist angle θ = 60° and 27.8°. The contributions of the
MoSe2 (black) and WSe2 (red) layer are denoted in band structures. (g) Electric-field-dependent PL spectra of K–Г interlayer
excitons (upper panel) and reflectance spectra of K–K intralayer excitons (lower panel) in different spots of t-BL MoSe2 at
4 K. (h) Shifted Brillouin zone corners of two constituent layers under a small twist in TMD heterostructure. The green
arrows denote the displacement vector between
and
corner. (i) An interlayer exciton formed with kinematic
momentum Q. The interlayer Coulomb interaction between the electron and the hole conserving their momentum sum is
marked by the gray wavy line. (a) Reproduced with permission from Ref. [68]. (b, c) Reproduced with permission from Ref. [45]. (d, e) Reproduced with permission from Ref. [72]. (f) Reproduced with permission from Ref. [70]. (g) Reproduced with
permission from Ref. [82]. (h, i) Reproduced with permission from Ref. [46]. has been theoretically predicted [46, 65] and experimentally
demonstrated [18, 78]. Q = ±Q0, ± ˆC3Q0, ± ˆC2
3Q0
Q0 ≡τK −τ ′K′
τ ′K′
τK
(τ, τ ′)
conversion with photons in the exciton phase-space. When the electrons and holes relax to the bottom of the
±K valleys in momentum space with Q = 0, their tran-
sitions must involve phonon or impurity scattering to
enter the finite-velocity light cones, resulting in long life-
times of the interlayer excitons due to this inefficiency
scattering process [102]. However, when the small
mismatch can be compensated by the exciton’s kinematic
momentum
, direct transition
for
radiative
recombination
is
allowed. Here,
represents the displacement from the
Brillouin zone corner
of one constituent layer to
the nearest Brillouin zone corner
of the other layer,
where
denotes the electron-hole valley configura-
tion. Such interlayer excitons are within the light cones,
whose transition dipole strength would decay rapidly Q ≡k′ + k
k′
k
Q
Such type II band alignment can easily promote the
formation of interlayer exciton in which electrons and
holes reside at K valleys from different layers. 2.2 Electronic band structures and relative twist in
momentum space The conduction band minimum (CBM) was
always located at the T point, while the valence band
maximum (VBM) was related to the stacking pattern Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-6 TOPICAL REVIEW FRONTIERS OF PHYSICS ¯
M ¯Γ
¯K
|+Γ⟩
τK
τ ′K′
Fig. 3 Electronic structure and optical properties in twist heterostructures. (a) Quasiparticle band structure of AA′ and
AB stacked bilayer TMDs. The green lines indicate the monolayer band structure. (b) Second-derivative plot of the ARPES
band map along
– –
of the MoS2 in Gr/MoS2 heterostructures under different twist angles. (c) Brillouin zone of Gr and
surface Brillouin zone of MoS2 under twist angle θ. (d) Scheme of band alignment (left panel) and two-dimensional band
structure (right panel) of twisted MoS2/WSe2 heterostructure. is the hybrid state of both layers and moves up as
hybridization increases. (e) Measured PL peak energies and calculated transition energies of different interlayer excitons
MoS2/WSe2 heterostructure with different twist angles. (f) Geometries (upper panel) and corresponding DFT-calculated
band structures (lower panel) of MoSe2/WSe2 heterostructures under twist angle θ = 60° and 27.8°. The contributions of the
MoSe2 (black) and WSe2 (red) layer are denoted in band structures. (g) Electric-field-dependent PL spectra of K–Г interlayer
excitons (upper panel) and reflectance spectra of K–K intralayer excitons (lower panel) in different spots of t-BL MoSe2 at
4 K. (h) Shifted Brillouin zone corners of two constituent layers under a small twist in TMD heterostructure. The green
arrows denote the displacement vector between
and
corner. (i) An interlayer exciton formed with kinematic
momentum Q. The interlayer Coulomb interaction between the electron and the hole conserving their momentum sum is
marked by the gray wavy line. (a) Reproduced with permission from Ref. [68]. (b, c) Reproduced with permission from Ref. [45]. (d, e) Reproduced with permission from Ref. [72]. (f) Reproduced with permission from Ref. [70]. (g) Reproduced with
permission from Ref. [82]. (h, i) Reproduced with permission from Ref. [46]. ¯
M ¯Γ
¯K
|+Γ⟩
τK
τ ′K′
Fig. 3 Electronic structure and optical properties in twist heterostructures. (a) Quasiparticle band structure of AA′ and
AB stacked bilayer TMDs. The green lines indicate the monolayer band structure. (b) Second-derivative plot of the ARPES
band map along
– –
of the MoS2 in Gr/MoS2 heterostructures under different twist angles. (c) Brillouin zone of Gr and
surface Brillouin zone of MoS2 under twist angle θ. Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 2.2 Electronic band structures and relative twist in
momentum space The interlayer
excitons presented long valley lifetimes (> 40 ns) due to
the indirect nature in both real and momentum space
[65, 103–106]. Michl et al. [83] reported a high degree of
intrinsic circular polarization of exciton PL driven by
non-resonant linearly polarized laser in nonmagnetic
twisted hBN/MoSe2/WSe2 heterostructure (θ = 5.3 ±
1.4°) at 1.8 K [Fig. 4(e)], which was attributed to the
kinetic spin polarization of excitons arising from new
pyromagnetic symmetry of chiral stack structures. Furthermore, it has been predicted theoretically that
valley splitting of WSe2 would be significantly enhanced
in twisted WSe2/CrI3 heterostructure in contrast to non-
twisted heterostructure, which was equivalent to the
case with a perpendicular external magnetic field of
about 20 T [107]. with an increase in the magnitude of momentum. Only
in commensurate stacking at θ = 0° or 60° with negligible
lattice mismatch, all the main and Umklapp light cones
can merge into a single cone at Q = 0 and get close
enough to the bottom of energy dispersion, leading to
strong interlayer exciton PL [46, 103]. g
y
[
,
]
Unlike the interlayer excitons with spatially separated
electron and hole both located at K valleys, Kunstmann
and co-workers [72] have identified a new type of interlayer
excitons in MoS2/WSe2 heterostructure, where the hole
and the electron reside at the hybridized Г valley and
localized K valley, respectively, as shown in Figs. 3(d)
and (e). This momentum-space indirect interlayer exciton
was revealed by combining temperature-dependent PL
spectroscopy and first-principle calculations. The emission
energy of this kind of interlayer excitons could be tuned
via relative twist by controlling the hybridization [72]. Likewise, Sung et al. [82] have also observed and investi-
gated the Г–K interlayer excitons at temperature T =
4 K. These momentum-indirect interlayer excitons
possessed opposite out-of-plane electric dipole moments
supported by broken mirror and inversion symmetry due
to the spatially alternating stacking domains, which
originated from atomic reconstruction. The electric
dipole moments of Г–K interlayer excitons were identified
by the linear Stark effect in PL spectra, measured in a
hexagonal boron nitride (hBN) encapsulated near-0°-
twisted MoSe2 bilayer under an out-of-plane electric field,
as shown in Fig. 3(g). Additionally, K–K intralayer excitons
with higher energy were also observed, which exhibited
field-asymmetric hybridization with interlayer K–K exci-
tons under large fields, manifesting as field-dependent
avoided crossings between the two corresponding peaks
in reflectance spectra [Fig. 3(g)]. 2.2 Electronic band structures and relative twist in
momentum space The electron
and hole valleys of interlayer excitons will shift away
from each other under a small twist and/or lattice
mismatch, leading to a momentum mismatch of the Bril-
louin zone, shown in Fig. 3(h). The exciton’s kinematic
momentum is defined as
, where
and
represent kinematic momentum of carriers from different
layers, as shown in Fig. 3(i). The transition dipole of
interlayer excitons is sensitive to
, forming sixfold
degenerate light cones at finite center-of-mass (COM)
velocities on the parabolic energy dispersion [46]. The so-
called “light cone” means the region for allowed inter- Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-7 TOPICAL REVIEW TOPICAL REVIEW FRONTIERS OF PHYSICS light cones merge into a single one at Q = 0 and interlayer
exciton PL gets enhanced, the brightness and the polar-
ization selection rules depend on the interlayer transla-
tion. This interlayer exciton system allows observation
of valley Hall and inverse valley Hall effects, which is
regarded as an ideal platform to investigate Berry phase
effects in the Bloch band [46]. Experimentally, Rivera
and collaborators [51] observed interlayer excitons with
valley-contrasting physics in WSe2/MoSe2 vertical
heterostructures at near-0° stacking with slight twist
and lattice mismatch at room temperature. In this case,
the existence of light cones at small kinematic momenta
allows interlayer excitons to interconvert with photons
and radiatively recombine through scattering such as
exciton‒phonon or exciton‒exciton interactions. Interlayer
hopping between valleys with the same electron spin
from two constituent layers becomes the dominant
relaxation channel under small momentum mismatch,
leading to the co-polarized optical selection rules [see
Figs. 4(b) and (c)]. Whereas, the interlayer hopping
between spin-contrasting valleys is strongly suppressed
under large momentum difference. Interlayer exciton
exhibits a long valley lifetime of about 40 nanoseconds, a
few orders of magnitude longer than that of intralayer
excitons in monolayer, enabling the visualization of the
valley-polarized interlayer exciton cloud expanding,
including lateral drift and diffusion over several microm-
eters [51]. Therefore, twisted van der Waals heterostruc-
tures act as a promising playground for exploring valley
exciton physics. In recent years, several works have
reported on the chiral optical properties and valley
polarization physics in vdW bilayers via twist modula-
tion. Scuri et al. [73] controlled the spin-valley properties
of interlayer excitons in twisted WSe2 bilayers encap-
sulated in hBN by tunning the momentum separation
between valleys of different layers [Fig. 4(d)]. 2.2 Electronic band structures and relative twist in
momentum space The electric field
dependence of these K–K interlayer excitons also varied
spatially under alternating crystal symmetry. This result
indicates the profound effects of crystal symmetry on
excitonic properties and potentials for realizing alternating
exciton arrays with high tunability. In TMD heterostructures, interlayer excitons with
spin-valley polarization inherited from the monolayers
can directly interconvert with a photon with elliptic
polarization [46, 54]. The interlayer exciton spin-valley
polarization originates from valley-polarized intralayer
excitons through interlayer charge transfer. Such valley-
contrasting physics can be modified by interlayer twist,
which can expand fascinating and complicated properties
through changing mismatch between the Brillouin zone
of constituent layers in momentum space. In exciton
momentum space, light cones of interlayer exciton
possess helicity determined by valley configurations of
electron and hole, which allow tunable resonant optical
injection of valley excitons and valley currents under
linearly polarized light excitation, as illustrated in
Fig. 4(a) [46]. For the commensurate case (i.e., θ = 0° or
60° with neglectable lattice mismatch ~0.1%) where all 2.3 Twist-angle-dependent exciton dynamics Lattice mismatch and relative twist between layers in
vdW heterostructures with the ability to modify the
interlayer interaction and electronic structure efficiently, Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-8 42501-8 TOPICAL REVIEW FRONTIERS OF PHYSICS σ+
Fig. 4 Spin-valley polarization physics in twisted vdW TMD heterostructure. (a) Light cones at finite velocities and injection
of pure valley current of excitons by linearly polarized light. Currents of different valleys are denoted by green and pink
arrows. (b) Schematic of the interlayer excitons in the +K valley. Intralayer excitons are excited by
polarized optical
pump (black wavy lines) and form interlayer excitons in the +K valley through fast interlayer charge transfer (blue dotted
arrows). (c) Circular polarization-resolved PL spectra of the interlayer exciton performing a strong valley polarization. (d) Polarization-resolved PL spectra (left panel) and degree of circular polarization (DOCP) (right panel) of intralayer (X0)
and interlayer (XI) excitons in WSe2 bilayers with varying twist angles. (e) PL (upper panel) and DOCP (lower panel) of
twisted MoSe2/WSe2 heterostructure under linearly polarized 532-nm laser pump and circular-polarized detection at 1.8 K. The splitting optical resonances are attributed to the moiré excitons. (a) Reproduced with permission from Ref. [46]. (b, c) Reproduced with permission from Ref. [51]. (d) Reproduced with permission from Ref. [73]. (e) Reproduced with
permission from Ref. [83]. σ+
Fig. 4 Spin-valley polarization physics in twisted vdW TMD heterostructure. (a) Light cones at finite velocities and injection
of pure valley current of excitons by linearly polarized light. Currents of different valleys are denoted by green and pink
arrows. (b) Schematic of the interlayer excitons in the +K valley. Intralayer excitons are excited by
polarized optical
pump (black wavy lines) and form interlayer excitons in the +K valley through fast interlayer charge transfer (blue dotted
arrows). (c) Circular polarization-resolved PL spectra of the interlayer exciton performing a strong valley polarization. (d) Polarization-resolved PL spectra (left panel) and degree of circular polarization (DOCP) (right panel) of intralayer (X0)
and interlayer (XI) excitons in WSe2 bilayers with varying twist angles. (e) PL (upper panel) and DOCP (lower panel) of
twisted MoSe2/WSe2 heterostructure under linearly polarized 532-nm laser pump and circular-polarized detection at 1.8 K. The splitting optical resonances are attributed to the moiré excitons. (a) Reproduced with permission from Ref. [46]. (b, c) Reproduced with permission from Ref. [51]. (d) Reproduced with permission from Ref. [73]. 2.3 Twist-angle-dependent exciton dynamics (e) Reproduced with
permission from Ref. [83]. are supposed to play a key role in exciton dynamics,
including ultrafast charge transfer, diffusion, and relax-
ation processes. With the help of transient spectroscopy
such as pump-probe technique, time-resolved SHG and
ultrafast electron diffraction (UED), a large range of
works have focused on how exciton dynamics in
heterostructures are affected by twist angle over these
years [52, 78, 79, 84, 85]. Physical properties of semicon-
ductor heterostructures are highly influenced by band
alignment, which can be classified into three types,
including type-I (straddling gap), type-II (staggered gap)
and type-III (broken gap) [108], as depicted in Fig. 5(a). Theoretical calculations have found that the type-II
interface band alignment is the most typical and
common case for TMD vdW heterostructures [13, 109]. Meanwhile, the type-II band alignment with CBM and VBM of the heterostructures located in different layers
supports the electron and hole to transfer to the spatial-
separated band edges [see Fig. 5(b)]. Early experiments have demonstrated the rapid inter-
layer charge transfer (within 100 fs) by various methods
such as micro-absorption spectroscopy, PL mapping and
femtosecond pump-probe spectroscopy [78, 110]. Such
efficient interfacial charge transfer (< 50 fs) dynamics in
TMD vdW heterostructures do not exhibit twist-angle
dependence [79, 111–113]. Ji et al. [79] found the charge
transfer in MoS2/WS2 heterostructures was robust in a
time scale of about 90 fs under varying twist angles with
different interlayer coupling strengths, measured by
optical two-color ultrafast pump-probe spectroscopy, as
shown in Figs. 5(c) and (d). The twist-angle-independent
charge transfer with high efficiency violates the common Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-9 TOPICAL REVIEW FRONTIERS OF PHYSICS Fig. 5 Exciton dynamics modulated by twist angle. (a) Three types of band alignment in semiconductor heterostructures. The VB and CB represent valence band and conduction band positions, respectively. (b) Scheme of the charge transfer in
type-II band alignment of TMD heterostructures. (c) Schematic illustration of interlayer hole transfer in of MoS2/WS2
heterostructures after optically pumping MoS2 A-exciton, while the electron remains in the MoS2 layer. (d) The transient
absorption spectra obtained by selectively probing the WS2 A-exciton resonance in MoS2 monolayer or MoS2/WS2
heterostructures with varying twist angles. (e) Sketch of the band alignment and ultrafast charge transfer dynamics for
twisted MoS2/WSe2 under 1.70 eV pump (left panel) and 1.85 eV pump (right panel). 2.3 Twist-angle-dependent exciton dynamics (f) The structure of the monolayer
Brillouin zone, where the red and blue closed curves indicate the energy contours of Qc valleys. The dashed circle denotes a
ring region with strong interlayer coupling of the conduction band. (g) Schematic illustrations of interlayer charge transfer
process mediated by strong layer mixed Г and Q valleys in the energy (upper two panels) and momentum (lower two panels)
spaces. The right two panels depict the electron transfer and those on the left correspond to the hole transfer. (h) Differential
reflection signal of WS2/WSe2 heterostructures under 30° (upper panel) and 60° (lower panel) twist, respectively. (b) Reproduced
with permission from Ref. [54]. (c, d) Reproduced with permission from Ref. [79]. (e) Reproduced with permission from Ref. [85]. (f, g) Reproduced with permission from Ref. [52]. (h) Reproduced with permission from Ref. [84]. Fig. 5 Exciton dynamics modulated by twist angle. (a) Three types of band alignment in semiconductor heterostructures. The VB and CB represent valence band and conduction band positions, respectively. (b) Scheme of the charge transfer in
type-II band alignment of TMD heterostructures. (c) Schematic illustration of interlayer hole transfer in of MoS2/WS2
heterostructures after optically pumping MoS2 A-exciton, while the electron remains in the MoS2 layer. (d) The transient
absorption spectra obtained by selectively probing the WS2 A-exciton resonance in MoS2 monolayer or MoS2/WS2
heterostructures with varying twist angles. (e) Sketch of the band alignment and ultrafast charge transfer dynamics for
twisted MoS2/WSe2 under 1.70 eV pump (left panel) and 1.85 eV pump (right panel). (f) The structure of the monolayer
Brillouin zone, where the red and blue closed curves indicate the energy contours of Qc valleys. The dashed circle denotes a
ring region with strong interlayer coupling of the conduction band. (g) Schematic illustrations of interlayer charge transfer
process mediated by strong layer mixed Г and Q valleys in the energy (upper two panels) and momentum (lower two panels)
spaces. The right two panels depict the electron transfer and those on the left correspond to the hole transfer. (h) Differential
reflection signal of WS2/WSe2 heterostructures under 30° (upper panel) and 60° (lower panel) twist, respectively. (b) Reproduced
with permission from Ref. [54]. (c, d) Reproduced with permission from Ref. [79]. (e) Reproduced with permission from Ref. [85]. (f, g) Reproduced with permission from Ref. [52]. (h) Reproduced with permission from Ref. [84]. 2.3 Twist-angle-dependent exciton dynamics τK
pz
τKc
τ ′K′c
dependence on the interlayer twist and translations, the
Гv (Г point of valence band as the VBM) and Qc (six
CBMs near the middle of the Г–
path) show strong
interlayer coupling comparable to the corresponding
band offsets in TMD heterostructures, as shown in
Fig. 5(f). Such strong interlayer mixing is attributed to
the nonignorable effects of
orbital of chalcogen atoms,
as well as the relatively larger hopping integral with
smaller momentums compared with those in ±K valleys
[52]. These highly layer-hybridized valleys allow the
twist-independent ultrafast interlayer charge transfer of
type-II TMD heterobilayers via a two-step mechanism
involving the Гv and Qc valleys as intermediary states. Specifically, the photoexcited electron with high energy
in
valley can be scattered to the Qc valleys through
coupling with phonons, defects, or other carriers, and
subsequently relax to the
, as depicted in Fig. 5(g). perception that the interlayer charge transfer dynamics
would sensitively depend on the relatively weak interlayer
coupling affected by twist angle [54]. Moreover, the
momentum mismatch between valleys from different
constituent monolayers is expected to reduce the efficiency
of interlayer charge transfer. Utilizing the STEM and
time-dependent DFT simulations, the atomic-level local
structural inhomogeneity caused by interlayer stretching
and shifting is considered to be the origin of the
observed robustness of ultrafast interlayer charge trans-
fer. Such interlayer sliding provides extra parallel multi-
channels for the efficient transfer process [79]. From the
perspective
of
momentum
mismatch
in
twisted
heterostructures, phonon scattering coupled with inter-
layer hybridization mediates the twist-insensitive ultrafast
interlayer charge transfer [52, 54]. Unlike the layer-localized
±K valleys whose coupling strengths exhibit sensitive Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-10 FRONTIERS OF PHYSICS TOPICAL REVIEW K′(K)
K(K′)
materials were excited, the transient SH response of
quasi-2H heterostructure presented a rapid recovery on
femtosecond timescale slightly slower than those in the
corresponding monolayer, which was not observed in the
quasi-3R and misaligned samples. This discrepancy was
explained by the competition between the coherent
(intralayer) radiative recombination and additional
relaxation tunnels, where holes from the MoS2 layer
could be scattered to the WSe2 layer in K valleys or
strongly hybridized states in Г valley. As shown in
Fig. 5(e), hole transfer from
in MoS2 to
in
WSe2 was spin forbidden in the quasi-2H case, and the
misaligned case had a larger momentum mismatch than
that in quasi-3R stacking sample. 2.3 Twist-angle-dependent exciton dynamics Therefore, the efficiency
of the hole transfer decreased from quasi-3R, misaligned
to quasi-2H, which corresponded to the increasing coherent
recombination [85]. This result indicates the possibility
of modifying the layer mixed states through stacking
configuration, thus affecting the exciton dynamics. Apart from the vdW heterostructures composed of the
TMD family, the charge transfer and relaxation dynamics
of TMD-Gr heterostructures are also strongly modulated
through the twist-angle-tunable overlap between interlayer
electronic states. Recently, Luo and co-workers [86] have
realized the simultaneous visualization of charge transfer
and electron–phonon coupling using the UED method
and demonstrated the sensitive dependence of interlayer
charge transfer on the twist angle [116]. τ ′K′v
τKv
K′
Q′
The high energy hole in
can similarly transfer to
the
mediated by the strongly layer mixed Гv valley. The efficient scatterings are supported by the strong
couplings between carriers in ±K valleys and the related
phonons. Wang and co-workers [52] predicted this two-
step interlayer electron (hole) transfer process assisted
by phonons, and the calculated intervalley scattering
rates accorded with experimental results. Twist in bilayers
does not influence interlayer coupling and band offset of
Гv valley, while Qc valleys are always within the
strongly interlayer hybridized ring region under arbitrary
twist angle. Therefore, the ultrafast (< 50 fs) interlayer
charge transfer in TMD heterostructures is unimpeded
by interlayer rotational mismatch [52]. This two-step
mechanism has been demonstrated by the observation of
ultrafast electron intervalley scattering in MoS2/WS2
heterostructure with a small twist angle of 5° using the
time- and angle-resolved photoemission spectroscopy
(TR-ARPES), where electrons in K valleys are scattered
to intermediary valleys (M, M/2 and Q) within 70 fs
and relaxed to
and
valleys on timescales of ~0.4
ps [114]. However, such robustness against interlayer twist in
TMD heterostructures does not persist in all circum-
stances. Recent experimental researches on how the
twist angle affects rapid charge transfer show diverse
results. For a longer timescale of hundreds of femtoseconds
in twisted WS2/WSe2 heterostructures, the interlayer
tunneling rate decreases significantly as the rotational
mismatch rises, affecting the formation and relaxation
dynamics of interlayer excitons [115]. On the other hand,
a more complicated stacking angle modulation mechanism
of ultrafast dynamics has been revealed by more precise
measurements (e.g., much higher time resolutions). Zimmermann et al. [85] investigated the ultrafast charge
transfer dynamics affected dramatically by stacking
angle in MoS2/WSe2 heterostructures at an ultrafast
time resolution of about 6 fs. Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 3 Moiré excitonic physics In the wake of experimental
technology progress, an increasing number of researchers
have set their sights on the moiré superlattices (MSL),
which are formed by heterostructures stacked with a
finite relative twist angle or lattice mismatch. MSL can
trigger various exotic properties, such as flat bands [49,
50, 64, 118–120], excitons modulated by moiré periodic
potential [57, 121] and many-body effects [122] in such
artificial superlattice. The magic angles in twisted
bilayer graphene generate fascinating properties including
correlated insulator states and unconventional supercon-
ductivity originating from the flat bands [59–61, 123]. Since then correlated electronic physics has been investi-
gated in various graphene-based structures such as ABC
trilayer graphene/BN [61, 124–126] and twisted double
bilayer graphene (TDBG) [127]. In the last few years,
moiré effects and correlated phenomena have also been
found in other 2D materials-based systems such as
TMDs [128–131]. TMDs possess unique advantages
compared with graphene due to their desirable proper-
ties, such as sizeable bandgap and large spin-orbit
coupling. Therefore, MSL in TMDs would perform thermal
stability and feasibility for topological band engineering
and spatially alternating spin-valley configuration [93]. There are several theoretical predictions on the moiré
modulated excitons and corresponding optical response
of TMDs-based heterostructures, like localized excitons
and nanodot quantum emission arrays [132]. In addition,
TMD MSLs have flat bands that exist over a wide range
of stacking angles instead of certain magic angles [133],
making MSL easier to realize and providing an extra
rotation degree of freedom for modulating the moiré
effects. When vertically stacked vdW layers present a
complex periodic pattern consisting of varying interlayer
atomic alignment, the in-plane moiré superlattice will
take shape, as shown in Figs. 6(a) and (b). Moiré super-
lattice will be formed in two ways mainly determined by
the magnitude of relative twist, including the rigid-
lattice and atomic reconstruction [132]. The rigid-lattice
moiré pattern would naturally occur under a relative
rotation angle or lattice mismatch between constituent
layers, and such misalignment serves as a crucial node
for controlling the electronic structure and optical prop-
erties [134–137]. The periodic MSL can be represented
by a moiré wavelength λm [138]: As the fabrication, detection and observation of moiré
structures become increasingly achievable, multiple
experimental studies on moiré excitons have been
performed through electronic and optical measurements
such as STEM [56], PL [63, 144, 145] and Reflectance
spectroscopy [145, 146]. 2.3 Twist-angle-dependent exciton dynamics They conducted the
measurement by the time- and polarization-resolved
second-harmonic (SH) imaging microscopy, which could
directly identify signals from different regions (two kinds
of monolayers and their overlap) and control the SH
intensity of different regions. For 1.70 eV pump which
only resonantly excited the intralayer A-excitons of
WSe2, the extracted transfer time ΔtCT from WSe2 to
MoS2 layer of probed electron sensitively depended on
stacking angle: which are comparable in quasi-3R (θ =
9°, ΔtCT = 65 ± 10 fs) and misaligned (θ = 16°, ΔtCT =
84 ± 10 fs) cases, while become much faster in quasi-2H
(θ = 52°, ΔtCT = 12 ± 6 fs) case [see Fig. 5(e)]. The
twist-angle dependence was attributed to the different
wave function overlap at K valleys between constituent
layers at different stackings. Such distinct wave function
overlap also influenced the recombination lifetimes,
about hundreds of picoseconds, where stronger spatial
coincidence was found in quasi-2H case [85]. When both In vdW heterostructures, intralayer and interlayer
Coulomb interaction leads to the formation of diverse
quasi-particle species such as intralayer excitons, interlayer
excitons and trions. Twist angles in vdW heterostructures
also influence the optical behaviors of these “many-body”
species. Recently, Shi et al. [84] observed increased lumi-
nescence ratio of positive trions compared with excitons
in steady-state PL spectra of WS2/WSe2 heterostructure
under twist angle of 30° and 60° but not 0°. They used
ultrafast transient absorption spectroscopy to reveal the
faster formation time for trions than intralayer excitons
at θ = 60°, and the higher formation probability of
trions due to accumulation of residual free carriers at θ =
30°, as illustrated in Fig. 5(h). There are several theoretical discussions on moiré
physics and a considerable part of experimental observa-
tions of the moiré quasi-periodic structures using high-
resolution methods such as annular dark-field scanning
transmission electron microscopy (ADF-STEM) [56]. Nevertheless, comprehensive investigations focusing on
moiré pattern remained scarce in early experimental
researches. Since the moiré pattern is easily disordered
in a heterostructure without hexagonal boron nitride
encapsulation [93], limitations of the quality of the
sample mainly account for this lack. Besides, the effects
arising from moiré potential would be submerged at
room temperature. Siwei Li, et al., Front. Phys. 3 Moiré excitonic physics a0′
aM ≈
a0
√
δ2+θ2
ˆC3
RA
h
(
HA
h
)
denote the lattice constants of the two constituent
layers, with θ as the relative twist angle. The corresponding
moiré period (also known as moiré lattice constant)
under small lattice constant mismatch δ can be approxi-
mately represented as
[65], with length
scales from a few nanometers to tens of nanometers [54,
93]. As mentioned above, there are different types of
commensurate
stacking
patterns,
which
can
be
subsumed under two categories: near-rhombohedral
stacking (R-stacking) and near-hexagonal stacking (H-
stacking). The high-symmetry local atomic registries
existing in one moiré supercell preserve three-fold rota-
tional symmetry
and generate charming properties
such as spatially alternating optical selection rules [49]. As depicted in Figs. 6(a) and (b), these high symmetry
points can be labeled by the notation
(A = h, X,
M) for R-(H-)stacking, indicating the “A” in the electron
layer aligned to the “h” in the hole layer. The superscript
“A” here refers to the lattice center (h), chalcogen atom
(X) or metal atom (M). Varying interlayer atomic
registries in the moiré supercell result in the spatial
modulation of interlayer distance, exciton potential
energy and spin-valley characteristics [49, 93, 132]. For
stacking order at near-commensurate twist angle (θ ≈ 0°
or 60°), atomic-level reconstruction would be induced
through interlayer coupling and lattice relaxation. Instead of a smoothly varying rigid-lattice moiré pattern,
discrete commensurate regions with narrow boundaries
will be formed [139–142]. These discrete domains with
constant conductivity could be observed using conductive
atomic force microscopy (CAFM), as shown in Fig. 6(c). Furthermore, the existence of strain variation across the
moiré supercell leads to substantial strain accumulations
at region boundaries and domains with relatively unstable
stacking orders, which introduces lattice deformation
and extra modulation on band structures [143]. DFT
calculated band structures revealed the spatial modulation
of the relative electronic band edges induced by atomic
reconstruction [140]. Consequently, such spatial distribu-
tion of considerable strain affects the formation of excitonic
species and optical properties, which are also highly
tunable by relative twist [92]. Twistronics investigates the physics including electronic,
optical and magnetic properties [117] of twisted 2D van
der Waals heterostructures, and has promoted various
studies as mentioned above. 2.3 Twist-angle-dependent exciton dynamics 19(4), 42501 (2024) 42501-11 42501-11 FRONTIERS OF PHYSICS FRONTIERS OF PHYSICS TOPICAL REVIEW TOPICAL REVIEW a0′
aM ≈
a0
√
δ2+θ2
ˆC3
RA
h
(
HA
h
)
denote the lattice constants of the two constituent
layers, with θ as the relative twist angle. The corresponding
moiré period (also known as moiré lattice constant)
under small lattice constant mismatch δ can be approxi-
mately represented as
[65], with length
scales from a few nanometers to tens of nanometers [54,
93]. As mentioned above, there are different types of
commensurate
stacking
patterns,
which
can
be
subsumed under two categories: near-rhombohedral
stacking (R-stacking) and near-hexagonal stacking (H-
stacking). The high-symmetry local atomic registries
existing in one moiré supercell preserve three-fold rota-
tional symmetry
and generate charming properties
such as spatially alternating optical selection rules [49]. As depicted in Figs. 6(a) and (b), these high symmetry
points can be labeled by the notation
(A = h, X,
M) for R-(H-)stacking, indicating the “A” in the electron
layer aligned to the “h” in the hole layer. The superscript
“A” here refers to the lattice center (h), chalcogen atom
(X) or metal atom (M). Varying interlayer atomic
registries in the moiré supercell result in the spatial
modulation of interlayer distance, exciton potential
energy and spin-valley characteristics [49, 93, 132]. For
stacking order at near-commensurate twist angle (θ ≈ 0°
or 60°), atomic-level reconstruction would be induced
through interlayer coupling and lattice relaxation. Instead of a smoothly varying rigid-lattice moiré pattern,
discrete commensurate regions with narrow boundaries
will be formed [139–142]. These discrete domains with
constant conductivity could be observed using conductive
atomic force microscopy (CAFM), as shown in Fig. 6(c). Furthermore, the existence of strain variation across the
moiré supercell leads to substantial strain accumulations
at region boundaries and domains with relatively unstable
stacking orders, which introduces lattice deformation
and extra modulation on band structures [143]. DFT
calculated band structures revealed the spatial modulation
of the relative electronic band edges induced by atomic
reconstruction [140]. Consequently, such spatial distribu-
tion of considerable strain affects the formation of excitonic
species and optical properties, which are also highly
tunable by relative twist [92] Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 3 Moiré excitonic physics The mechanisms of how moiré
structure modulates intralayer and interlayer excitons
have attracted much attention but still lack a compre-
hensive explanation. For the moiré superlattice with a
periodicity of about 10 nm in a twisted semiconducting
vdW heterostructure, excitons have the Bohr radius
between the moiré wavelength and the lattice constant
of monolayer [132]. Therefore, exciton in superlattice can
be regarded as a composite quasi-particle modulated by λm=
(1 + δ) a0
√
2 (1 + δ) (1 −cos θ) + δ2 ,
(3.1) (3.1) δ = |a0
′−a0|
a0
a0
where lattice mismatch is presented as
, and , δ = |a0
′−a0|
a0
a0
where lattice mismatch is presented as
, and , Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-12 42501-12 FRONTIERS OF PHYSICS TOPICAL REVIEW through changing moiré period [147]. This harmonic-
potential model is widely accepted in most TMD
heterostructures but cannot dominate all systems [132]. In the case of band alignment under small band-edge
offset, the near-resonant hybridization between interlayer
and intralayer excitons would occur through interlayer
tunneling
in
moiré
minibands,
which
generates
hybridized excitons with intriguing features [146,
148–150]. 3.1 Theoretical predictions and experimental
observations of new excitons in moiré structure τK τ ′K′
A Rh
h
B RX
h
C RM
h
ˆC3
ˆC3
As mentioned in the previous section, early experimental
methods and sample quality cannot provide sufficient
conditions for the observation and further investigation
of moiré modulated exciton properties. Whereas, several
theoretical prediction works [49, 52, 64–66, 118] on the
new physics of excitons in moiré superlattice have been
reported. In the superlattice with a large-scale moiré
period, a mini Brillouin zone (BZ) would be formed,
whose corners are located at
–
and its π/3 rotations
[52], as depicted in Fig. 7(a). The original monolayer
bands are folded into a series of closely spaced mini
bands inside the mini BZ, where interlayer hopping can
happen between states with the same wave vector k
from different layers [Fig. 7(b)]. The local atomic
registry is spatially varying in a moiré supercell much
larger than the monolayer unit cell, resulting in a periodic
modulation of the local band structure [52]. Yu et al. [49]
described the moiré modulated exciton as an exciton
wave packet moving adiabatically in the periodic poten-
tial, which was constructed by the basis of kinematic
momentum eigenstates under misaligned heterobilayer. In the near R-type stacking case, exciton wave packets
at the three high symmetry points [ –
,
–
and
–
in Fig. 7(c)] have distinct
transformations due
to the different
eigenvalues of the electron Bloch
function: Fig. 6 MSLs in twisted TMD heterostructures. High
symmetry points in a moiré supercell of near R-stacking
(a) and near H-stacking (b) MSLs. (c) CAFM image of near-
0° (left panel) and near-60° (right panel) heterostructures
presenting alternating triangular and hexagonal domains
with constant conductivity and narrow boundaries. (a,
b) Reproduced with permission from Ref. [93]. (c) Reproduced
with permission from Ref. [140]. Fig. 6 MSLs in twisted TMD heterostructures. High
symmetry points in a moiré supercell of near R-stacking
(a) and near H-stacking (b) MSLs. (c) CAFM image of near-
0° (left panel) and near-60° (right panel) heterostructures
presenting alternating triangular and hexagonal domains
with constant conductivity and narrow boundaries. (a,
b) Reproduced with permission from Ref. [93]. (c) Reproduced
with permission from Ref. [140]. exciton potential with spatially periodical variation,
manifesting in the locally changing oscillator strength
and optical transition dipole [49]. 3.1 Theoretical predictions and experimental
observations of new excitons in moiré structure The center-of-mass
motion could be described by [147]: H = ℏΩ0 + ℏ2k2
2M + ∆(r) ,
(3.2) (3.2) ˆC3χA,s =e−i 2π
3 sχA,s, ˆC3χB,s =ei 2π
3 sχB,s, ˆC3χC,s =χC,s. (3.4) χC,s
(3.4) ℏΩ0
ℏ2k2
2M
∆(r)
M
∆(r)
where
is a constant representing the energy of the
lowest exciton state without moiré potential energy,
is the center-of-mass kinetic energy, and
denotes
the exciton moiré potential energy with
as the exciton
effective mass. varies smoothly and can be approxi-
mated by the lowest-order harmonic expansion [64, 65]: s = +(−)
Here, χ is the exciton wave packets and
denotes
the spin-valley index for exciton at K (–K) valley with
up (down) spin. As shown in Figs. 7(d) and (e), the
convertible photons must possess the same rotational
symmetry, which leads to the spatially alternating optical
selection rules. The interlayer exciton emissions are
circularly polarized at A and B points with opposite
helicity and forbidden at C, while the emissions are
elliptically polarized at other locals in the moiré super-
cell. The distinct rotational symmetries in the three
points also make them the extrema of exciton potential
[Fig. 7(e)]. The strong deep traps of exciton potential
(~100 meV) around the global minima at A points can s = +(−)
Here, χ is the exciton wave packets and
denotes
the spin-valley index for exciton at K (–K) valley with
up (down) spin. As shown in Figs. 7(d) and (e), the
convertible photons must possess the same rotational
symmetry, which leads to the spatially alternating optical
selection rules. The interlayer exciton emissions are
circularly polarized at A and B points with opposite
helicity and forbidden at C, while the emissions are
elliptically polarized at other locals in the moiré super-
cell. The distinct rotational symmetries in the three
points also make them the extrema of exciton potential
[Fig. 7(e)]. The strong deep traps of exciton potential
(~100 meV) around the global minima at A points can ∆(r) ≈
6
∑
j=1
Vj exp (ibj · r),
(3.3) (3.3) bj = θGj × ˆz
Rh
h
∆(r)
where
defines the reciprocal lattice vectors
of the moiré superlattice under a finite θ [65]. Near
points,
has the form of a harmonic oscillator with
quantized energy levels modulated by twist angle Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-13 TOPICAL REVIEW FRONTIERS OF PHYSICS σ+
σ−
Fig. 3.1 Theoretical predictions and experimental
observations of new excitons in moiré structure 7 Theoretical predictions of excitons in moiré superlattices. (a) The monolayer Brillouin zones (solid blue and red
hexagons) and the mini Brillouin zone of moiré superlattice (dash black hexagon). (b) The corresponding interlayer hopping
terms (green double arrows) between different mini bands near the band edges of the 1D moiré mini BZ. (c) Exciton wave
packets at the three high symmetry locals (left column) and the corresponding transformations of electron Bloch wave function
with hexagon center of the hole layer as a rotation center. (d) Oscillator strength of the interlayer exciton (left panel) and
optical selection rule (right panel) for the spin-up interlayer exciton at the K valley. The insert denotes the major axis of
polarization with the length proportional to ellipticity. (e) Contrasted potential landscapes for the intralayer and interlayer
excitons, with the optical selection rules for the spin-up species at the energy minima. (f) Upper panel: A perspective view of
an STM image zoomed in on one moiré supercell. Lower panel: A height profile under varying stacking order along the diagonal
line. (g) The site-dependent electronic structures in MoS2/WSe2 heterobilayers. Upper panel: The experimental values and
calculated DFT results of energy differences between KW and ГW of valence band at four different local atomic registries. Lower panel: The experimental and calculated DFT results of local bandgap formed between the CBM of MoS2 and the
VBM of WSe2. (h) Optical spectrum of K-valley interlayer excitons under different twist angles of WS2/MoS2 heterobilayer
with AA stacking. The red and blue curves show the optical absorption in response to
- and
-polarized light at frequency
ω, respectively. (i) The real-space probability function of the interlayer excitons responsible for the first three absorption
peaks at θ = 1°. (a, b) Reproduced with permission from Ref. [52]. (c‒e) Reproduced with permission from Ref. [49]. (f,
g) Reproduced with permission from Ref. [56]. (h, i) Reproduced with permission from Ref. [56]. σ+
σ−
Fig. 7 Theoretical predictions of excitons in moiré superlattices. (a) The monolayer Brillouin zones (solid blue and red
hexagons) and the mini Brillouin zone of moiré superlattice (dash black hexagon). (b) The corresponding interlayer hopping
terms (green double arrows) between different mini bands near the band edges of the 1D moiré mini BZ. 3.1 Theoretical predictions and experimental
observations of new excitons in moiré structure (c) Exciton wave
packets at the three high symmetry locals (left column) and the corresponding transformations of electron Bloch wave function
with hexagon center of the hole layer as a rotation center. (d) Oscillator strength of the interlayer exciton (left panel) and
optical selection rule (right panel) for the spin-up interlayer exciton at the K valley. The insert denotes the major axis of
polarization with the length proportional to ellipticity. (e) Contrasted potential landscapes for the intralayer and interlayer
excitons, with the optical selection rules for the spin-up species at the energy minima. (f) Upper panel: A perspective view of
an STM image zoomed in on one moiré supercell. Lower panel: A height profile under varying stacking order along the diagonal
line. (g) The site-dependent electronic structures in MoS2/WSe2 heterobilayers. Upper panel: The experimental values and
calculated DFT results of energy differences between KW and ГW of valence band at four different local atomic registries. Lower panel: The experimental and calculated DFT results of local bandgap formed between the CBM of MoS2 and the
VBM of WSe2. (h) Optical spectrum of K-valley interlayer excitons under different twist angles of WS2/MoS2 heterobilayer
with AA stacking. The red and blue curves show the optical absorption in response to
- and
-polarized light at frequency
ω, respectively. (i) The real-space probability function of the interlayer excitons responsible for the first three absorption
peaks at θ = 1°. (a, b) Reproduced with permission from Ref. [52]. (c‒e) Reproduced with permission from Ref. [49]. (f,
g) Reproduced with permission from Ref. [56]. (h, i) Reproduced with permission from Ref. [56]. Brillouin zone (MBZ) corners, resulting in new exciton
eigenstates with rotational symmetries of the moiré
pattern. Thus, the circular optical selection rule
remained, which was sensitive to the spatially varying
potential energy and local optical absorption strength,
but not locked to the valley. The moiré potential energy
mixed COM eigenstates, and consequently split the
spectral weight associated with interlayer excitons across
a variety of different states. Such energy splitting can be
tuned by twist angles through changing moiré periodicity
and the MBZ [Fig. 7(h)]. Second, the exciton states
could be confined in potential minima [Fig. 7(i)], allowing
the realization of nanoscale 2D arrays of identical quantum
dots. 3.1 Theoretical predictions and experimental
observations of new excitons in moiré structure The calculated exciton band structure in the moiré
Brillouin zone had flat-band features for the first few
exciton bands, showing the mixing of excitons with
different momenta and strong confinement for these
Wannier excitons states. The exciton moiré band structure realize perfect arrays of nanodot confinement, which can
act as uniform quantum emitters tunable by perpendicular
electric field and strain [49]. The moiré pattern with
periodic varying interlayer separation was observed
experimentally by scanning tunneling microscope (STM)
measurement in MoS2/WSe2 heterobilayer, as shown in
Fig. 7(f) [56]. The STM and scanning tunneling spectra
(STS) also revealed the formation of an electronic super-
lattice and detailed information on the local electronic
structures affected by interlayer coupling. The experi-
mentally observed and DFT-calculated site-dependent
electronic structures showed consistent trends as illust-
rated in Fig. 7(g). Wu and co-workers [65] adopted the
theory of moiré potential modulated interlayer exciton
and predicted that moiré potential affected interlayer
excitons in two aspects: First, the periodic potential
mixed and split the three independent exciton COM
eigenstates that were otherwise degenerate at the moiré Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-14 42501-14 FRONTIERS OF PHYSICS TOPICAL REVIEW resonances I, II and III), as shown in Fig. 8(e). Different
moiré exciton peaks in WSe2 exhibited distinct dependences
on electron doping in WS2 [Fig. 8(g)]. The discrepancy
indicates that peaks I, II and III arose from exciton
states with different real-space distributions in the moiré
superlattice. These phenomena were well explained by
the theoretical model in which the periodic moiré potential
was much stronger than the exciton kinetic energy and
generated multiple flat exciton minibands [151]. Besides,
excitons modulated by moiré potential have also been
observed in TMD vdW homostructures [63, 141, 145,
152]. Wu et al. [63] observed moiré intralayer excitons in
WSe2/WSe2 bilayers with small twist angles in the PL
spectrum at 8 K. The extracted moiré potential depth
(inversely proportional to the moiré superlattice period)
increased with the twist angle, manifesting as more
splitting and broader energy peaks [Fig. 8(h)]. As the
excitation power increased, moiré excitons turned into a
main peak dominated by intralayer excitons with a
larger peak width in the PL spectrum. The power
dependence of moiré exciton peaks was attributed to the
saturation of flat minibands filled by trapped excitons
[63]. 3.1 Theoretical predictions and experimental
observations of new excitons in moiré structure Moiré excitons were also identified in the twisted
WS2/WS2 homostructure with a twist angle of 1.46° ±
0.07° determined by SHG [145]. The multiple emergent
peaks in the PL spectrum at 8 K exhibited a nearly
constant peak spacing of 19 ± 3 meV and showed similar
pump power dependence, which also resulted from the
flat electronic minibands generated by the reconstructed
moiré potential. ˆC3
would exhibit Dirac dispersion within the light cone
when the moiré potential was tuned to have an additional
two-fold rotational symmetry besides the
symmetry
[65]. Wu and co-workers also focused on the intralayer
excitons under periodic moiré potentials and found that
moiré superlattice in the TMD bilayer generated the
twist-angle-dependent satellite excitonic peaks in the
optical absorption spectrum [64]. These theoretical
works also point out the topological features of the
moiré exciton bands that would further relate to the
unique Hubbard models with twist-angle-dependent
hopping and interaction strength [118]. Twisted bilayers
with moiré structure provide an accessible platform to
simulate the Hubbard models for exploring many-body
phenomena such as spin-liquid states and quantum
anomalous Hall insulators [49, 64]. With the development of experimental methods,
multiple
observations
of
moiré
effects
in
vdW
heterostructures have been reported and showed diverse
characteristics and fascinating physics. In most cases,
the periodic moiré potential was regarded to play a
crucial role in the emergence and modulation of excitons
in a twisted heterostructure, supporting the harmonic-
potential model based on perturbation theory. Pan et al. [57] observed the moiré pattern in MoS2/WSe2 on
epitaxial graphene using STM and STS at temperatures
of 5 and 80 K. STS showed significant energy shifts of
about 0.2 eV between the maxima and minima of the
moiré corrugation [Fig. 8(a)]. Sharp peaks (linewidth
<10 meV) occurred near band-edge energies of the spectra
for both the valence band (VB) and the conduction
band (CB) at 5 K, which were attributed to the quan-
tum-confined states associated with the ГW–VB and
KMo–CB under moiré potential. These confined states
showed obvious temperature dependence and the corre-
sponding narrow peaks vanished at 80 K. The spatially
periodic in-plane potential leads to splittings of both
intralayer exciton and trion energies, which was
observed in the emission and absorption spectra of
MoSe2/MoS2 heterostructure encapsulated in hBN at
5 K, as shown in Figs. 8(b)‒(d) [121]. The energy differences
between the split peaks were consistent with the theoretical
predictions mentioned above. Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 3.1 Theoretical predictions and experimental
observations of new excitons in moiré structure The intensities of the split
peaks of excitons and trions also exhibited temperature
dependence, suggesting that excitons can be detrapped
from moiré potential minima through thermally activated
phonon scattering. Moiré exciton states were also
observed from the multiple emergent peaks around the
original WSe2 A exciton resonance in the absorption
spectra of closely aligned WSe2/WS2 heterostructures at
10 K [151]. These peaks were considered to arise from
the strongly coupled heterobilayer rather than from
several separated domains, since the photoluminescence
excitation (PLE) spectrum showed the strong enhancement
of the interlayer exciton emission under excitation at the
energies of each of the three emergent peaks (labeled as Although the effective harmonic-potential model has
been utilized to describe moiré effects successfully in
TMD heterostructures with considerable interlayer band
edges offsets like WS2/WSe2 and MoSe2/MoS2, this
theoretical model cannot explain moiré excitonic behaviors
in all systems. In the case of TMD heterostructures with
a slight band-edges offset featuring nearly degenerate
carrier bands, such as MoSe2/WS2 and some twisted
homobilayers, resonant interlayer hybridization must be
explicitly considered. The interlayer hybridization would
enhance the moiré superlattice effects on the electronic
structures and lead to the formation of hybridized excitons
(hXs) through interlayer carrier tunneling [146, 150]. The hybridized excitons arise out of the band hybridization
effects [Fig. 8(j)], consisting of strongly mixed electron
or hole states involved in the formation of intralayer
excitons (Xs) and interlayer excitons (IXs). The hXs
inherit the brightness of intralayer excitons and the
polarity of interlayer excitons, leading to their identical
optical selection rules to those of intralayer excitons in
constituent monolayer and sensitivity to vertical electrical
bias, respectively [132, 150]. The theoretical absorption
spectra of resonant heterobilayers MoSe2/WS2 varied
sharply with twist angle, as shown in Fig. 8(l), in which
a bright line at higher energies appeared when closely
aligned due to moiré umklapp electron-photon interac- Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-15 TOPICAL REVIEW FRONTIERS OF PHYSICS Fig. 8 Experimental observations of moiré excitons. (a) STM image with different moiré locations of MoS2/WSe2
heterostructure (upper panel) and constant-height conductance map for voltage in the valence band-edge region (lower panel),
where confined states occurred at B and C. (b) PL and reflectivity spectra of MoSe2 measured in monolayer and MoSe2/
MoS2 heterostructure. (c) Spatial map showing the presence of MoSe2 transition splitting overlaid with the areas of most
intense PL of MoS2 and MoSe2. 3.1 Theoretical predictions and experimental
observations of new excitons in moiré structure (b) PL and reflectivity spectra of MoSe2 measured in monolayer and MoSe2/
MoS2 heterostructure. (c) Spatial map showing the presence of MoSe2 transition splitting overlaid with the areas of most
intense PL of MoS2 and MoSe2. (d) The energy of the observed MoSe2 transitions (blue and red dots) extracted along the
horizontal dashed line of (c). Green diamonds denote the MoS2 PL intensity. (e) Comparison between the interlayer exciton
PLE spectrum (black dots) and the reflection spectrum (blue curve) of WSe2/WS2 moiré superlattice. (f) Twist-angle dependent
PL calculated for i) anti-parallel and ii) parallel stacking twisted WSe2 on SiO2 at 4 K. (g) Reflection contrast spectra in the
range of the WSe2 A exciton on the electron-doping side. (h) Optical characteristics of moiré intralayer excitons in WSe2/
WSe2 twisted homobilayer. Left panel: Comparing the PL spectra of WSe2 monolayer and homobilayer at 8 K. Right panel:
PL spectra of homobilayers with twist angles of 1.36° and 3°. (i) Left panel: Moiré mini Brillouin zone (purple) defined by
the moiré Bragg vectors (purple arrows). Right panel: Electronic band structures and Brillouin zone alignment for twisted
MoSe2/WS2 heterobilayers. Spin-down (spin-up) bands are colored red or green (grey). (j) Band alignments with almost resonant
conduction band states of the twisted bilayer of TMD MoX2 and WX′2 (X, X′ = S, Se, Te) for near parallel (θ ≈ 0°, left panel)
and near antiparallel (θ ≈ 60°, right panel) cases. (k) Exciton hybridization in twisted bilayers TMDs. Upper panel:
Schematic electronic band structure at the K and Λ valley of the two twisted layers (red and blue, respectively) as well as
possible intra- (X) and interlayer excitons (IX). Lower panel: Schematic exciton center-of-mass dispersion without hybridization
as well as hybridized K–Λ state hX (green). (l) Twist-angle dependent low-energy absorption spectra near the energy of A
exciton in MoSe2/WS2 with resonant conduction band edges. The hXs form at closely alignment cases, leading to the avoided
crossings marked by green arrows. The white arrows denote the absorption lines enabled by moiré Umklapp processes. (a) Reproduced with permission from Ref. [57]. (b‒d) Reproduced with permission from Ref. [121]. (e, g) Reproduced with
permission from Ref. [151]. (f, k) Reproduced with permission from Ref. [149]. (h) Reproduced with permission from Ref. [63]. (i) Reproduced with permission from Ref. [146]. (j, l) Reproduced with permission from Ref. [150]. tions. 3.1 Theoretical predictions and experimental
observations of new excitons in moiré structure (d) The energy of the observed MoSe2 transitions (blue and red dots) extracted along the
horizontal dashed line of (c). Green diamonds denote the MoS2 PL intensity. (e) Comparison between the interlayer exciton
PLE spectrum (black dots) and the reflection spectrum (blue curve) of WSe2/WS2 moiré superlattice. (f) Twist-angle dependent
PL calculated for i) anti-parallel and ii) parallel stacking twisted WSe2 on SiO2 at 4 K. (g) Reflection contrast spectra in the
range of the WSe2 A exciton on the electron-doping side. (h) Optical characteristics of moiré intralayer excitons in WSe2/
WSe2 twisted homobilayer. Left panel: Comparing the PL spectra of WSe2 monolayer and homobilayer at 8 K. Right panel:
PL spectra of homobilayers with twist angles of 1.36° and 3°. (i) Left panel: Moiré mini Brillouin zone (purple) defined by
the moiré Bragg vectors (purple arrows). Right panel: Electronic band structures and Brillouin zone alignment for twisted
MoSe2/WS2 heterobilayers. Spin-down (spin-up) bands are colored red or green (grey). (j) Band alignments with almost resonant
conduction band states of the twisted bilayer of TMD MoX2 and WX′2 (X, X′ = S, Se, Te) for near parallel (θ ≈ 0°, left panel)
and near antiparallel (θ ≈ 60°, right panel) cases. (k) Exciton hybridization in twisted bilayers TMDs. Upper panel:
Schematic electronic band structure at the K and Λ valley of the two twisted layers (red and blue, respectively) as well as
possible intra- (X) and interlayer excitons (IX). Lower panel: Schematic exciton center-of-mass dispersion without hybridization
as well as hybridized K–Λ state hX (green). (l) Twist-angle dependent low-energy absorption spectra near the energy of A
exciton in MoSe2/WS2 with resonant conduction band edges. The hXs form at closely alignment cases, leading to the avoided
crossings marked by green arrows. The white arrows denote the absorption lines enabled by moiré Umklapp processes. (a) Reproduced with permission from Ref. [57]. (b‒d) Reproduced with permission from Ref. [121]. (e, g) Reproduced with
permission from Ref. [151]. (f, k) Reproduced with permission from Ref. [149]. (h) Reproduced with permission from Ref. [63]. (i) Reproduced with permission from Ref. [146]. (j, l) Reproduced with permission from Ref. [150]. Fig. 8 Experimental observations of moiré excitons. (a) STM image with different moiré locations of MoS2/WSe2
heterostructure (upper panel) and constant-height conductance map for voltage in the valence band-edge region (lower panel),
where confined states occurred at B and C. 3.1 Theoretical predictions and experimental
observations of new excitons in moiré structure Under moiré potential with a relatively
large period, the moiré intralayer excitons and trions in
MoS2/MoSe2 heterostructure exhibited unchanged decay
times compared with those in monolayer at 5 K due to
the relatively weak localization effect [121]. In the recon-
structed moiré patterns of WSe2/WSe2 homobilayer with
spatially alternating AB- and BA-stacked domains, a
higher-energy intralayer exciton peak emerged in the p-
doped regime of PL spectra (T = 4 K) at regions with
larger λm, in contrast to those with λm < 10 nm. As
shown in Fig. 9(b), the two peaks showed different
doping dependence and distinct valley coherence repre-
sented by the degree of linear polarization (DOLP) [141]. These results could be explained by the formation of two
exciton species (type I and II excitons). The two
intralayer exciton species were distributed in different
intralayer locations with different bandgaps in the
reconstructed superlattice, as depicted in Fig. 9(c). Type
II excitons could only be observed in perfectly recons-
tructed moiré patterns with aligned domains size larger
than the exciton diffusion length (estimated to be
10–100 nm), because the interlayer tunneling at the K
point was symmetry forbidden in AB/BA-stacked
domains, which would prevent the excitons from relaxing
to the lower-energy state (type I) before recombining. Type I excitons could bind strongly to extra holes under
p-doped to form charged excitons due to the higher
VBM of exciton I than that of type II excitons. Whereas, the less-layer-localized electrons were expected
to interact with both exciton species under n-doping, angle-dependent mini BZ, as depicted in Fig. 8(i). In
closely aligned (or anti-aligned) MoSe2/WS2 heterobilay-
ers, the minimized momentum mismatch for interlayer
conduction-band
tunneling
enabled
hybridization
between the interlayer excitons and the MoSe2 intralayer
excitons (both A and B excitons), leading to shared hole
band of the hybridized excitons (hXs) [Fig. 8(i)] [146]. The resulting hXs (labeled as hX1) and higher-energy
peaks arising from the moiré mini-band states of hX
(labeled as hX2 and hX3) appeared at closely aligned
case in reflectance contrast spectra at low temperatures
(T = 10 – 105 K). Apart from the bright excitons,
momentum-dark excitons in moiré superlattice have also
been reported to be affected by interlayer hybridization. Berm et al. [149] investigated the twisted WSe2 bilayers
and
showed
the
twist-angle-dependent
electron
hybridization at the Λ point of momentum-indirect K–Λ
exciton in phonon-assisted PL (T = 4 K). 3.1 Theoretical predictions and experimental
observations of new excitons in moiré structure The umklapp processes fold exciton states with
finite-momentum onto zero-momentum, regarded as the
evidence for moiré superlattice minibands for the exci-
tons. Such features were absent in non-resonant cases
due to the weak mixing between IXs and X [150]. Alexeev
et al. [146] investigated the interplay between moiré
superlattice effects and twist-angle dependent hybridiza-
tion of X and IX in MoSe2/WS2 heterostructures with
near-degenerate conduction band edges. The exciton
energy and linewidth in PL spectra change periodically
with the interlayer twist angle at a period of θ ≈ 60°, ∆K =KWS2 −KMoSe2
∆K′ = K′WS2 −KMoSe2
GMoSe2
n
GWS2
n
kWS2 −kMoSe2 = ∆K + bn
kWS2
kMoSe2
bn = GWS2
n
−GMoSe2
n
suggesting a connection to the valley rotational
mismatch between the Brillouin zones of two constituent
monolayers
(
). The conduction-band states had plane-wave projections
onto all Bragg vectors of their corresponding reciprocal
lattices
and
. As a result, the interlayer
tunneling between states satisfied momentum conservation
. Here,
and
were the
corresponding first-Brillouin-zone wave vectors from the
K valley. The
denoted the first Bragg
vectors of moiré superlattice, which defined the twist- Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-16 42501-16 FRONTIERS OF PHYSICS TOPICAL REVIEW twisting in vdW heterostructures, the spatial distribu-
tions, resonant energies and oscillation strengths of
moiré excitons can also be continuously tunned by twist
angle. Guo et al. [50] theoretically predicted the distri-
butions of moiré excitons in twisted MoS2/WSe2
heterostructures under different angles. Based on time-
dependent density functional theory (TDDFT), they
predicted that exciton with lower energy could be more
localized and would experience deeper moiré potential
under a small twist angle relative to θ = 0° or 60°, as
shown in Fig. 9(a). The holes showed stronger in-plane
localization than electrons, consistent with the narrower
bandwidth of VBM than that of CBM [50]. The calculated
charge density showed that the hole with stronger in-
plane localization extended to both layers (modulated by
interlayer moiré potential), while the delocalized electron
is confined at the MoSe2 layer (modulated by intralayer
moiré potential). This result suggested a more localized
interlayer exciton than intralayer exciton because the
bandgap modulation of interlayer exciton was larger
than that of the intralayer exciton [50]. Numbers of
studies have observed the intralayer excitons in moiré
superlattices. 3.1 Theoretical predictions and experimental
observations of new excitons in moiré structure The K–Λ
excitons exhibited a prominent hybridization effect due
to their strong delocalization over both layers, in
contrast to the K-K excitons, as shown in Figs. 8(f) and
(k). Recently, several experimental methods apart from
optical measurements for observation of moiré superlattice
and moiré exciton have been reported. Andersen and co-
workers [141] realized the direct stacking domains imaging
in vdW heterostructure devices. They developed a
secondary electron microscope (SEM) technique to
investigate reconstructed moiré patterns in twisted
WSe2/WSe2 bilayers. They directly demonstrated the
considerable variation of local moiré periodicity in a
small twist angle bilayer and correlated the local structure
with distinct optical properties. Karni et al. [153]
utilized the time-resolved and angle-resolved photoemis-
sion spectroscopy (TR-μ-ARPES) to capture images of
the electron and hole distributions of interlayer exciton
in WSe2/MoS2 heterostructure. The interlayer exciton
was found to have a diameter comparable with the
moiré period, whereas its center of mass was highly
confined in a region much smaller than the size of the
exciton itself. 3.2 Localization and modulation of excitons by moiré
superlattice In the left panel with θ = 6°,
the green arrows indicate vectors of momentum shift between the Brillouin zone corners of the two monolayers. In the middle
(right) panel, a commensurate moiré reciprocal lattice is formed with the corresponding moiré Brillouin zone denoted by the
black hexagons. The yellow arrow represents the moiré reciprocal lattice base vector that connects KM and
(KW). (i) Twist-angle dependence of normalized PL spectra at 290 K (left panel) and reflectance contrast spectra at 10 K (right
panel) of MoSe2/WS2 heterobilayers near the A-exciton energy in an isolated MoSe2 monolayer (black). (a) Reproduced with
permission from Ref. [50]. (b‒d) Reproduced with permission from Ref. [141]. (e, f) Reproduced with permission from Ref. [134]. (g) Reproduced with permission from Ref. [158]. (h) Reproduced with permission from Ref. [135]. (i) Reproduced with
permission from Ref. [146]. K′
W
Fig. 9 Exciton properties and dynamics modulated by moiré superlattice. (a) Localized moiré excitons with different twist
angles and energies in the MoS2/WS2 heterostructure. The charge densities of the electron and the hole are colored in red
and blue, respectively. (b) Gate-dependent PL spectra (left panel) and DOLP (right panel) of the two exciton species in
WSe2 bilayers with reconstructed moiré superlattice. Inset in the left panel: SEM image of the measured spot. (c) Upper
panel: Band structure schematic and wavefunction distribution at important points in k-space. Lower panel: Side views of
AB and BA domains, where type I and II locations are denoted by maroon and orange boxes, respectively. (d) Schematic of
alternating properties in the 2D triangular exciton array, showing only excitons in the top layer for brevity. (e) Anomalous
diffusion of interlayer excitons in WS2/WSe2 heterobilayers with exciton density at time zero N0 = 6.0 × 1012 cm–2 at 295 K,
compared with the normal exciton diffusion in 1L-WSe2 and 1L-WS2 with linear temporal dependence. (f) Twist-angle- and
temperature-dependent K–K and K–Q interlayer exciton dynamics. Left panel: Schematic of the probing electron (reflecting
K–K interlayer exciton population) and hole (reflecting the sum of the K–K and K–Q exciton populations) dynamics. Right
panel: The fitted decay time of the K–K exciton as a function of temperature under exciton density at 4.1 × 1012 cm–2. (g) Spatially resolved PL images of the interlayer excitons for CVD-grown sample A-1, mechanical exfoliation and transfer
(MET) sample B-1, and MET sample C with different twist angles θ, respectively. 3.2 Localization and modulation of excitons by moiré
superlattice Excitons in TMD moiré superlattices under the spatially
varying potentials show in-plane localization [153] and
possess the flat-band feature. The flat bands are related
to the in-plane localization due to the vanishing of group
velocity [154]. The moiré potential traps are analogous
to the localized defect levels in semiconductors, where
the moiré periodicity corresponds to the defect–defect
distance. The “defect” bands would become less dispersive
as the periodicity increased and their interactions
reduced [50]. Since the amplitude and period of moiré
potential are sensitive to the interlayer stacking and Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-17 FRONTIERS OF PHYSICS TOPICAL REVIEW FRONTIERS OF PHYSICS K′
W
Fig. 9 Exciton properties and dynamics modulated by moiré superlattice. (a) Localized moiré excitons with different twist
angles and energies in the MoS2/WS2 heterostructure. The charge densities of the electron and the hole are colored in red
and blue, respectively. (b) Gate-dependent PL spectra (left panel) and DOLP (right panel) of the two exciton species in
WSe2 bilayers with reconstructed moiré superlattice. Inset in the left panel: SEM image of the measured spot. (c) Upper
panel: Band structure schematic and wavefunction distribution at important points in k-space. Lower panel: Side views of
AB and BA domains, where type I and II locations are denoted by maroon and orange boxes, respectively. (d) Schematic of
alternating properties in the 2D triangular exciton array, showing only excitons in the top layer for brevity. (e) Anomalous
diffusion of interlayer excitons in WS2/WSe2 heterobilayers with exciton density at time zero N0 = 6.0 × 1012 cm–2 at 295 K,
compared with the normal exciton diffusion in 1L-WSe2 and 1L-WS2 with linear temporal dependence. (f) Twist-angle- and
temperature-dependent K–K and K–Q interlayer exciton dynamics. Left panel: Schematic of the probing electron (reflecting
K–K interlayer exciton population) and hole (reflecting the sum of the K–K and K–Q exciton populations) dynamics. Right
panel: The fitted decay time of the K–K exciton as a function of temperature under exciton density at 4.1 × 1012 cm–2. (g) Spatially resolved PL images of the interlayer excitons for CVD-grown sample A-1, mechanical exfoliation and transfer
(MET) sample B-1, and MET sample C with different twist angles θ, respectively. (h) Schematics of the Brillouin zone of
twisted bilayers. The red (blue) hexagons denote the Brillouin zone of MoSe2 (WS2) monolayers. 3.2 Localization and modulation of excitons by moiré
superlattice A larger energy
difference between the K and Q valleys of the conduction
band
in the 0°-twisted bilayer suppressed the effi-
cient electron scattering from Q to K, compared with
the 60° twisted case. Consequently, WS2/WSe2 heterobi-
layers presented a shorter K–K exciton decay time and
higher temperature for thermally excited phonons to
assist the Q–K scattering at near-0° than at near-60°, as
shown in Fig. 9(f) [134]. Choi and co-workers [158] also
reported that interlayer exciton diffusion in WSe2/
MoSe2 heterobilayers could be highly controlled by twist
angle, using spatially and time-resolved PL measurements
at T = 4 K, as shown in Fig. 9(g). The presence of
moiré superlattice with a large supercell and deep potential
at small twisting could completely localize interlayer
exciton and lead to a reduced lifetime and velocity. Consequently, the diffusion length was decreased in
comparison with that in commensurate heterostructure. At a larger twist angle leading to a reduced moiré period,
excitons may tunnel between supercells and have a
longer interlayer exciton lifetime [46, 158]. Apart from
the low-temperature case, the temperature-activated
diffusivity is also dependent on the twist angle due to
the deep moiré potentials [136]. Moiré potential effects
can be effectively screened at a sufficiently high exciton
density or temperature [134]. The moiré interlayer excitons In addition to interlayer twist and temperature, various
factors in twisted vdW heterostructures have shown the
ability to modulate the localization of moiré excitons in
recent researches. Cai and co-workers [161] focused on
the formation mechanism of numerous PL peaks from
localized interlayer excitons in moiré superlattices. They
constructed an interlayer donor-acceptor pair exciton
(DAP IX) dynamic model to explain the origins of these
narrow excitonic peaks, where the localized DAP exciton
has an electron trapped in the donor site and a hole
trapped in the acceptor site. They also fabricated an
hBN-encapsulated MoSe2/WSe2 bilayer (θ ≈ 60° ± 1°)
on a nanopillar array, and experimentally confirmed the
DAP IX model through observations of the emission
energy-lifetime correlation and unique nonmonotonic
power dependence of the energy-resolved lifetime. The
nanopillars here produced strain to provide PL emission
enhancement through strain gradient-induced funnelling
as well as the increased coupling between the band-edge
and DAP IX, indicating the modulation of moiré excitons
from out-of-plain strain. Coincidentally, Lin et al. [143]
have demonstrated the influence of atomic-level in-plane
strain on moiré potential. 3.2 Localization and modulation of excitons by moiré
superlattice The observed diffusion length of the moiré
exciton was one order of magnitude smaller than that in
the untwisted WSe2/MoSe2 heterostructure [26, 50]. Using transient absorption (TA) microscopy, Yuan et al. [134] investigated interlayer exciton dynamics and trans-
port in WS2/WSe2 heterobilayers under twist angles of 0°
and 60° with a 4% lattice mismatch, which had a moiré
period of about 7.6 nm. The diffusions of interlayer excitons
with non-linear temporal dependence of mean square
distance
deviated from the normal diffusions of
intralayer excitons in the constituent monolayers
[Fig. 9(e)]. The deviations suggested the interplay
between
moiré
potentials
and
strong
repulsive
exciton–exciton interactions of interlayer excitons. Diffu-
sions of interlayer exciton exhibited twist-angle depen-
dence, corroborating the twist-angle tunable moiré
potentials. Specifically, the DFT calculation indicated
deeper potential and stronger localization for interlayer
excitons at θ ≈ 0° than those at θ ≈ 60°. A larger energy
difference between the K and Q valleys of the conduction
band
in the 0°-twisted bilayer suppressed the effi-
cient electron scattering from Q to K, compared with
the 60° twisted case. Consequently, WS2/WSe2 heterobi-
layers presented a shorter K–K exciton decay time and
higher temperature for thermally excited phonons to
assist the Q–K scattering at near-0° than at near-60°, as
shown in Fig. 9(f) [134]. Choi and co-workers [158] also
reported that interlayer exciton diffusion in WSe2/
MoSe2 heterobilayers could be highly controlled by twist
angle, using spatially and time-resolved PL measurements
at T = 4 K, as shown in Fig. 9(g). The presence of
moiré superlattice with a large supercell and deep potential
at small twisting could completely localize interlayer
exciton and lead to a reduced lifetime and velocity. Consequently, the diffusion length was decreased in
comparison with that in commensurate heterostructure. At a larger twist angle leading to a reduced moiré period,
excitons may tunnel between supercells and have a of MoSe2/WSe2 heterobilayers showed a transition
temperature, above which the confined IXs turned to be
delocalized, manifesting as significantly decreased PL
intensity. The temperature dependence of IXs was
considered to result from IX–IX exchange interactions
exceeding the moiré potential traps at a sufficiently high
temperature [159]. Brem and Malic [155] have newly
reported the theoretical prediction that the strong delo-
calization of moiré-confined interlayer excitons would
occur at intermediate densities (1011‒1012 cm–2) due to
the combination of strong repulsive IX–IX interactions
and their bosonic nature. 3.2 Localization and modulation of excitons by moiré
superlattice (h) Schematics of the Brillouin zone of
twisted bilayers. The red (blue) hexagons denote the Brillouin zone of MoSe2 (WS2) monolayers. In the left panel with θ = 6°,
the green arrows indicate vectors of momentum shift between the Brillouin zone corners of the two monolayers. In the middle
(right) panel, a commensurate moiré reciprocal lattice is formed with the corresponding moiré Brillouin zone denoted by the
black hexagons. The yellow arrow represents the moiré reciprocal lattice base vector that connects KM and
(KW). (i) Twist-angle dependence of normalized PL spectra at 290 K (left panel) and reflectance contrast spectra at 10 K (right
panel) of MoSe2/WS2 heterobilayers near the A-exciton energy in an isolated MoSe2 monolayer (black). (a) Reproduced with
permission from Ref. [50]. (b‒d) Reproduced with permission from Ref. [141]. (e, f) Reproduced with permission from Ref. [134]. (g) Reproduced with permission from Ref. [158]. (h) Reproduced with permission from Ref. [135]. (i) Reproduced with
permission from Ref. [146]. would be located in a valley (Q/Q′) different from that
of the exciton (K/K′ ) [141]. The results indicate the
possibility of realizing exciton arrays with tunable quantum
coherence properties based on reconstructed moiré
superlattices. |K′⟩h
|K⟩h
The orthogonal
and
made linearly polarized
emission impossible because of the broken symmetry
caused by correlated resident holes. On the contrary, the
resident hole of type II excitons was in the other layer
and carried no information about the exciton valley,
allowing a non-zero DOLP. DOLP was non-zero for both
exciton species in the n-doped regime, because the electron Compared with intralayer excitons, interlayer excitons
with spatial separation and permanent dipole are Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-18 FRONTIERS OF PHYSICS TOPICAL REVIEW σ2
t −σ2
0
∆EK−Q
expected to be more deeply affected by moiré potential,
which could serve as interacting bosonic lattices suitable
for experimental realization of Bose–Hubbard model and
study on bosonic supersolid and superfluid states
[155–157]. Given the relatively long lifetime and diffusion
length of interlayer excitons, modulation of the interlayer
excitons diffusion dynamics by moiré superlattice is of
great interest [50, 54]. The exciton diffusion in the moiré
system can be described as a range of incoherent hops
between sites with local minimum of moiré potential. The confined moiré excitons are expected to have
shorter diffusion lengths compared with the delocalized
excitons. 3.2 Localization and modulation of excitons by moiré
superlattice The delocalization causes a
blueshift and the collapse of the split series of optical
moiré resonances, which would merge into a single free
exciton peak. The interlayer exciton lifetime has also
been demonstrated to be influenced by moiré potential
[160]. Based on a low-energy continuum mode, the twist-
angle-dependent IX lifetimes in twisted TMD bilayers
should consider two main mechanisms: One is the shift
to indirect transitions due to the rotation momentum
mismatch under an increasing twist angle. The other
mechanism is that the presence of moiré potential intro-
duces an interaction among the IXs with different COM
momenta and opens additional radiative recombination
channels, resulting in the relaxed momentum conservation
and consequently a softened twist angle dependence
[160]. σ2
t −σ2
0
∆EK−Q
expected to be more deeply affected by moiré potential,
which could serve as interacting bosonic lattices suitable
for experimental realization of Bose–Hubbard model and
study on bosonic supersolid and superfluid states
[155–157]. Given the relatively long lifetime and diffusion
length of interlayer excitons, modulation of the interlayer
excitons diffusion dynamics by moiré superlattice is of
great interest [50, 54]. The exciton diffusion in the moiré
system can be described as a range of incoherent hops
between sites with local minimum of moiré potential. The confined moiré excitons are expected to have
shorter diffusion lengths compared with the delocalized
excitons. The observed diffusion length of the moiré
exciton was one order of magnitude smaller than that in
the untwisted WSe2/MoSe2 heterostructure [26, 50]. Using transient absorption (TA) microscopy, Yuan et al. [134] investigated interlayer exciton dynamics and trans-
port in WS2/WSe2 heterobilayers under twist angles of 0°
and 60° with a 4% lattice mismatch, which had a moiré
period of about 7.6 nm. The diffusions of interlayer excitons
with non-linear temporal dependence of mean square
distance
deviated from the normal diffusions of
intralayer excitons in the constituent monolayers
[Fig. 9(e)]. The deviations suggested the interplay
between
moiré
potentials
and
strong
repulsive
exciton–exciton interactions of interlayer excitons. Diffu-
sions of interlayer exciton exhibited twist-angle depen-
dence, corroborating the twist-angle tunable moiré
potentials. Specifically, the DFT calculation indicated
deeper potential and stronger localization for interlayer
excitons at θ ≈ 0° than those at θ ≈ 60°. Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 3.2 Localization and modulation of excitons by moiré
superlattice They measured the moiré
potential for excitons in a large amount of MoSe2/MoS2
heterobilayers with various twist angles. Surprisingly,
the moiré potential depth for intralayer excitons was
found to be up to ~130 meV, which was larger than the
predicted value of most DFT calculations and comparable
to the potential depth for interlayer excitons. The deepened
moiré potential depth for intralayer excitons was Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-19 TOPICAL REVIEW TOPICAL REVIEW FRONTIERS OF PHYSICS decreased due to a flattened conduction valley. These
high-angle commensurate twisted bilayers exhibit opto-
electronic properties different from those in the small
twist angle cases and are commonly found in as-
prepared samples without deliberate alignment, which
would motivate further studies into dynamics and valley
properties of these high-angle moiré excitons [137]. attributed to the remarkable in-plane strain distribution
caused by atomic reconstruction, which would induce
band gap variation up to ~100 meV in MoSe2. Besides
vdW heterobilayers, multilayer-based systems can
provide new degree of freedom for moiré superlattice
regulations. Zheng and collaborators reported that inter-
layer excitons in both twisted WSe2/WSe2/WSe2 and
WSe2/WS2/WSe2 trilayers were confined by deeper
moiré potentials compared with bilayer systems, mani-
festing as multiple dense and sharp emission peaks in PL
spectra [162, 163]. Moiré system based on twisted TMD heterostructures
can form large-scale superlattice with spatially modulated
bandgap confining excitons in localized orbitals [52],
providing a strongly interacting regime [122]. The moiré
superlattice offers a compelling platform for exotic many-
body interactions phenomena, such as exciton BEC,
especially for the indirect interlayer excitons with interlayer
electric dipole and relatively longer lifetimes [122]. Recently, Remez et al. [122] have shown that the strong
interactions in twisted TMD bilayers can drive “leaky
emission” originate from “dark condensate” where
momentum-indirect excitons form a Bose–Einstein
condensate. These “leaky emissions” originate from
strong interactions and dominate the photoluminescence
of TMD moiré excitons at low temperatures with
distinctive features. 1
/√
7
For the circumstance suitable for the resonant
hybridization model, twist angle modulate the properties
and dynamics of interlayer and intralayer hybrid excitons
(hXs) by changing moiré reciprocal lattices [135]. The
properties of the hXs such as oscillator strengths, resonance
energies, and mixing of intralayer and interlayer compo-
nents, are sensitively dependent on the moiré period
(twist angle) [135, 146], as shown in Fig. 9(i). 3.2 Localization and modulation of excitons by moiré
superlattice It is
notable that though large detuning between interlayer
and intralayer states would suppress hybridization over
a range of angles deviating from aligned or anti-aligned
cases, pronounced hybridized moiré excitons with strong
interlayer tunneling occur under twist angles near 21.8°
and 38.2°, These high-angle moiré excitons were
observed in WS2/MoSe2 bilayers by reflectance contrast
spectra at 4 K. The features and valley configurations of
moiré hXs at θ = 21.8° and θ = 38.2° resembled those at
θ = 0° and 60°, respectively [135]. Moiré reciprocal
lattice at these angles are commensurate with the mono-
layer lattices in Brillouin zones and therefore the angularly
mismatched valleys of constituent monolayers are
connected by primitive moiré reciprocal lattices vectors. The commensurate moiré lattices allow nearly resonant
tunneling between the intra- and interlayer states, which
contributes to the observable hX features, as depicted in
Fig. 9(h) [135]. Furthermore, the moiré reciprocal
lattices at θ = 21.8° and 38.2° have the largest reciprocal
lattice constant equivalent to
of the monolayer
reciprocal lattice constant, corresponding to the smallest
moiré lattice with Σ = 7. Σ is the coincidence site lattice
(CSL), defined as the smallest unit cell volume shared
by the commensurately twisted bilayers. The unit cell
maintains the in-plane rotational and translational gliding
symmetry [137]. Zhao and co-workers [137] systematically
investigated the optical properties of TMD homobilayers
with various twist angles. For the MoS2 homobilayers
with Σ = 7 (θ = 21.8°), an intense peak of indirect
moiré excitons (1.6 eV) appeared in the PL spectra at
10 K. The indirect moiré excitons showed PL intensity
stronger than that of direct intravalley excitons (1.9 eV),
which was attributed to a smaller effective mass at the
multiple valley points in the conduction bands. Similar
commensurate structures at Σ = 13 (θ = 27.8°) also
showed a remarkable moiré exciton peak, but its relative
intensity with respect to the intravalley exciton 3.3 Spin-valley configuration of moiré excitons In this work,
PLE and resonant pump-probe spectroscopy were
utilized to detect the relative oscillator strengths and
spin-valley configurations of the moiré interlayer exci-
tons. The conception of exciton quasi-angular momentum
(QAM) was adopted here, which describes the phase
change of the exciton wave function after the
trans-
formation [Eq. (3.4)]. The total QAM of an interlayer /
[
]
The presence of moiré confinement for interlayer excitons
can realize perfect arrays of quantum emitters, which
are highly tunable by several external methods [49]. Seyler et al. [62] studied the valley-polarized nature of
the trapped interlayer excitons of MoSe2/WSe2 heterobi-
layers at T = 1.6 K. The moiré trapped IXs exhibited
strong valley polarizations with selection rules depending
on the twist angle, as shown in Fig. 10(d), corroborating
the retention of three-fold rotational symmetry under a
sufficient smooth moiré potential. They performed
magneto-photoluminescence spectroscopy to determine
the Landé g-factor of the trapped interlayer excitons
through the valley Zeeman effect [Fig. 10(e)]. The
extracted g-factors were homogeneous across the same
sample and took two values, –15.89 ± 0.03 and 6.72 ±
0.02 for near 60° (57°) and 0° (2°), respectively. The g-
factor of an interlayer exciton represents its valley
configuration and valley magnetic moment. Thus the
two different values indicated the distinct valley-pairing
configurations at the two twist angles. At θ ≈ 60°, the
spin-flip optical transitions would lead to emissions with
much larger Zeeman shifts, whereas the spin-singlet exci-
tonic Zeeman splitting at θ ≈ 0° was similar to that of
the monolayer. In particular, the trapped interlayer exci-
tons at θ = 20° (near the commensurate stacking angle
21.8° providing the shortest superlattice periodicity Σ =
7) had a g-factor close to that at θ = 57° because of the
same valley configurations, while the emission intensities
at θ = 20° were reduced by two orders of magnitude
compared with those at θ = 57° due to the weak optical
dipole of the umklapp recombination [62]. Beyond the moiré system based on heterobilayer,
introducing the layer degree of freedom would open up
more possibility of tuning the spin-valley configuration
through spin-layer locking. Brotons-Gisbert and co-
workers [165] fabricated a trilayer (TL) heterostructure
consisting of a monolayer (ML) WSe2 and bilayer (BL)
2H-MoSe2 (anti-aligned) and observed the spin-layer
locking of moiré-trapped excitons at cryogenic tempera-
ture. 3.3 Spin-valley configuration of moiré excitons In contrast to the monolayer TMD where
the spin-triplet excitons are considered dark, the optical
transition dipole of the spin-triplet interlayer exciton is
expected to be comparable to that of the spin-singlet
exciton in TMD heterobilayer due to the breaking of the
out-of-plane symmetry [66]. Yu et al. [66] reported that
as the local interlayer atomic registry changes, the optical
transition dipole of interlayer exciton wave packet varies
between two in-plane transition dipoles of opposite
circular polarizations and the out-of-plane one, which
enables the spatial modulation of IX light-coupling
including the strength of optical transition dipole and
the optical selection rule in a large scale moiré pattern. Additionally, the spin-triplet and spin-singlet excitons
have distinct valley polarization selection rules at a
certain atomic registry, as shown in Fig. 10(f). As a
result, the selective optical addressing of both the valley
and spin configurations of IXs would be achievable. Zhang and collaborators [164] identified the spin-singlet
and spin-triplet exciton states with very high valley
polarizations (over 80%) and opposite helicities in high-
quality hBN encapsulated WSe2/MoSe2 heterobilayers
with near 60° (58.7° ± 0.7°) twist angle at 4 K
[Fig. 10(g)]. The two exciton states with high valley
polarization and short lifetimes originated from direct
band-gap transition, which were regarded to be localized
at the
atomic registry (local point A as the potential
minimum). In addition, the long interlayer exciton
valley depolarization time suggested strongly suppressed
intervalley exchange interactions, on account of both
spatial separation of interlayer exciton as well as the
discretized spin singlet and triplet states [164]. Jin et al. [67] also observed that the coexisting multiple interlayer
exciton states localized at different sites in moiré supercell
and exhibited opposite optical selection rules in a near-0°
twisted WSe2/WS2 heterostructure at 10 K. In this work,
PLE and resonant pump-probe spectroscopy were
utilized to detect the relative oscillator strengths and
spin-valley configurations of the moiré interlayer exci- exciton state directly determines its optical selection rule,
which is related to the spin, valley, and moiré pattern. The spatially varying moiré QAM associated with different
interlayer lattice registrations explained the opposite
selection rules of multiple interlayer excitons in the
WSe2/WS2 heterostructure [67]. σ+(σ )
ˆC3
Hh
h
ˆC3
at local A (B) only couples to light with
circular
polarization, while light coupling at C is forbidden. At
other locals without
symmetry, optical selection rules
manifest as elliptical polarization [49]. Wu et al. 3.3 Spin-valley configuration of moiré excitons [63]
investigated the valley polarization of interlayer excitons
in WSe2/WSe2 twisted homobilayer by measuring the
polarized-resolved PL spectra at 8 K. Here, interlayer
excitons showed two emission peaks with opposite polar-
izations, as shown in Figs. 10(b) and (c). The distinct
behaviours of the two IX peaks were attributed to the
spin–orbit coupling effect. The conduction band split
into spin-up and spin-down energy levels, where the two
kinds of interlayer excitons with different transition
energies exhibit distinct spin configurations, depicted in
Fig. 10(c) [63]. In contrast to the monolayer TMD where
the spin-triplet excitons are considered dark, the optical
transition dipole of the spin-triplet interlayer exciton is
expected to be comparable to that of the spin-singlet
exciton in TMD heterobilayer due to the breaking of the
out-of-plane symmetry [66]. Yu et al. [66] reported that
as the local interlayer atomic registry changes, the optical
transition dipole of interlayer exciton wave packet varies
between two in-plane transition dipoles of opposite
circular polarizations and the out-of-plane one, which
enables the spatial modulation of IX light-coupling
including the strength of optical transition dipole and
the optical selection rule in a large scale moiré pattern. Additionally, the spin-triplet and spin-singlet excitons
have distinct valley polarization selection rules at a
certain atomic registry, as shown in Fig. 10(f). As a
result, the selective optical addressing of both the valley
and spin configurations of IXs would be achievable. Zhang and collaborators [164] identified the spin-singlet
and spin-triplet exciton states with very high valley
polarizations (over 80%) and opposite helicities in high-
quality hBN encapsulated WSe2/MoSe2 heterobilayers
with near 60° (58.7° ± 0.7°) twist angle at 4 K
[Fig. 10(g)]. The two exciton states with high valley
polarization and short lifetimes originated from direct
band-gap transition, which were regarded to be localized
at the
atomic registry (local point A as the potential
minimum). In addition, the long interlayer exciton
valley depolarization time suggested strongly suppressed
intervalley exchange interactions, on account of both
spatial separation of interlayer exciton as well as the
discretized spin singlet and triplet states [164]. Jin et al. [67] also observed that the coexisting multiple interlayer
exciton states localized at different sites in moiré supercell
and exhibited opposite optical selection rules in a near-0°
twisted WSe2/WS2 heterostructure at 10 K. 3.3 Spin-valley configuration of moiré excitons Excitons in moiré superlattice experience spatially varying
periodic potential due to the local bandgaps, determined
by the atomic registry. As shown in Fig. 7(c) and (e),
intralayer excitons and interlayer excitons confined in
the local energy minima have different landscapes,
accompanied with distinct spin-valley optical selection
rules [49]. In near-0° twisted heterostructures and
homostructures, valley optical selection rules for moiré
intralayer excitons were maintained, presenting a degree
of circular polarization (DOP) very similar to that in
the monolayer [Fig. 10(a)]. In conclusion, the moiré
pattern has no influence on the intralayer optical transi-
tions due to the unchanged rotational symmetry in the
plane of the monolayer [63, 121, 145]. ˆC3
On the contrary, optical selection rules for interlayer
excitons exhibit extra complexity. The intralayer excitons
excited by circularly polarized light will then relax
through the interlayer transfer process to form interlayer
excitons. The spin and valley indices of carriers would
be conserved in this process [65]. Unlike intralayer exci-
tons, interlayer excitons with certain spin-valley configu-
rations may generate emissions with circular polarizations
opposite to those of the excitation light. Take R-type
stacking MoX2/WX2 heterobilayer (X denotes chalcogen)
as an example. As mentioned in Section 3.1, moiré inter-
layer exciton wave packets at the high-symmetry locals
have distinct
transformations described by Eq. (3.4). Hence the corresponding convertible photons must have
the same rotational symmetry [65]. Therefore, the calcu-
lated optical selection rules would vary spatially, as
depicted in Fig. 7(e): The spin-up exciton wave packet Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-20 42501-20 TOPICAL REVIEW FRONTIERS OF PHYSICS σ+(σ−)
ˆC3
Hh
h
at local A (B) only couples to light with
circular
polarization, while light coupling at C is forbidden. At
other locals without
symmetry, optical selection rules
manifest as elliptical polarization [49]. Wu et al. [63]
investigated the valley polarization of interlayer excitons
in WSe2/WSe2 twisted homobilayer by measuring the
polarized-resolved PL spectra at 8 K. Here, interlayer
excitons showed two emission peaks with opposite polar-
izations, as shown in Figs. 10(b) and (c). The distinct
behaviours of the two IX peaks were attributed to the
spin–orbit coupling effect. The conduction band split
into spin-up and spin-down energy levels, where the two
kinds of interlayer excitons with different transition
energies exhibit distinct spin configurations, depicted in
Fig. 10(c) [63]. 3.3 Spin-valley configuration of moiré excitons Electrons (red shadows) at the ±K valleys localized either in the bottom or top layer MoSe2, are strongly bound to holes
(blue shadows) in the WSe2, creating two species of IX (green circles): IXH and IXR, respectively. (a) Reproduced with
permission from Ref. [121]. (b, c) Reproduced with permission from Ref. [63]. (d, e) Reproduced with permission from Ref. [62]. (f) Reproduced with permission from Ref. [66]. (g) Reproduced with permission from Ref. [164]. (h) (i, j) Reproduced
with permission from Ref. [165]. σ+
σ+
σ−
σ+ σ−
Rh
h
Fig. 10 Spin-valley configurations of moiré excitons. (a) Circular-polarization-resolved PL spectrum of MoSe2 measured in
(left panel) and out of (right panel) the MoSe2/MoS2 heterostructure region at T = 5 K. The green bars represent the DOP
of the peaks. (b) Interlayer excitons valley polarization in WSe2/WSe2 twisted homobilayer at T = 8 K. (c) The illustration
of the interlayer excitons transitions for IX1 and IX2 showing opposite circular polarizations. (d) Valley polarization of
trapped interlayer excitons of MoSe2/WSe2 heterobilayers with twist angles of 57° (co-circularly polarized), 20° (cross-circularly
polarized) and 2° (co-circularly polarized) excited by
-polarized light at T = 1.6 K. The
and
components of the
photoluminescence are shown in red and blue, respectively. (e) Dependence of Zeeman splitting of trapped interlayer excitons
on the twist angle in MoSe2/WSe2 heterobilayer. Top: Helicity-resolved PL spectra at 3 T under linearly polarized excitation. Bottom: Plot of total PL intensity as a function of magnetic field. (f) The spin-conserved and spin-flip momentum matrix
elements for R-type and H-type MoSe2/WSe2 heterobilayers. The red, blue and black colors correspond to the
,
and out-
of-plane (z) polarized components, respectively. (g) Valley-polarized singlet and triplet interlayer exciton emissions for hBN
encapsulated WSe2/MoSe2 twisted bilayer. (h) Circular-polarization-resolved PL spectra of representative IXR excitons of TL
heterostructures (ML-WSe2 and BL 2H-MoSe2) trapped in the site
under linearly polarized excitation at 2.33 eV and
different applied magnetic fields. (i) Schematics of the selection rules for optical transitions involving the K-point valence
band for both spin-singlet and spin-triplet IXs trapped in moiré potential sites with different atomic registries. (j) Sketch of
the TL heterostructures consisting of a 2H BL-MoSe2 crystal with ML- and BL-thick terraces stacked on top of an ML-WSe2. 3.3 Spin-valley configuration of moiré excitons Two types of interlayer excitons IXH and IXR were
identified, with electrons localized in the lower and
upper MoSe2 layer, respectively [Fig. 10(j)]. In magneto-
optical spectroscopy measurements, the two IX species
exhibited distinct spin-valley-locked magnetic moments
and exhibited different optical selection rules determined
by the local atomic registry of the moiré trapping sites,
as shown in Figs. 10(h) and (i). Such spin-layer locking
induces the layer index as a potential knob to engineer
few-level quantum systems in vdW heterostructures,
providing new opportunities for quantum information
applications [165]. Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-21 42501-21 TOPICAL REVIEW FRONTIERS OF PHYSICS σ+
σ+
σ−
σ+ σ−
Rh
h
Fig. 10 Spin-valley configurations of moiré excitons. (a) Circular-polarization-resolved PL spectrum of MoSe2 measured in
(left panel) and out of (right panel) the MoSe2/MoS2 heterostructure region at T = 5 K. The green bars represent the DOP
of the peaks. (b) Interlayer excitons valley polarization in WSe2/WSe2 twisted homobilayer at T = 8 K. (c) The illustration
of the interlayer excitons transitions for IX1 and IX2 showing opposite circular polarizations. (d) Valley polarization of
trapped interlayer excitons of MoSe2/WSe2 heterobilayers with twist angles of 57° (co-circularly polarized), 20° (cross-circularly
polarized) and 2° (co-circularly polarized) excited by
-polarized light at T = 1.6 K. The
and
components of the
photoluminescence are shown in red and blue, respectively. (e) Dependence of Zeeman splitting of trapped interlayer excitons
on the twist angle in MoSe2/WSe2 heterobilayer. Top: Helicity-resolved PL spectra at 3 T under linearly polarized excitation. Bottom: Plot of total PL intensity as a function of magnetic field. (f) The spin-conserved and spin-flip momentum matrix
elements for R-type and H-type MoSe2/WSe2 heterobilayers. The red, blue and black colors correspond to the
,
and out-
of-plane (z) polarized components, respectively. (g) Valley-polarized singlet and triplet interlayer exciton emissions for hBN
encapsulated WSe2/MoSe2 twisted bilayer. (h) Circular-polarization-resolved PL spectra of representative IXR excitons of TL
heterostructures (ML-WSe2 and BL 2H-MoSe2) trapped in the site
under linearly polarized excitation at 2.33 eV and
different applied magnetic fields. (i) Schematics of the selection rules for optical transitions involving the K-point valence
band for both spin-singlet and spin-triplet IXs trapped in moiré potential sites with different atomic registries. (j) Sketch of
the TL heterostructures consisting of a 2H BL-MoSe2 crystal with ML- and BL-thick terraces stacked on top of an ML-WSe2. 3.3 Spin-valley configuration of moiré excitons Electrons (red shadows) at the ±K valleys localized either in the bottom or top layer MoSe2, are strongly bound to holes
(blue shadows) in the WSe2, creating two species of IX (green circles): IXH and IXR, respectively. (a) Reproduced with
permission from Ref. [121]. (b, c) Reproduced with permission from Ref. [63]. (d, e) Reproduced with permission from Ref. [62]. (f) Reproduced with permission from Ref. [66]. (g) Reproduced with permission from Ref. [164]. (h) (i, j) Reproduced
with permission from Ref. [165]. 3.4 Moiré system under external tunning Tang et al. [148] employed a dual-gate device [Fig. 11(b)] to inde-
pendently apply a vertical electric field and doping to
the WSe2/WS2 (type II band alignment) and MoSe2/
WS2 (type I band alignment) heterobilayers at
5 K. They identified layer-hybridized moiré excitons
through an emergent feature iX in the doping-dependent
reflectance contrast spectrum, which was only observed
in anti-aligned (θ = 60°) WSe2/WS2 heterostructures. The configuration of anti-aligned WSe2/WS2 bilayers
[Fig. 11(b)] enabled extra Bragg reflection from the
moiré potential to help conserve quasi-momentum and
promote strong coupling. Therefore, the resonant interlayer
tunneling was regarded as spin-conserving. Under an out-
of-plane electric field, strong exciton coupling was
observed in WSe2/WS2 heterobilayers with coupling
constants comparable to the intralayer moiré potential
(~25 meV), manifesting as energy-level anticrossing and
electric-field-dependent oscillator redistribution between
the interlayer and intralayer resonances, as shown in
Fig. 11(c). The IX peak exhibited linear electric-field
response and weak oscillator strength when it was
decoupled from other excitons under a sufficiently large
positive perpendicular electric field, indicating the inter-
layer exciton nature of IX. The angle-aligned MoSe2/
WS2 heterobilayers had much weaker coupling with the
extracted coupling constants smaller than the exciton
linewidths (about 5–10 meV), but still showed discer-
nible multiple layer-hybridized moiré excitons with two
distinct electric-field dispersions. In addition to layer
hybridization, electric fields are also expected to control
moiré exciton transport. Based on the Förster theory,
the relative orientation of the dipole moments plays an
important role in the modulation of moiré exciton diffusion
[50]. Lately, several works have reported the electric-
field-tunable spatial distribution and transport of excitonic
species in 2D vdW heterostructures. Tagerlli et al. [170]
have achieved electrostatic control over layer-hybridized
exciton states represented by effective out-of-plane
dipole lengths and revealed the dipole-dependent
micrometre-scale transport of hybrid exciton. They
modulated the layer hybridization and many-body inter-
actions of excitonic species in 2H-stacked WSe2 homobi-
layers by applying a vertical electric field at 4 K. The
moiré-less 2H-WSe2 structures eliminated the potential
traps and type-II band alignment, which allowed tunable
exciton–exciton interactions and long-range propagating. 3.4 Moiré system under external tunning Under an out-
of-plane electric field, strong exciton coupling was
observed in WSe2/WS2 heterobilayers with coupling
constants comparable to the intralayer moiré potential
(~25 meV), manifesting as energy-level anticrossing and
electric-field-dependent oscillator redistribution between
the interlayer and intralayer resonances, as shown in
Fig. 11(c). The IX peak exhibited linear electric-field
response and weak oscillator strength when it was
decoupled from other excitons under a sufficiently large
positive perpendicular electric field, indicating the inter-
layer exciton nature of IX. The angle-aligned MoSe2/
WS2 heterobilayers had much weaker coupling with the
extracted coupling constants smaller than the exciton
linewidths (about 5–10 meV), but still showed discer-
nible multiple layer-hybridized moiré excitons with two
distinct electric-field dispersions. In addition to layer
hybridization, electric fields are also expected to control
moiré exciton transport. Based on the Förster theory,
the relative orientation of the dipole moments plays an
important role in the modulation of moiré exciton diffusion
[50]. Lately, several works have reported the electric-
field-tunable spatial distribution and transport of excitonic
species in 2D vdW heterostructures. Tagerlli et al. [170]
have achieved electrostatic control over layer-hybridized
exciton states represented by effective out-of-plane
dipole lengths and revealed the dipole-dependent
micrometre-scale transport of hybrid exciton. They
modulated the layer hybridization and many-body inter-
actions of excitonic species in 2H-stacked WSe2 homobi-
layers by applying a vertical electric field at 4 K. The
moiré-less 2H-WSe2 structures eliminated the potential
traps and type-II band alignment which allowed tunable sites, and generate a mixture of these interlayer excitons
as a superposition moiré states, which has been demon-
strated in a trilayer WSe2/monolayer WS2 heterostructure
reported by Lian and co-workers [171]. Besides, in the R-
stacked TMD vdW heterostructures accompanied with
breaking out-of-plane mirror symmetry, the electric field
can act as a switch to flip the out-of-plane polarization
through in-plane sliding motion, which would bring
about interfacial ferroelectricity [81, 172, 173]. Utilizing
piezoelectric force microscopy (PFM), Wang et al. [81]
have achieved the visualization of moiré ferroelectric
domains and observed the electric-field-induced domain
wall motion in several nearly parallel-stacked R-type
bilayers. the twisted MoS2/WS2 heterostructure, as shown in
Fig. 11(a). Furthermore, the shifts of CBM and VBM
from different constituent monolayers would control the
interlayer resonant tunneling of the electron or hole and
therefore the formation of hybridized excitons. In addi-
tion, an electric field can change both the electric dipole
orientations and dipole moments of hXs [50]. 3.4 Moiré system under external tunning p = eh
e
h
An applied external electric field can effectively tune
the properties of interlayer excitons due to their static
electric dipole perpendicular to the plane (
, where
is the charge quantity and
is the vector denoting
charge separation from negative charge to positive
charge) [50, 54]. The coupling between the perpendicular
electric field and the electric dipole would shift energy
levels due to the Stark effect, which can realize modulations
of the resonant energy and polarity of interlayer exciton. Consequently, the in-plane distributions of moiré interlayer
excitons are controllable by external electric fields. Guo
et al. [50] used first-principle calculations to predict the
charge density distributions tuned by the electric field in As the novel electronic and optical physics of moiré
superlattice based on van der Waals heterostructures
has been widely investigated theoretically and experi-
mentally, several researches on control of moiré systems
by external tunning methods have been reported in
recent years, such as electric fields [148], carriers doping
[154, 166], magnetic field [62, 165], temperature [136,
159], strain [144, 167, 168] and so on. Thanks to the
remarkable sensitivity to external factors, modulations
on moiré systems based on 2D vdW heterostructures
have shown desirable performance and novel physics. Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-22 42501-22 TOPICAL REVIEW FRONTIERS OF PHYSICS the twisted MoS2/WS2 heterostructure, as shown in
Fig. 11(a). Furthermore, the shifts of CBM and VBM
from different constituent monolayers would control the
interlayer resonant tunneling of the electron or hole and
therefore the formation of hybridized excitons. In addi-
tion, an electric field can change both the electric dipole
orientations and dipole moments of hXs [50]. Tang et al. [148] employed a dual-gate device [Fig. 11(b)] to inde-
pendently apply a vertical electric field and doping to
the WSe2/WS2 (type II band alignment) and MoSe2/
WS2 (type I band alignment) heterobilayers at
5 K. They identified layer-hybridized moiré excitons
through an emergent feature iX in the doping-dependent
reflectance contrast spectrum, which was only observed
in anti-aligned (θ = 60°) WSe2/WS2 heterostructures. The configuration of anti-aligned WSe2/WS2 bilayers
[Fig. 11(b)] enabled extra Bragg reflection from the
moiré potential to help conserve quasi-momentum and
promote strong coupling. Therefore, the resonant interlayer
tunneling was regarded as spin-conserving. Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 3.4 Moiré system under external tunning (d, e) Reproduced with permission from Ref. [154]. (f) Reproduced with permission from Ref. [166]. (g,
i) Reproduced with permission from Ref. [167]. (h) Reproduced with permissions from Refs. [144, 168]. FRONTIERS OF PHYSICS
TOPICAL REVIEW Nm
TAB
Fig. 11 Moiré system tunning by external methods. (a) The charge density distribution for the lowest-energy moiré exciton
in the twisted MoS2/WS2 heterostructure under different electric fields. Red and blue colors represent the charge density of
the electron and the hole, respectively. (b) Upper panel: Schematic of the dual-gate device structure for WSe2/WS2 bilayer. Lower panel: Type II band alignment for 60°-aligned and 0°-aligned samples. Intralayer (interlayer) dipole-allowed optical
transitions are represented by solid (dashed) double-headed arrows. (c) Electric-field dependence of layer-hybridized excitons
in 60°-aligned WSe2/WS2 moiré superlattice at fixed doping densities. Top row: Energy derivative of the reflectance contrast
spectrum. Bottom row: The extracted exciton resonance energies, where solid red lines and dashed black lines represent
experimental data and the best fit three-level model, respectively. The blue dotted lines show the dispersion of uncoupled
exciton states. (d) Moiré trions in a twisted bilayer MoS2. The left panel is the color plot of PL spectra under varying gate
voltage. Right panel: Peak intensities of the two trion peaks as a function of gate voltage. (e) Phase diagram of moiré trion
formation as a function of exciton density and electron density under positive gating, where
denotes moiré trap density. represent different types of trions, where the subscripts A and B denote the phase of the exciton and the electron
involved in the trion formation, respectively. (f) PL spectra as a function of doping. Insets depict the charge configuration of
both neutral and charged excitons. (g) Left two panels: Schematic of quasi-1D moiré superlattices formed with a uniaxial
strain S of 8% under different twist angle ∆θ, where the arrows highlight the primary 1D moiré structures and the dashed
rectangles of the secondary ones. Right two panels: PFM images at different locations of the twisted hBN/WSe2/MoSe2. Insets: FFTs of the PFM images. (h) Schematic of dynamic tunning of moiré excitons in WSe2/WS2 heterostructure by optical
fiber tip (upper panel) and DAC (lower panel). (i) Direct correlation between 1D moiré patterns and linearly polarized PL
emissions for WSe2/MoSe2 heterobilayers under different twist angles. Top row: Angular dependence of the PL emission. Bottom row: Corresponding angular distribution in the 2D FFTs of the PFM images. 3.4 Moiré system under external tunning In the presence of moiré superlattice, the electric-field-
dependent hybridization would allow continuous tuning
of interlayer exciton hopping between different moiré y
|Vg|
Doping introduces charged moiré excitons that obey
fermionic statistics and exhibit distinct properties such
as the long-range Coulomb interaction in contrast to
bosonic neutral excitons, providing an accessible platform
to study fermionic many-body effects [166]. Recently,
Dandu et al. [154] have realized the control of carrier
concentration by gate-induced doping in H-type stacked
twisted MoS2 bilayer, and investigated the coexisting
localized and delocalized intralayer moiré excitonic
species at 4.5 K. As
increased, the energies of
intralayer exciton peaks stayed relatively unchanged,
whereas their intensities (except the localized exciton XI)
quenched rapidly accompanied with emergent peaks
with lower energy, as shown in Fig. 11(d). The results
suggested
the
unchanged
spatial
modulation
of
intralayer moiré potential and the formation of trions
from the delocalized higher energy excitons. Through a
combination of gating (modulation of carrier density and
potential depth) and varying excitation power (exciton
density), multiple intralayer moiré trion species would
be formed with tunable localization phases, depicted in
Fig. 11(e). They also demonstrated the gate tunable
valley coherence and exciton–phonon coupling strength
for the moiré excitons. The charging effects would influence
the spin-valley physics of interlayer moiré exciton as
well. Wang and collaborators [166] observed moiré trions
as the moiré traps were filled with either electrons or
holes in H-stacked WSe2/MoSe2 heterobilayer at 1.6 K,
manifesting as new sets of narrow peaks in PL spectra,
as shown in Fig. 11(f). These emergent peaks were about
7 meV below the energies of the neutral interlayer moiré
excitons. The similar g-factors of these excitons and
trions (about ‒16) implied their same spin-triplet config-
uration and the smooth moiré potential traps. When
positively charged, the valley polarization of moiré trion
was inverted to cross-circular polarization under the σ+
excitation at WSe2 monolayer exciton resonance, in
contrast to the co-circularly PL in the neutral and negative
cases. Such inversion was explained by the competition
between spin-flip valley-conserved and spin-conserved
valley-flip relaxation channels of electrons during the
formation of interlayer trions. In both negative and Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-23 TOPICAL REVIEW FRONTIERS OF PHYSICS Nm
TAB
Fig. 11 Moiré system tunning by external methods. (a) The charge density distribution for the lowest-energy moiré exciton
in the twisted MoS2/WS2 heterostructure under different electric fields. 3.4 Moiré system under external tunning Red and blue colors represent the charge density of
the electron and the hole, respectively. (b) Upper panel: Schematic of the dual-gate device structure for WSe2/WS2 bilayer. Lower panel: Type II band alignment for 60°-aligned and 0°-aligned samples. Intralayer (interlayer) dipole-allowed optical
transitions are represented by solid (dashed) double-headed arrows. (c) Electric-field dependence of layer-hybridized excitons
in 60°-aligned WSe2/WS2 moiré superlattice at fixed doping densities. Top row: Energy derivative of the reflectance contrast
spectrum. Bottom row: The extracted exciton resonance energies, where solid red lines and dashed black lines represent
experimental data and the best fit three-level model, respectively. The blue dotted lines show the dispersion of uncoupled
exciton states. (d) Moiré trions in a twisted bilayer MoS2. The left panel is the color plot of PL spectra under varying gate
voltage. Right panel: Peak intensities of the two trion peaks as a function of gate voltage. (e) Phase diagram of moiré trion
formation as a function of exciton density and electron density under positive gating, where
denotes moiré trap density. represent different types of trions, where the subscripts A and B denote the phase of the exciton and the electron
involved in the trion formation, respectively. (f) PL spectra as a function of doping. Insets depict the charge configuration of
both neutral and charged excitons. (g) Left two panels: Schematic of quasi-1D moiré superlattices formed with a uniaxial
strain S of 8% under different twist angle ∆θ, where the arrows highlight the primary 1D moiré structures and the dashed
rectangles of the secondary ones. Right two panels: PFM images at different locations of the twisted hBN/WSe2/MoSe2. Insets: FFTs of the PFM images. (h) Schematic of dynamic tunning of moiré excitons in WSe2/WS2 heterostructure by optical
fiber tip (upper panel) and DAC (lower panel). (i) Direct correlation between 1D moiré patterns and linearly polarized PL
emissions for WSe2/MoSe2 heterobilayers under different twist angles. Top row: Angular dependence of the PL emission. Bottom row: Corresponding angular distribution in the 2D FFTs of the PFM images. The light-golden sectors represent the
angular distributions of the primary oval structures in the strained moiré landscapes, while the green sectors denote the
angular distributions of the secondary pseudo-1D stripes formed by the primary structures. The blue double arrows represent
the corresponding PL polarization directions. (a) Reproduced with permission from Ref. [50]. (b, c) Reproduced with permission
from Ref. [148]. Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 3.4 Moiré system under external tunning The PL
peaks of HS-1.15° showed continuous redshift with
approximately linear dependence for intralayer excitons,
which could be explained by the increased moiré potential
under smaller interlayer spacing. The exciton dispersion
band of the mini-Brillouin zone shifted to lower energy
levels under the deepened moiré potential. At pressure
higher than 1.1 GPa, effective enhancement of interfacial
coupling in all twisted heterostructures dominated the
exciton response in PL spectra [144]. However, conventional
experiment methods to realize in-situ deformation like
DAC are challenging to apply to moiré heterostructures
at low temperatures. Zhao et al. [168] utilized an optical
fiber tip in a cryogenic scanning near-field optical micro-
scope (SNOM) to locally deform the sample and
measure its background-free near-field optical response
simultaneously, as illustrated in Fig. 11(h). The moiré
potential
of
the
near-zero-twisted
WSe2/WS2
heterostructure increased under compressive deformation
at 25 K, resulting in redshift of both intralayer and
interlayer excitons. Under the maximum stress of 1.4
GPa, the sample obtained a modulation of 7 meV on the
moiré potential coefficient
in Eq. (3.3). The maximum
stress was limited by the damage threshold of the optical
fiber tip. Therefore, this method requires a tip made
from stronger materials to achieve a higher limit [168]. Cai et al. [161] utilized a nanopillar array placed under
the hBN-encapsulated MoSe2/WSe2 heterostructures to
introduce strain-enhanced coupling and strain gradient-
induced funneling, which helped them to corroborate
and develop the DAP IX dynamic model for understanding
the primary origin of moiré localized interlayer excitons. y
y
ˆC3
The excellent inherent mechanical flexibility and
remarkable strength of two-dimensional materials make
strain engineering an ideal tuning method for manipulating
their electronic and optical properties. For example,
Both graphene and TMD possess maximum elastic
strains larger than 10% [29]. Strain can realize continuous
band structure modulation [30, 32, 174, 175], and inho-
mogeneous strain can creat local variation of bandgap
[31, 176–178]. The stacking structures of twisted 2D
vdW heterostructures enable sensitive and dynamic
strain engineering of moiré potential landscape [167] and
topological properties [33]. Moreover, the moiré system
only requires small strains to achieve considerable modu-
lation. By applying proper differential (inequivalent
strains on the two constituent layers in the order of the
lattice mismatch) and uniaxial tensile strain to twisted
WSe2/MoSe2 heterobilayers, Bai et al. 3.4 Moiré system under external tunning The light-golden sectors represent the
angular distributions of the primary oval structures in the strained moiré landscapes, while the green sectors denote the
angular distributions of the secondary pseudo-1D stripes formed by the primary structures. The blue double arrows represent
the corresponding PL polarization directions. (a) Reproduced with permission from Ref. [50]. (b, c) Reproduced with permission
from Ref. [148]. (d, e) Reproduced with permission from Ref. [154]. (f) Reproduced with permission from Ref. [166]. (g,
i) Reproduced with permission from Ref. [167]. (h) Reproduced with permissions from Refs. [144, 168]. M −
T
M 0
X
neutrally charged cases (
and
) where the optical
selection rules were determined by the spin-valley index
of the photo-excited hole, the excited K-valley-polarized
hole could form a spin-triplet recombining pair with –K′
valley electron. The resulting high co-circular PL polar-
ization suggested that the valley polarization of the
excited hole was well-protected by spin-valley locking
and that these multiple election–hole pairs were all Hh
h
M +
T
M +
T
trapped at
locals. Under hole-doping (
), the emission
polarization was determined by the electron valley
configuration since the doped holes would distribute at
both opposite valleys. The relaxation channels of photo-
excited electrons involved either a spin flip or a valley
flip, where the latter was found to dominate the process,
resulting in the smaller cross-circular polarization of
[166]. Moreover, the properties of layer-hybridized excitons Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-24 42501-24 FRONTIERS OF PHYSICS TOPICAL REVIEW Vj
lation of moiré potential for twisted WS2/WSe2
heterostructures by employing diamond anvil cell (DAC)
hydrostatic pressure engineering at room temperature
[Fig. 11(h)]. When the pressure was lower than 1.1 GPa,
the exciton response of heterostructures (HS) had
distinct pressure dependence under different twist angles. For HS-7.5°, PL peaks exhibited blue shift trends due to
the changed band structure, in which the energy of
direct transition from the K valley would increase under
larger pressure. Whereas in HS-1.15°, the predominant
moiré effect led to different exciton responses. The PL
peaks of HS-1.15° showed continuous redshift with
approximately linear dependence for intralayer excitons,
which could be explained by the increased moiré potential
under smaller interlayer spacing. The exciton dispersion
band of the mini-Brillouin zone shifted to lower energy
levels under the deepened moiré potential. Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 3.4 Moiré system under external tunning At pressure
higher than 1.1 GPa, effective enhancement of interfacial
coupling in all twisted heterostructures dominated the
exciton response in PL spectra [144]. However, conventional
experiment methods to realize in-situ deformation like
DAC are challenging to apply to moiré heterostructures
at low temperatures. Zhao et al. [168] utilized an optical
fiber tip in a cryogenic scanning near-field optical micro-
scope (SNOM) to locally deform the sample and
measure its background-free near-field optical response
simultaneously, as illustrated in Fig. 11(h). The moiré
potential
of
the
near-zero-twisted
WSe2/WS2
heterostructure increased under compressive deformation
at 25 K, resulting in redshift of both intralayer and
interlayer excitons. Under the maximum stress of 1.4
GPa, the sample obtained a modulation of 7 meV on the
moiré potential coefficient
in Eq. (3.3). The maximum
stress was limited by the damage threshold of the optical
fiber tip. Therefore, this method requires a tip made
from stronger materials to achieve a higher limit [168]. Cai et al. [161] utilized a nanopillar array placed under
the hBN-encapsulated MoSe2/WSe2 heterostructures to
introduce strain-enhanced coupling and strain gradient-
induced funneling, which helped them to corroborate
and develop the DAP IX dynamic model for understanding
the primary origin of moiré localized interlayer excitons. Moiré system combined with other elements would
f
ili
f
il
h
i
d d
f
h
i
i including energies, coupling constants and effective
dipole moment can also be significantly modulated by
doping through electron-exciton interactions under
considerable localization of moiré potential [148]. The
rich and complex phenomena of moiré excitonic species
under electrical modulation indicate the promising
potential of engineering both bosonic and fermionic
many-body
physics. Furthermore,
their
dynamic
adjustable features suggest the possibilities of photonic
and valleytronic devices based on moiré systems. Vj
lation of moiré potential for twisted WS2/WSe2
heterostructures by employing diamond anvil cell (DAC)
hydrostatic pressure engineering at room temperature
[Fig. 11(h)]. When the pressure was lower than 1.1 GPa,
the exciton response of heterostructures (HS) had
distinct pressure dependence under different twist angles. For HS-7.5°, PL peaks exhibited blue shift trends due to
the changed band structure, in which the energy of
direct transition from the K valley would increase under
larger pressure. Whereas in HS-1.15°, the predominant
moiré effect led to different exciton responses. FRONTIERS OF PHYSICS [118] have presented that TMD moiré flat
bands could exist over a range of twist angles (smaller
than around 3.5°), enabling continuous tuning of correla-
tion effects. They used the continuum model to describe
the low-energy electrons in the moiré superlattice, in
which the Hamiltonians were spatially periodic under
effective moiré potential [64, 118]. The moiré band
Hamiltonian for twisted TMDs heterobilayers had only
two low-energy valence bands when the chemical potential
was within the topmost valence bands, and could be
mapped to triangular-lattice single-band Hubbard
models with the Wannier wave function localized in one
of the moiré potential extremum positions. The estimated
hopping parameters (related to the bandwidth
) and
on-site Coulomb repulsion energy
(sensitive to the
effective dielectric constant
) both varied with the
moiré period, resulting in a correlation strength that
could be modulated over a wide range by twist angle. The relatively strong on-site repulsion would suppress
double occupation of one moiré site when satisfying
, and therefore the Mott insulator ground state
was predicted at hole half-filling (filling factor
considering the Hubbard model spin degeneracy, signifying
one electron per moiré unit cell) [118]. Hubbard model is
a simple theoretical model of interacting quantum particles
in a lattice, which is considered to be paradigmatic in
the physics of complex quantum many-body effects [129,
189, 190]. The TMDs moiré superlattices are regarded as
an analogue quantum simulator of the Hubbard model
with widely tunable parameters like bandwidth, interaction
strength and band filling which could play an important FRONTIERS OF PHYSICS The moiré band
Hamiltonian for twisted TMDs heterobilayers had only
two low-energy valence bands when the chemical potential
was within the topmost valence bands, and could be
mapped to triangular-lattice single-band Hubbard
models with the Wannier wave function localized in one
of the moiré potential extremum positions. The estimated
hopping parameters (related to the bandwidth
) and
on-site Coulomb repulsion energy
(sensitive to the
effective dielectric constant
) both varied with the
moiré period, resulting in a correlation strength that
could be modulated over a wide range by twist angle. The relatively strong on-site repulsion would suppress
double occupation of one moiré site when satisfying
, and therefore the Mott insulator ground state
was predicted at hole half-filling (filling factor
considering the Hubbard model spin degeneracy, signifying
one electron per moiré unit cell) [118]. Hubbard model is
a simple theoretical model of interacting quantum particles
in a lattice, which is considered to be paradigmatic in
the physics of complex quantum many-body effects [129,
189, 190]. The TMDs moiré superlattices are regarded as
an analogue quantum simulator of the Hubbard model
with widely tunable parameters like bandwidth, interaction
strength and band filling, which could play an important
role in revealing the physics of various strongly interacting
quantum particles [118, 128–131, 183, 191]. Wang et al. p
[
,
,
,
]
λ
m∗
e ≈0.5m0
m0
t
W
U
ε
U ≫t
v = 1
A real moiré system based on van der Waals
heterostructures possesses more complexity beyond the
simplified model shown above. Flat bands in the
graphene-based heterostructures appear only at a
discrete set of magic angles [123, 127], limiting the
tunability of the strongly correlated systems. A sufficiently
strong moiré effects require effective interlayer coupling
between the constituent materials. Besides, a small
enough twist angle provides a large but simultaneously
brings about atomic reconstruction [132, 140], which
would decrease the homogeneity of the moiré superlat-
tice. Given these factors, TMD moiré heterostructures
are supposed to offer an ideal system for the exploration
of quantum many-body phenomena thanks to their
strong light–matter interactions, considerable moiré
potential and the reduced degeneracy arising from the
coupled spin-valley index [128, 131, 181]. The electronic
band structures of TMDs with relatively large effective
mass (
, with
as the free electron mass)
facilitate flat band features and many-body effects [181]. Wu et al. 3.4 Moiré system under external tunning [167] transformed
the zero-dimensional (0D) moiré pattern into a one-
dimensional (1D) structure with parallel stripes and
presented real-space imaging of these two patterns using
PFM, as shown in Fig. 11(g). In the 1D moiré pattern,
the photoluminescence emission at 4 K from interlayer
exciton showed a single broad peak and significantly
higher intensity in contrast to the 0D case. The
enhanced broad peak in PL spectra was attributed to
the higher density of states with densely spaced excitonic
levels and radiative recombination from multiple levels. The emissions of 1D structures were linear polarized and
their directions were aligned with the direction of one of
the primary structures obtained from 2D-FFT (Fast
Fourier Transforms) image analysis [Fig. 11(i)]. The
linear polarization here was qualitatively attributed to
the hybridization of the two valley configurations
through electron-hole exchange interaction, because the
electron-hole wave functions were highly anisotropic in
the 1D moiré potential from the breaking
rotational
symmetry. The strain-field-induced 1D moiré potential,
analogous to the quantum wires, is attractive for studying
strongly correlated and anisotropic charge transport
[167]. Apart from the differential strain, in-situ deformation
is another approach to realize dynamic control of the
moiré potential and correlated states by directly altering
the interlayer separation of the vdW heterostructures,
which can help gain a clear understanding of the exciton
physical behaviour in moiré system [144, 168]. Li and co-
workers [144] have performed the in situ dynamic modu Moiré system combined with other elements would
facilitate more fertile physics and deserve further investi-
gation. Anđelković et al. [169] discussed the supermoiré
(SM) resulting from two slightly misaligned interfering
moiré patterns in the trilayer system. They concentrated
on graphene encapsulated by hBN and quantitatively
described the angle-dependent SM effects that led to
electronic spectrum reconstruction, velocity suppression
and spatial electron redistribution. Fitzgerald et al. [34]
studied twist-angle-tunable moiré exciton polaritons
resulting from hybridization between moiré intralayer
excitons and photons in the strong coupling regime. They focused on twisted AA-stacked MoSe2/WSe2
heterostructures integrated within a Fabry‒Pérot cavity. Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-25 42501-25 FRONTIERS OF PHYSICS TOPICAL REVIEW They theoretically predicted the delocalized nature of
the new hybrid light-exciton states and the optical
responses of moiré exciton polaritons including exciton-
light coupling, polariton energy and the number of
polariton branches, which could be modulated by twist
angle and cavity [34]. Moiré patterns have also
prompted new progress in diverse fields. For example,
moiré phonon physics derived from the atomic stress
redistribution and folding effects of phonon dispersion in
twisted 2D materials can drastically regulate the lattice
vibration modes and thermal transport properties [91]. Besides, a variety of researches involved with moiré
structures have been reported like gap solitons in
parity–time symmetric moiré optical lattice [179], and
moiré bilayer hyperbolic metasurface with highly
controllable electromagnetic responses [180]. These
research efforts are expected to pave the way to realize
flat bands and localized states at arbitrary frequencies
for multifunctional devices. Cao and co-workers, a wealth of moiré-system-based
correlated electronic states in twisted vdW heterostruc-
tures has been reported [61, 129–131, 181, 183–188]. tures has been reported [61, 129–131, 181, 183–188]. λ
m∗
e ≈0.5m0
m0
t
W
U
ε
U ≫t
v = 1
A real moiré system based on van der Waals
heterostructures possesses more complexity beyond the
simplified model shown above. Flat bands in the
graphene-based heterostructures appear only at a
discrete set of magic angles [123, 127], limiting the
tunability of the strongly correlated systems. A sufficiently
strong moiré effects require effective interlayer coupling
between the constituent materials. Besides, a small
enough twist angle provides a large but simultaneously
brings about atomic reconstruction [132, 140], which
would decrease the homogeneity of the moiré superlat-
tice. Given these factors, TMD moiré heterostructures
are supposed to offer an ideal system for the exploration
of quantum many-body phenomena thanks to their
strong light–matter interactions, considerable moiré
potential and the reduced degeneracy arising from the
coupled spin-valley index [128, 131, 181]. The electronic
band structures of TMDs with relatively large effective
mass (
, with
as the free electron mass)
facilitate flat band features and many-body effects [181]. Wu et al. [118] have presented that TMD moiré flat
bands could exist over a range of twist angles (smaller
than around 3.5°), enabling continuous tuning of correla-
tion effects. They used the continuum model to describe
the low-energy electrons in the moiré superlattice, in
which the Hamiltonians were spatially periodic under
effective moiré potential [64, 118]. 4 Correlated electronic states in moiré
superlattice Furthermore, TMDs moiré superlattice are
expected to provide a promising platform for engineering
novel correlated states, such as emerging correlated
valley and spin physics thanks to the layer-valley-spin
locking in TMDs [165, 185, 195]. v = 1/2
utilized a new optical sensing method based on dielectric-
sensitive excited-state excitons in WS2 monolayer as a
probe of correlated states. As depicted in Fig. 12(f), the
cascade insulating states with energy ordering nearly
symmetric about filling factor
(half a particle
per moiré supercell) were attributed to a series of charge-
ordered states ranging from generalized Wigner crystals
to charge density waves, which further corroborated the
remarkable long-range interactions in TMD-based moiré
systems. rs
rs ⩾14
v = 1/2
Shimasaki et al. [131] have reported the incompressible
electronic states probed by the exciton-polariton reso-
nances in a long-period moiré superlattice. They fabricated
a MoSe2/hBN/MoSe2 homobilayer with λ = 24 nm and
a large
parameter (
, the ratio of interaction
energy to kinetic energy). The inserted hBN layer weakened
the moiré potential and increased the on-site interaction
strength, making the homobilayer moiré system a unique
regime beyond the standard Fermi-Hubbard model. They controlled the hybridization as well as the doping
of the top and bottom layers independently. The
hybridization between intralayer and interlayer excitons
with opposite dipole moments was regulated through
electric-field-dependent coherent carrier tunneling. The
charging configuration of the two layers could be simul-
taneously detected by the attractive polaron (AP) and
repulsive polaron (RP) branches in the differential
reflectance spectrum due to the dynamical screening of
excitons by itinerant carriers. Negative compressibility
was observed as the top gate voltage increased while the
back gate fixed, manifesting as the depletion of electrons
from the bottom layer due to the intralayer exchange
interaction [Fig. 12(g)], indicating the prominent electron
–electron interactions at high electron densities [131]. In
the low-electron-density regime at half-filling of the
lowest moiré subband (
), abrupt collective inter-
layer transfer of electrons was observed with resilience
towards electric-field-induced charge redistribution, as
shown in Fig. 12(i). This result suggested the existence
of Mott-like incompressible electronic states, which
could be accommodated in either the top or the bottom
MoSe2 layer. Similar interaction-induced incompressible
electronic states with abrupt phase transitions also
appeared for other doping levels equal to an integer
multiple of the moiré supercell density. 4 Correlated electronic states in moiré
superlattice U
W
λ
U ≈
e2
4πελ
W ≈
ℏ2π
2m∗
eλ2
ε
m∗
e
U/W > 1
m∗
eλ
λ
W
Moiré superlattice in vdW heterostructures provides a
general and promising solid-state platform for exploring
and engineering correlated electronic states, supported
by the strong periodic moiré potential with the flat elec-
tronic band. The core of the flat band lies in the compa-
rability between the electron–electron interaction and
the electronic bandwidth
under sufficient narrow elec-
tronic bands, enabling the effects of Coulomb interactions
to become pronounced in the system [181]. We can
consider a simplified model of a two-dimensional electron
gas in a periodic potential with a period , accompanied
with the formation of a set of electronic minibands. For
the lowest electronic miniband, it can be estimated that
and
, where and
are the effective
dielectric constant and electron effective mass, respec-
tively. Strong correlation would become achievable when
the ratio of electron-electron interaction to electronic
bandwidth satisfies
, which needs sufficient
large
[181]. The theoretical analysis of such systems
must consider the correlated electronic effects instead of
one electron approximation, which can trigger abundant
quantum many-body states such as quantum anomalous
Hall insulators and high-temperature superconductivity
[118, 182]. A comprehensive understanding of such
exotic physics based on strong electronic correlations
requires both in-depth theory and experiment researches
that complement each other. In a moiré superlattice, the
electronic correlated effects would be enhanced under a
large
with reduced bandwidth
, which can be
achieved by a small twist angle θ. Since the correlated
insulator [59] and unconventional superconductivity [60]
were observed in magic-angle graphene superlattice by Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-26 42501-26 FRONTIERS OF PHYSICS TOPICAL REVIEW U/W
[128] have reported the observations of the correlated
insulator and zero-resistance regions in bilayer WSe2
under twist angles ranging from 4 to 5.1°, indicating the
continuously tunable collective phases in the TMD-
based moiré system. Utilizing transport measurements,
the effective electronic interactions
here were
demonstrated to be tunable by external electric field and
doping, driving a continuous metal-insulator transition
(MIT) at near half-filling of the first moiré subband (one
hole/electron per superlattice site) [183, 191]. The real-
ization of the highly controllable triangular-lattice
Hubbard model offers new avenues in exploring the
correlation insulating phase transitions [191] that would
potentially relate to exotic states such as quantum spin
liquids [192, 193] and unconventional superconductivity
[194]. 4 Correlated electronic states in moiré
superlattice (m) The phase diagram of the correlated interlayer exciton insulator
in the double-layer system detected by the WSe2 monolayer 2s exciton signal. (n) MIM spectra as a function of gate voltage
for the moiré superlattice of 1L/1L, 2L/1L and 3L/1L WSe2/WS2 at 10 K, respectively. (a‒c) Reproduced with permission
from Ref. [181]. (d, e) Reproduced with permission from Ref. [129]. (f) Reproduced with permission from Ref. [130]. (g,
i) Reproduced with permission from Ref. [131]. (h) Reproduced with permission from Ref. [187]. (j, k) Reproduced with
permission from Ref. [186]. (l, m) Reproduced with permission from Ref. [188]. (n) Reproduced with permission from Ref. [185]. FRONTIERS OF PHYSICS
TOPICAL REVIEW ∆˜V
n0/2
v = −1/3
(vtop, vbot)
Fig. 12 Correlated electronic states in moiré systems. (a) Illustration of a near-zero twisted WSe2/WS2 heterostructure
device used for an ODRC measurement. A small a.c. bias (
) leads to charge redistribution between the region covered by
top gate and the uncovered region, which is detected via the change in the optical reflectivity of the WSe2 exciton in the
uncovered region. (b) The doping-dependent Mott insulating states and Wigner crystal of devices in (a) probed by optical
contrast reflectance ΔOC. (c) Illustrations of generalized Wigner crystal and Mott insulator states in a WSe2/WS2 moiré
superlattice. (d) Two-terminal resistance of 60° aligned WSe2/WS2 bilayers as a function of filling factor at different temper-
atures. The inset shows the temperature-dependent resistance at two different filling factors. (e) Dependence of the magnetic
susceptibility g–g0 (left axis, black filled symbols) on the filling factor at 1.65 K and Weiss constant θ (right axis, red empty
symbols) in 60° aligned WSe2/WS2 bilayers, with
as the density of moiré supercell. Here g–g0 is the difference of Landé g-
factor between excitons in the moiré superlattice and bare excitons. (f) Nearly two dozen insulating states and their energy
ordering in a WSe2/WS2 moiré heterostructure at 1.6 K probed by the 2s exciton in the sensor. The top axis shows the
proposed filling factor for the insulating states. (g) Gate dependence of differential reflectance spectrum of MoSe2/hBN/
MoSe2 with back gate fixed at 4 V. (h) Observation of optically induced ferromagnetism at near
filling of WS2/
WSe2 measured by power-dependent RMCD at 1.6 K. 4 Correlated electronic states in moiré
superlattice This kind of
moiré system was expected to act as a new platform for
studying the interaction between dipolar excitons and
electrons confined to flat bands, like Bose–Fermi
mixtures [131]. v = 1
n = n0 n0
v = 1/3
n = n0/3
v = 2/3
n = 2n0/3
U/t ≫1
The
strong
light–matter
interactions
of
TMD
heterostructures enable optical spectroscopy measure-
ments to detect the strongly correlated phases, comple-
mentary to the traditional transport measurements [93,
181]. Regan et al. [181] have realized the optical detection
of strongly correlated phases in near-zero twisted WSe2/
WS2 heterostructures. They developed a novel measuring
technique called optically detected resistance and capaci-
tance (ODRC) [Fig. 12(a)], which could avoid the large
contact resistances that impeded direct electrical transport
measurements. As shown in Fig. 12(b), they observed
the Mott insulating state at hole doping factor
(
;
was the moiré supercell density) and additional
insulating states at
(
) and
(
) in the heterostructures with moiré period λ ≈
8 nm at 3 K. The additional insulating states were
attributed to generalized Wigner crystallization of holes
in the moiré superlattice, where holes tried to avoid
simultaneous occupation of adjacent sites [Fig. 12(c)]. The emergence of generalized Wigner crystallization
arises from the relatively large inter-site interaction
compared with the moiré miniband bandwidth, indicating
the necessary of an extended Hubbard model that
considers inter-site (long-range) interactions in addition
to on-site (short-range) interactions in TMDs moiré
superlattice [181]. Tang et al. [129] have also reported
the observation of Mott insulating state in aligned
WSe2/WS2 heterobilayers using the close-cycle cryostat
magneto-optical measurements. The Mott insulating
state occurred at half-filling of the first hole moiré band
in
the
limit
of
with
antiferromagnetic
Curie–Weiss behaviour [Figs. 12(d) and (e)], suggesting
the antiferromagnetic interaction between the moiré
band holes. Xu and co-workers [130] have observed
nearly two dozen correlated insulating states at several
fractional fillings of WSe2/WS2 moiré superlattice. They U
t
As mentioned above, TMDs moiré superlattice have
emerged as an ideal system for studying correlated elec-
tronic physics with high tunability, where the onsite
Coulomb interaction
and the nearest-neighbour
hopping parameter are controllable by varying twist
angle [128] and electric field [183, 191]. Apart from these
methods, Wang et al. [187] and Chen et al. [185] have Siwei Li, et al., Front. Phys. 4 Correlated electronic states in moiré
superlattice 19(4), 42501 (2024) 42501-27 42501-27 TOPICAL REVIEW FRONTIERS OF PHYSICS ∆˜V
n0/2
v = −1/3
(vtop, vbot)
Fig. 12 Correlated electronic states in moiré systems. (a) Illustration of a near-zero twisted WSe2/WS2 heterostructure
device used for an ODRC measurement. A small a.c. bias (
) leads to charge redistribution between the region covered by
top gate and the uncovered region, which is detected via the change in the optical reflectivity of the WSe2 exciton in the
uncovered region. (b) The doping-dependent Mott insulating states and Wigner crystal of devices in (a) probed by optical
contrast reflectance ΔOC. (c) Illustrations of generalized Wigner crystal and Mott insulator states in a WSe2/WS2 moiré
superlattice. (d) Two-terminal resistance of 60° aligned WSe2/WS2 bilayers as a function of filling factor at different temper-
atures. The inset shows the temperature-dependent resistance at two different filling factors. (e) Dependence of the magnetic
susceptibility g–g0 (left axis, black filled symbols) on the filling factor at 1.65 K and Weiss constant θ (right axis, red empty
symbols) in 60° aligned WSe2/WS2 bilayers, with
as the density of moiré supercell. Here g–g0 is the difference of Landé g-
factor between excitons in the moiré superlattice and bare excitons. (f) Nearly two dozen insulating states and their energy
ordering in a WSe2/WS2 moiré heterostructure at 1.6 K probed by the 2s exciton in the sensor. The top axis shows the
proposed filling factor for the insulating states. (g) Gate dependence of differential reflectance spectrum of MoSe2/hBN/
MoSe2 with back gate fixed at 4 V. (h) Observation of optically induced ferromagnetism at near
filling of WS2/
WSe2 measured by power-dependent RMCD at 1.6 K. (i) Electric field dependence of differential reflectance spectrum of
MoSe2/hBN/MoSe2 at two fixed filling factors, with charge configuration of the top and bottom layer indicated by
in green. (j) Schematic of moiré exciton I and III under hole and electron doping. (k) Doping-dependent reflection contrast
spectrum of a WSe2/WS2 moiré superlattice shows distinct behaviour for WSe2 moiré excitons I, II and III. (l) The schematic
of hole distributions for the double-layer system of (left panel) the Mott insulator at point A of (m), (middle panel) interlayer
exciton insulator at point B of (m), and (right panel) the particle-hole transformation of the doped Mott insulator state at
point B of (m) to form the interlayer exciton insulator. 4 Correlated electronic states in moiré
superlattice The moiré excitons behaviours under optical measure-
ments with limited spatial resolutions are commonly
described by a continuum model [118, 197], which
considers the center-of-mass motion of the pristine exciton
modulated by the effective moiré potential, assuming
that the internal spatial electron-hole correlations from
the pristine single layers are retained [186]. The prevailing
effective continuum model is limited to large moiré periods
and cannot capture the microscopic nature of exciton
states in real moiré superlattice, where electrons and
holes could experience different moiré effects and exhibit
distinct spatially modulated moiré flat bands [133, 139,
198]. Combining large-scale first-principles GW-BSE
predictions with micro-reflection spectroscopy, Naik and
co-workers [186] have identified various exciton states
with distinct electron–hole correlations in rotationally
aligned WSe2/WS2 reconstructed moiré superlattice at
T = 1.6 K, including a Wannier-type exciton with
lowest energy and a new-found intralayer charge-transfer
exciton with higher resonance energy. Different from the
Wannier-type moiré exciton with tightly correlated elec-
tron and hole, the intralayer charge transfer exciton had
electron and hole densities distributed with about
5 nm separation in the WSe2 layer. The distinct spatial
electron–hole distributions resulted in different carrier
densities and magnetic-field dependence. As shown in
Fig. 12(k), the moiré peak I (Wannier exciton) was
almost unaffected by doping except for the formation of
Mott state at one doped hole per moiré unit cell, while
the intralayer charge exciton resonance (the moiré peak
III) was strongly modulated by carrier doping due to the p
pmono
pbi
p = pbi + pmono
p
pbi = p0
p0
p/p0 = 1
p
Taking advantage of the rich excitonic physics in
TMD vdW heterostructures, several new correlated insu-
lating phases of excitons have been reported in recent
years. Zhang et al. [188] integrated the moiré superlattice
into a “double-layer system” composed of a near-aligned
WS2/WSe2 heterobilayer (moiré superlattice) and a
WSe2 monolayer separating by an ultrathin hBN layer,
aiming at realizing flat electronic bands and strong
interlayer electron–hole interactions simultaneously. They controlled the total hole concentration and the
average electric field independently. The electric field
determined the hole distribution between the WSe2
monolayer
and the WSe2 from moiré bilayer
,
following a relation of
. 4 Correlated electronic states in moiré
superlattice The systematic changes including
emergent exciton resonances and increased resonance
energy separations in the differential reflectance spectra
suggested that moiré coupling was strongly confined at
the WSe2/WS2 interface with limited out-of-plane exten-
sion. In the multilayer WSe2/1L WS2, the added WSe2
layer(s) could affect the hybridization between moiré
excitons
and
the
interlayer-like
hybrid
exciton. Microwave impedance microscopy (MIM) measurements
demonstrated the tunability of the electron correlation
by layer degree of freedom, as illustrated in Fig. 12(n). As the layer number of WSe2 increased, the correlated
insulating states became fewer and less pronounced in
the MIM spectra accompanied with reduced Mott transi-
tion temperature. The reduction of transition temperature
indicated the weaker inter-site and on-site electron inter-
action strengths, which could be explained by reduced
interfacial Coulomb interaction under enhanced dielectric
screening and the increased kinetic energy originated
from the larger bandwidth of the flat band in multilayer
WSe2/1L WS2. It is worth noting that even the weakened
electron correlation (with the Mott transition temperature
about 60 K for 3L WSe2/1L WS2) reported here was still
stronger than that in the graphene moiré system (with
transition temperature of about 4 K). pmono
monolayer from occupying the same sites with holes
from the WS2/WSe2 moiré bilayer. This correlated insu-
lating phase was interpreted as an interlayer exciton
insulator through particle-hole transformation, where
holes in the WSe2 monolayer and the doped electrons in
the WS2/WSe2 moiré bilayer could form tightly bound
interlayer excitons, as depicted in Fig. 12(l). This interlayer
exciton insulator would evolve to other correlated phases
such as metal-like optical response under sufficient high
, and could potentially form exciton condensate and
counterflow superfluidity [188]. Recently, Xiong et al. [196] have also reported an exciton incompressible state
as a bosonic correlated insulator in WSe2/WS2 moiré
superlattices persisting above 30 K. Using a unique
pump-probe spectroscopy method to independently tune
the charge and exciton density through electrostatic
gating and pump light respectively, they achieved
continuous transitions between the bosonic correlated
insulating phase and the electron correlated insulator
where excitons avoid electron-occupied sites under on-
site electron‒exciton repulsion. These excitonic correlated
states observed in semiconducting moiré superlattices
open up new ways for manipulating emergent bosonic
phases and many-body interactions involving both
fermions and bosons [196]. 4 Correlated electronic states in moiré
superlattice (i) Electric field dependence of differential reflectance spectrum of
MoSe2/hBN/MoSe2 at two fixed filling factors, with charge configuration of the top and bottom layer indicated by
in green. (j) Schematic of moiré exciton I and III under hole and electron doping. (k) Doping-dependent reflection contrast
spectrum of a WSe2/WS2 moiré superlattice shows distinct behaviour for WSe2 moiré excitons I, II and III. (l) The schematic
of hole distributions for the double-layer system of (left panel) the Mott insulator at point A of (m), (middle panel) interlayer
exciton insulator at point B of (m), and (right panel) the particle-hole transformation of the doped Mott insulator state at
point B of (m) to form the interlayer exciton insulator. (m) The phase diagram of the correlated interlayer exciton insulator
in the double-layer system detected by the WSe2 monolayer 2s exciton signal. (n) MIM spectra as a function of gate voltage
for the moiré superlattice of 1L/1L, 2L/1L and 3L/1L WSe2/WS2 at 10 K, respectively. (a‒c) Reproduced with permission
from Ref. [181]. (d, e) Reproduced with permission from Ref. [129]. (f) Reproduced with permission from Ref. [130]. (g,
i) Reproduced with permission from Ref. [131]. (h) Reproduced with permission from Ref. [187]. (j, k) Reproduced with
permission from Ref. [186]. (l, m) Reproduced with permission from Ref. [188]. (n) Reproduced with permission from Ref. [185]. introduced other factors that could modulate the electronic
correlation in TMDs moiré superlattice. The former
studied the impact of optical excitation on the correlated
electronic states in WS2/WSe2 heterobilayers. They
showed that optical excitation enhanced magnetic inter- v = −1/3
action between moiré-trapped carriers and consequently
led to ferromagnetic order [187]. The hysteresis loop at
low filling factor
measured by the reflective
magnetic circular dichroism (RMCD) at T = 1.6 K,
signified the emergence of ferromagnetic order due to Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-28 FRONTIERS OF PHYSICS TOPICAL REVIEW long-range spin-spin interaction in generalized Wigner
crystal state. The power dependence of RMCD responses
[Fig. 12(h)] indicated the remarkable optical tuning of
spin–spin interaction strength between moiré-trapped
holes mediated by itinerant photoexcited excitons, which
could realize the modulation of magnetic phases [187]. Chen and co-workers [185] introduced the layer degree of
freedom in the angle-aligned WSe2/WS2 heterostructure
with WSe2 varying from monolayer to trilayer, in order
to systematically tune both electronic flat bands and
moiré exciton bands. Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) FRONTIERS OF PHYSICS Coulomb interactions and large dipole of charge-transfer
exciton [Fig. 12(j)]. The attractive electron–hole interac-
tion between doped electrons at local A led to the emergent
peak IIIA close to peak I. As electron doping increased,
the peak IIIA showed blueshifts and consequently
reduced the resonant hybridization between peak I and
peak IIIA, resulting in the weakened oscillator strength. The brief enhanced oscillator strength of the peak IIIA at
around 1/3 and 2/3 filling was ascribed to the reduced
screening of correlated states. The peak II exhibited a
mixed character. The non-trivial intralayer charge-transfer
exciton state with specific spatial characters can be more
easily modified and dissociated, suggesting a new
approach to engineering many-body physics and desirable
optoelectronic nanodevices based on moiré system [186]. Such charge-transfer excitons with long lifetimes have
lately been reported in monolayer WSe2/twisted bilayer
graphene (TBG) by Hu et al. [199], due to strongly
asymmetric interlayer Coulomb interactions under a
relatively large moiré wavelength compared to the exciton
size. At a quite small twist angle (θ = 0.6°), they
observed Rydberg moiré excitons where the accumulated
charges in TBG strongly attracted the loosely bound 2s
excitons in WSe2, resembling the Rydberg atom trapped
by arrays of optical tweezers. The moiré system brings
about highly tunable properties and mutual interactions
of the spatially confined Rydberg excitons, demonstrating
great potential for the applications of quantum information
processing and quantum simulations based on the excitonic
Rydberg states in solid-state systems [199]. energy and wave function symmetry, which will lead to
some novel phenomena such as splitting resonance ener-
gies, spatially modulated optical selection rules and
localization of moiré excitons, etc. [49, 52]. The
harmonic moiré potential model [65] describes the moiré
excitons as particles moving under approximated potentials
with spatial periodicity. This model is suitable for
heterostructures with relatively large band edge offsets,
but is not sufficient to capture the excitonic physics in
systems with near-resonant band edges, where interlayer
and intralayer hybridization through carrier tunneling
should be considered [146]. Apart from the moiré super-
lattice under small twist angles, the enhanced moiré
exciton features also appear in high-angle twisted
heterostructures due to the umklapp processes in
commensurate moiré reciprocal lattice [135, 137]. FRONTIERS OF PHYSICS Moiré
systems
based
on
vdW
heterostructures
exhibit
expanded properties and dynamic tunability in conjunction
with external factors, such as electric fields, carrier
doping and strain, which would promote an in-depth
understanding of moiré physics and pave the way to
promising
applications. Recently,
the
enhanced
electron–electron interactions and flat band features in
moiré vdW heterostructures have facilitated research on
strongly correlated electronic states at a few certain filling
factors, such as Mott insulating states, generalized
Wigner crystal and other correlated phases. These studies
are expected to experimentally realize novel quantum
phenomena like fractional quantum anomalous Hall
effects and spin-liquid states [118]. In this review, we mainly focus on the excitonic
behaviours in twisted vdW heterostructures revealed by
optical spectroscopy, which only covers a part of new
physics in twistronics and moiré superlattice. There are
still a large variety of controversies and challenges in
current research that need to be tackled, even within
this certain topic. A comprehensive model to understand
the excitonic physics modulated by moiré pattern has
yet to be established. Excitons presented distinct
behaviours in different twisted heterostructure systems. On one hand, such variations are possibly caused by the
randomness of twisted heterostructures, which require
preparation and detection technology with high precision. On the other hand, the impacts of the moiré pattern on
electronic band structures and optical properties in vdW
heterostructures are still not clear, such as strain distri-
butions and atomic reconstructions under lattice
mismatches [92, 143]. Besides, the twist-angle-dependent
electron-phonon coupling strength in combination with
moiré phonon physics in twisted 2D vdW materials
might deserve further study [91]. The carrier dynamics
and transport influenced by interlayer twist show much
more complicated mechanisms other than interlayer
momentum mismatch. For example, the ultrafast interlayer
charge transfer processes are relatively robust due to the
mixed valley serving as an intermediate in some TMD 4 Correlated electronic states in moiré
superlattice As finite hole
density was distributed in both WSe2 layers, a higher
total should be required to achieve the moiré bilayer
Mott insulator state (
, with
denoting one hole
per moiré lattice site), while actually a stable correlated
insulator state appeared at
[Fig. 12(m)]. The
unexpectedly low originated from the strong interlayer
Coulomb interaction preventing the holes in the WSe2 Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-29 Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) FRONTIERS OF PHYSICS TOPICAL REVIEW TOPICAL REVIEW 5 Conclusions and outlook However, the microscopic mechanisms of the
interplay between correlated electrons and excitons
remain obscure. The nature of various correlated states
identified at certain filling factors needs further investi-
gation. Moiré system based on vdW heterostructure inherits
the high tunability and ability to be customized in
multiple ways. Moiré vdW heterostructures combined
with various external factors such as cavity and strain
are interesting and intriguing for further investigations. For instance, strain engineering of twisted vdW
heterostructures can cause efficient tunning of optoelec-
tronic properties due to the nano-scale structures of the
moiré pattern [167]. Moiré superlattice can also couple
to multiple 2D vdW materials with different stacking
sequences and orders to construct vdW heterostructure
possessing new electronic and photonic physics, including
controllable ferroelectricity [20, 81, 173, 205] and
magnetism [206]. Beyond the most studied TMDs and
graphene reviewed here, the material choice to fabricate
a vdW heterostructure is wider regardless of lattice
constants matching. The research on twisted vdW
heterostructures will prompt engineering artificial mate-
rials with moiré superlattice, providing a fascinating
platform for exploring emerging quantum phenomena
and valuable applications. Declarations The authors declare that they have no competing
interests and there are no conflicts. Acknowledgements The authors are grateful for financial support
from the National Natural Science Foundation of China (Nos. 62105364 and 62075240); the Science and Technology Innovation
Program of Hunan Province (No. 2021RC2068); and the Scientific
Researches Foundation of National University of Defense Technology
(No. ZK22-16). 5 Conclusions and outlook This review systematically summarizes the recent
progress in twistronic and moiré exciton physics in van
der Waals heterostructures that consist of 2D semicon-
ducting materials. The interlayer twist angle can act as
an effective knob to modulate the electronic structures
and optical properties through interlayer interactions
and momentum-space mismatches. The most concerning
aspect is exciton physics, where the exciton properties
such as binding energy, static electric dipole, emission
intensities of different excitonic species [87, 88] and
degree of spin-valley polarization [77], are all controlled
by the interlayer twist angle. Moreover, the interlayer
twist in momentum space also affect the excitonic
dynamics including formation, transport and recombina-
tion processes with complex mechanisms that involve
localized and hybridized valleys [52, 79, 85]. The twist-
angle-tunable physics is significantly enriched when the
moiré effects are considered, which still lacks a compre-
hensive model to understand moiré excitons in various
systems. Early theoretical analyses have predicted the
Moiré Brillouin zone of superlattice structures as well as
the moiré patterns-induced spatial modulations of exciton Siwei Li, et al., Front. Phys. 19(4), 42501 (2024) 42501-30 42501-30 TOPICAL REVIEW FRONTIERS OF PHYSICS heterostructures, whereas they show obvious twist angle
dependence in other cases [85, 86]. Interlayer stretching
and shift caused by atomic reconstructions are also
expected to affect the carrier dynamics through generating
extra channels [79]. In addition, moiré excitons exhibit
splitting resonance energies and localization introduced
by moiré periodic potential. The cascade transitions
between moiré interlayer excitons with different energies
have been reported recently, suggesting the spatial overlap
between the multiple IX minibands in TMDs-based
moiré superlattice [200]. The links between excitons
distributed in different moiré sites and different energies
still need in-depth exploration. Moreover, the study on
strong moiré excitonic features observed in high-angle
commensurately twisted bilayers at several twist angles
is still preliminary but interesting, since these cases are
widely found in randomly stacked TMD bilayers without
the need for deliberate alignment [137]. Apart from the
moiré exciton properties and dynamics, correlated elec-
tronic states in TMDs moiré superlattice have spurred
great interest and generated many open questions in
recent years. The TMDs-based moiré systems working as
designed quantum simulation platforms provide many
opportunities for new quantum states. It is inspiring
that the strong light–matter interactions of TMDs allow
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Recollection-Based Retrieval Is Influenced by Contextual Variation at Encoding but Not at Retrieval
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PloS one
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Recollection-Based Retrieval Is Influenced by
Contextual Variation at Encoding but Not at
Retrieval Eyal Rosenstreich*, Yonatan Goshen-Gottstein
Tel-Aviv University, Ramat Aviv, Israel Eyal Rosenstreich*, Yonatan Goshen-Gottstein
Tel-Aviv University, Ramat Aviv, Israel * eyal@pac.ac.il OPEN ACCESS Citation: Rosenstreich E, Goshen-Gottstein Y (2015)
Recollection-Based Retrieval Is Influenced by
Contextual Variation at Encoding but Not at Retrieval. PLoS ONE 10(7): e0130403. doi:10.1371/journal. pone.0130403 Citation: Rosenstreich E, Goshen-Gottstein Y (2015)
Recollection-Based Retrieval Is Influenced by
Contextual Variation at Encoding but Not at Retrieval. PLoS ONE 10(7): e0130403. doi:10.1371/journal. pone.0130403 Received: June 6, 2014
Accepted: May 19, 2015
Published: July 2, 2015 Received: June 6, 2014
Accepted: May 19, 2015
Published: July 2, 2015 Copyright: © 2015 Rosenstreich, Goshen-Gottstein. 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 Abstract In this article, we investigated the effects of variations at encoding and retrieval on recollec-
tion. We argue that recollection is more likely to be affected by the processing that informa-
tion undergoes at encoding than at retrieval. To date, manipulations shown to affect
recollection were typically carried out at encoding. Therefore, an open question is whether
these same manipulations would also affect recollection when carried out at retrieval, or
whether there is an inherent connection between their effects on recollection and the encod-
ing stage. We therefore manipulated, at either encoding or retrieval, fluency of processing
(Experiment 1)—typically found not to affect recollection—and the amount of attentional
resources available for processing (Experiments 2 and 3)—typically reported to affect recol-
lection. We found that regardless of the type of manipulation, recollection was affected
more by manipulations carried out at encoding and was essentially unaffected when these
manipulations were carried out at retrieval. These findings suggest an inherent dependency
between recollection-based retrieval and the encoding stage. It seems that because recol-
lection is a contextual-based retrieval process, it is determined by the processing informa-
tion undergoes at encoding—at the time when context is bound with the items—but not at
retrieval—when context is only recovered. Encoding Influences Recollection-Based Retrieval Over the past 40 years, numerous recognition-memory studies examined the effects of a
variety of manipulations on memory performance. For example, deeply processed items, such
as to-be-solved anagrams of words presented at study, were more likely to be remembered than
shallowly-processed items, such as the words in their standard form (i.e., a levels-of-processing
effect; [5] [6]). Similar patterns were observed using full- versus divided-attention manipula-
tions (e.g., [7]). In addition, several demonstrations showed that manipulating the speed, or fluency, at
which an item was processed, increased the probability that the item would subsequently be
judged as studied. For example, Whittlesea [8] [9] (see also [10]) presented participants with a
list of to-be-remembered words at study. At test, both studied and unstudied words were pre-
sented as terminal words in two types of sentences. In one type, the sentence predicted the target
word (i.e., The stormy sea tossed the BOAT; the fluent condition). In the second type of sen-
tence, the sentence did not predict the target word (i.e., She saved her money and bought a
BOAT; the non-fluent condition). Results revealed that words presented in the fluent condition
were more likely to be judged as studied, as compared to words presented in the non-fluent con-
dition, thereby producing both more hits and more false alarms (FAs). Whittlesea explained
this effect by an automatic, heuristic, misattribution of the fluent processing of the terminal
word to a previous encounter with the target word. Indeed, when participants were aware of
purpose of the fluency manipulation, no effect was found [11] [12]. The manipulations that have been found to affect recognition memory can be roughly clas-
sified into two categories (cf., the fluency-distinctiveness model [13]). The first category con-
sists of procedures designed to manipulate the extent to which an item or its meaning are
processed. This category includes manipulations as the Read-Anagram (in which words are
presented in their normal form as compared to scrambled. For example, TABLE vs. ELBAT),
Read-Generate (in which words are read as compared to generated. For example, TABLE vs. TA__E) and Divided Attention (in which words are studied without performing a secondary
task, as compared to studied while performing a different task). The second category consists
of manipulations designed to control the speed at which information is processed, with
research programs reflecting Whittlesea’s fluency manipulation. Introduction Memory processes are often characterized by the nature of the manipulations affecting them. For example, the demonstration that certain tests of memory are affected by a levels-of-pro-
cessing manipulation has been used as evidence for the semantic/ conceptual nature of memory
[1] [2] [3] [4]. Guided by this notion, in three experiments described in this article, we suggest
that a more accurate understanding of the processes underlying memory performance may be
achieved by considering the stage in which these manipulations are carried out (i.e., encoding
versus retrieval). Consequently, we present a modified interpretation for the nature of the pro-
cesses underlying recognition—namely recollection and familiarity—which focuses on mem-
ory stage as an important factor in predicting them. Competing Interests: The authors have declared
that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 1 / 22 This category also includes
priming manipulations [14] [15], known to influence processing times, and hence, predicted to
affect the fluency of processing. Interestingly, the classification of the different manipulations into two categories elegantly
maps onto the two types of subjective experience that participants are able to associate with the
recognition of an item. The first type of subjective experience entails remembering contextual
information related to the target item. The second type entails knowing that the target item
was studied, in the absence of conscious recollection of context detail [16] [17] (for a similar
idea in free recall, see [18]). One way to tap these two different processes is the Remember-
Know (RK) task ([17]; other techniques include dual-process ROC curves and the process-dis-
sociation paradigm), wherein participants are instructed to respond “Remember” (R) if they
judge an item as studied because they have clear recollection of the item and its study context. In contrast, participants are asked to respond “Know” (K) if they judge an item as studied with-
out the recollection of episodic features from the study stage. Critically, it has been demonstrated that manipulations belonging to the first category
(manipulating the extent of item's processing) primarily affect R responses whereas those
belonging to the second category (manipulating the speed of item's processing) primarily affect
K responses (cf., [13]). To illustrate, Yonelinas reported that a divided-attention manipulation at
study affected the proportion of R responses, yet hardly affected the proportion of K responses
[19]. Similarly, promoting deeper processing of words at study yielded higher rates of R as com-
pared to shallow processing [20]. In contrast, preceding the presentation of a test item (i.e., the PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 2 / 22 Encoding Influences Recollection-Based Retrieval recognition probe) with a semantically-related item (e.g., study: "NURSE"; Test: "doctor-
NURSE"; Semantic-priming paradigm), yielded higher proportion of K responses as compared
to preceding the item with an unrelated item (e.g., study: "NURSE"; Test: "phone- NURSE"). This manipulation, however, did not affect the proportion of R responses [21] [22]. Different models of recognition have been proposed to explain the way in which different
manipulations affect recognition memory, with a particular focus on the RK procedure. Single-
process models explain the effect of different manipulations by a shift in a subjective decision
criterion located on a hypothetical axis of memory strength (or mnemonic evidence), with dif-
ferent levels of the manipulation producing different levels of strength of the memory trace
[23] [24] [25]. Accordingly, the R and K responses reflect the different levels of strength, with
R responses reflecting higher levels of strength than K responses. In contrast, dual-process models of recognition memory postulate that two distinct pro-
cesses underlie retrieval from recognition memory, Recollection and Familiarity. Based on
behavioral data [21] [26] [27] [28] [29] [30] and on a neuropsychological double dissociations
between recollection and familiarity[31] [32] [33] [34], it has been argued that recollection and
familiarity do not index the retrieval of different levels of strength, but rather the retrieval of
different forms of memory. For sake of simplicity, the studies in this article are described in
terms of an influential version of dual-process model [27] [29] [35]. According to Yonelinas, recollection entails the conscious retrieval of an event including
contextual detail related to the event. Familiarity, in contrast, is considered to be a non-contex-
tual, automatic, retrieval process that induces the feeling that an event was encountered before
[27] [35] (see [36], for a comprehensive review of these and other differences between the two
processes). If so, the nature of the manipulation carried out should be a major determinant of
the memory process affected by the manipulation. Thus, manipulations designed to affect the
depth to which an item was processed—or in other words, the extent to which contextual infor-
mation is available—should affect only recollection but are not likely to affect familiarity. In
contrast, manipulations designed to induce some sort of unconscious sensation that could be
attributed to past occurrence, should affect familiarity but not recollection. Here, our goal was to further understand how recollection and familiarity are affected by
different manipulations. Our thesis is based on the straightforward notion that the process of
recollection entails retrieving information that was part of the encoding context. Because
encoding is the memory stage in which context is bound with the item[37] [38] [39]; and
because the process of recollection is intimately related to the encoding context, it follows that
manipulations undertaken at encoding should affect recollection and that the identical manip-
ulations should not affect recollection when carried out at retrieval. PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 Encoding Influences Recollection-Based Retrieval variables. Specifically, we suggest that variations in an item's processing during the encoding
stage may elicit different amounts of contextual cues for later retrieval, thus promoting differ-
ences in recollection-based retrieval. In contrast, similar variations in processing during
retrieval may not produce helpful contextual cues, hence should not promote an effect on rec-
ollection-based retrieval. Support for the notion that memory stage can predict the contribution to recollection can
be found in [41]. These researchers manipulated the amount of attention allotted to the study
and test stages. Their findings indicated that estimates of recollection were more susceptible to
an attentional manipulation at encoding than at retrieval. In contrast, familiarity was affected
at both encoding and retrieval by the manipulation. In three experiments, we used identical manipulations, materials and procedures at encod-
ing and retrieval to test our hypothesis. In Experiment 1, we tested the conceptual fluency
manipulation [9], in which target words are presented in predictive as compared to not predic-
tive sentences. The fluency manipulation has typically been used at retrieval without affecting
recollection. We asked whether when manipulated at encoding, this manipulation would now
affect recollection. Then, in Experiments 2 and 3, we asked the reverse question, whether a
manipulation typically shown to affect recollection when carried out at encoding will not do so
when manipulated at retrieval. Experiment 2 was designed to replicate—using the Experiment-
1 materials—a finding by Knott and Dewhurst, who showed an effect of divided-attention for
recollection when attention was manipulated at encoding but not when manipulated at
retrieval [40] [41] [42]. In Experiment 3, we attempted to extend this finding using a different
attentional manipulation and a different set of participants and materials (i.e., faces). Thus, we argue that, irre-
spective of the characteristics of the manipulation, a major predictor for when recollection
should be affected is the stage at which the manipulation is carried out, encoding or retrieval. In reviewing the literature, we have not found any suggestion that knowledge of the memory
stage (encoding, retrieval) in which a manipulation was carried out can largely predict perfor-
mance—irrespective of other details regarding the design, procedure and the, most importantly,
the nature of the manipulation. Instead, we found two alternative proposals for predictors of
performance. The first proposal, as described above, focuses on the nature of the manipulation
(e.g., directing participants' attention to the processing of the item or to its meaning as com-
pared to manipulations designed to affect the speed of processing; cf., [13]). The second propo-
salfocuses on study-test compatibility, highlighting the importance of such compatibility to
recollection [40]. While not pitting these suggestions with ours, here we endorse the contribu-
tion of a third, hitherto, ignored variable—memory stage. Thus, we argue that memory-stage may be a helpful construct for understanding the vari-
ability found in the literature on differential effects on recollection and familiarity by numerous 3 / 22 PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 Method Participants. Forty-eight Tel-Aviv University students (mean age = 22.56 years, SD = 1.31
years) participated in the experiment in exchange for course credit. This study was approved
by the ethics committee for studies in psychology at Tel-Aviv University. Also, all participants
gave their consent to participate in the experiments by pressing a key when they were prompt
to declare that they agree to participate. They were informed that they can terminate their par-
ticipation at anytime by pressing the "escape" key on the keyboard. Participants' consent was
recorded in the data file of each experiment. Design, Apparatus and Materials. Fluency of processing (fluent, non-fluent) was manip-
ulated within subject in two separate blocks, with block-order counterbalanced across partici-
pants. The two blocks were the encoding-manipulation block (with fluency manipulated at
encoding) and the retrieval-manipulation block (with fluency manipulated at retrieval). In all, 120 Hebrew nouns served as target stimuli. Following Whittlsea [9], each target word
was chosen so as to be the terminal word of each two types of sentences—fluent and non-fluent. Thus, a total of 240 sentences were created. Results of a pilot study of 10 participants confirmed
that in the fluent sentences, the target word could be anticipated with a high probability, whereas
in the non-fluent sentences, the target word was unlikely to be anticipated. Also, reading laten-
cies of the fluent (M = 2440 ms, SD = 236.65) and non-fluent (M = 2366 ms, SD = 389.48) sen-
tences were found to be equal (t(9) = 0.795, p = .447). Four additional words served as buffers
and corresponded to only one of the two types of sentence. Target words were presented in gray
Arial font (bold), size 60. In the encoding-manipulation block, the study list comprised 30 words, corresponding to
15 fluent and 15 non-fluent sentences. At test, 60 stand-alone words (i.e., not assigned to any
sentences), half old and half new, were presented. In the retrieval-manipulation block, the
study list comprised 30 stand-alone words. At test, 60 words, half studied and half unstudied,
equally distributed between the fluent and non-fluent conditions, were presented as terminal
words within sentences. All conditions were counterbalanced across participants, such that
each word appeared an equal number of times as old and new, and an equal number of times
in the fluent and non-fluent conditions. In addition, in each block, items—single-words or sen-
tences—were presented randomly. Procedure. Experiment 1 In Experiment 1, we used a fluency manipulation [8] [9]. This manipulation was originally
designed to affect familiarity when carried at retrieval. We explored whether, when carried out
at encoding, this very same manipulation would affect recollection. Participants were presented
with sentences that either predicted terminal target words by constraining the possible candi-
dates—the fluent condition—or did not predict target words—the non-fluent condition [8] [9]. For example, the target word “BOAT”, is predictable to a greater extent when it is embedded in
the sentence “The stormy sea tossed the _____” (i.e., the fluent condition), than when embed-
ded in the sentence “She saved her money and bought a _____” (i.e., the non-fluent condition). We assumed that target words in the fluent condition would be processed faster than target
words in the non-fluent condition and would, therefore, be perceived as more familiar. To date, this fluency manipulation has been carried out to examine overall recognition (but
see [22]) not only at retrieval [9] but also at encoding [43]. However, the effects of this manipu-
lation on recollection and familiarity have never been examined at encoding. Therefore, the
results of our experiment may inform us not only regarding the effects of the fluency manipula-
tion on recollection and familiarity at retrieval [22], but also on its contribution to recollection
and familiarity when performed at encoding as compared to retrieval. Because judgments based on fluency are considered to be automatic, they should influence
familiarity, which is considered an automatic, heuristic process. Therefore, we predicted that
when manipulated at retrieval, a fluency effect would be found for familiarity but not for recol-
lection (cf. [21]). Critically, because recollection entails retrieval of contextual information
from the encoding stage, when manipulated at encoding, an effect of fluency should be found
for recollection. 4 / 22 PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 Encoding Influences Recollection-Based Retrieval Encoding Influences Recollection-Based Retrieval To increase the effect of the fluency manipulation on familiarity (cf. [44] [45]), a distractor
task preceded the recognition test in both the encoding- and the retrieval-manipulation blocks. In the distractor task, participants classified tones to “Low”, “Medium” and “High” for two
minutes, by pressing designated keys on a computer keyboard. At test, in both the encoding-manipulation block and the retrieval-manipulation block, tar-
get words were presented until a response was made. Participants read the target words out
loud, judged whether the word was old or new, and then, if an 'old' judgment was made, per-
formed a RKG judgment. After the judgments were typed in by the participants, a blank screen
was presented for 200 milliseconds, followed by the next test trial. The encoding-manipulation
and the retrieval-manipulation blocks were identical, with the exception that only in the
retrieval-manipulation block, target words were preceded by a fluent or non-fluent sentence. When a sentence was presented, participants read it out loud, pressed the space bar, and then,
after an interval of 250 ms, the target word appeared as terminal word in the sentence. Method Participants were first introduced to the general structure of the experiment
and with the characteristics of remember, know and guess (RKG) responses, as described by
Gardiner et al. [16]. Specifically, following Gardiner et al., participants were also informed
that they could give a 'guess' response, if the judgment of the item as ‘old’ could be attributed
by the participant to neither recollection nor familiarity. After a short practice session,
participants were prompted to declare whether they agree or not agree to participate in
the study. In the encoding-manipulation block, each study trial began with a '+' sign presented for 300
milliseconds, followed by a blank screen for 150 milliseconds. The fluent and non-fluent sen-
tence then appeared on the screen, and participants were to first read the sentence, then to
press the space bar on a computer keyboard. Only then–following a 250ms interval–the target
word appeared in the terminal position. The terminal word remained on the screen for 1000
milliseconds, during which participants were to read it out loud and study it for the subsequent
memory test. Also in the retrieval-manipulation block, each study trial began with a '+' sign presented for
300 milliseconds, followed by a blank screen for 150 milliseconds. Then, the to-be-remembered
words appeared at study without the preceding sentence. In both blocks, the offset of the target
word was followed by a blank screen for 150 milliseconds. 5 / 22 PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 Results and Discussion We present the data corrected for the estimates of recollection and familiarity: We first calcu-
lated, for each participant, the proportion of Remember (R) and Know (K) responses by divid-
ing a given response by the total number of non-guess responses, that is, by the total number of
responses excluding guess responses. To illustrate, given that each experimental condition con-
tained 20 items, the R proportion was calculated as: PðRÞ ¼
R
20G PðRÞ ¼ where R and G are
the raw number of R and G responses. The subtraction of G responses from the total number
of trials was performed after post-test debriefing revealed that G responses were mainly due to
experimental noise. For example, participants typically responded with G when they pressed
the "old" button by mistake, or when they could not decide when an item was old or new. Over-
all G rates were very small (M = .016, SD = .035 in the encoding-manipulation block; M = .038,
SD = .071 in the retrieval-manipulation block), and were equally distributed over the experi-
mental conditions (t(47) = 0.275, p = .785 in the encoding-manipulation block; F(3,141) =
0.828, p = .480 in the retrieval-manipulation block) (cf., [46]). Thus, given that each experimental condition contained 20 items, Proportion Rs and Ks
were calculated as: PðRÞ
¼
R
20 G P
PðKÞ
¼
K
20 G P PðRÞ
¼
R
20 G P
PðKÞ
¼
K
20 G P where R, K and G are the raw frequencies of R, K and G responses, respectively. where R, K and G are the raw frequencies of R, K and G responses, respectively. Then, R and K proportions were applied to the correction for the estimates of recollection
and familiarity according to Yonelinas and Jacoby's correction formulas [47] [48]. Recollection
was estimated by the R proportion: Recollection ¼ PðRÞRecollection and familiarity was estimated by: Familiarity ¼
PðKÞ
1 PðRÞ Familiarity Familiarity ¼
PðKÞ
1 PðRÞ Familiarity The raw proportions of R and K responses are presented in S1 Table. Effect sizes throughout
the article were calculated as Cohen's d. 6 / 22 PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 Encoding Influences Recollection-Based Retrieval To examine whether our manipulation replicated the well-documented fluency advantage
for total recognition scores carried out at retrieval [9], we conducted two directional t-tests of
dependent samples. Results and Discussion E
i
i
f h
i
l d
fl
ff
f
f
ili
i
h
h
i
l i For hit rates, the mean estimates of recollection and familiarity are presented in Table 1. Examination of the estimates revealed a fluency effect for familiarity when the manipulation For hit rates, the mean estimates of recollection and familiarity are presented in Table 1. Examination of the estimates revealed a fluency effect for familiarity when the manipulation
was carried out at retrieval, but not at encoding. Most important, as predicted, the recollection
data showed the opposite pattern. The fluency effect was found for recollection only when the
manipulation was carried out at encoding, but not at retrieval. y
y
p
was carried out at retrieval, but not at encoding. Most important, as predicted, the recollection
data showed the opposite pattern. The fluency effect was found for recollection only when the
manipulation was carried out at encoding, but not at retrieval. We submitted each of the interactions to an ANOVA, with both memory stage (Encoding,
Retrieval) and fluency (Fluent, Non-fluent) manipulated within subject. For familiarity, the flu-
ency X memory stage interaction was significant, F(1, 47) = 5.52, MSE = 0.04, p < .05. At
encoding, no significant effect of fluency on familiarity was found, F < 1. In contrast, at
retrieval an effect was found, with higher estimates of familiarity under the fluent condition
than under the non-fluent condition, F(1, 47) = 5.70, MSE = 0.03, p < .01. For recollection too, the fluency X memory stage interaction was significant, F(1, 47) = 9.47,
MSE = 0.01, p < .01. Critically, however, this interaction was opposite in direction to the one
found for familiarity. As predicted, the source of this interaction was an increase in estimated
of recollection in the fluent, as compared to the non-fluent, condition, but only at encoding,
F(1, 47) = 10.98, MSE = 0.01, p < .01. At retrieval, however, no significant effect of fluency of
recollection was found, F(1, 47) = 2.14, MSE = 0.01, p > .05. Two aspects of the results are noteworthy. First, Whittlesea's fluency manipulation was
found to affect familiarity hits when carried out at retrieval, with no effect found on recollection. Results and Discussion Specifically, we compared recognition scores for items that were studied in
the fluent condition, which were higher (mean = 0.80, SE = 0.02) than those studied under the
non-fluent condition (mean = 0.75, SE = 0.02). This difference was significant, t(47) = 1.84, p
< .05, d = 0.36. Also for unstudied items, recognition scores for items that were processed flu-
ently (mean = 0.33, SE = 0.03) were higher than recognition scores for items that were not pro-
cessed fluently (mean = 0.23, SE = 0.02). This difference, too, was significant, t(47) = 4.56, p <
.01, d = 0.58. Therefore, the original fluency effect on overall recognition was replicated. Next, we turned to examine the influence of the fluency manipulation, carried out at
retrieval, on the estimates of recollection and familiarity. Separate analyses are reported for hits
and for FA. Note that the influence of study stage could not be assessed for the FA data because
when the fluency manipulation was manipulated at encoding, it could not—by definition—be
applied to unstudied items. Thus, here and in Experiment 3, FAs are only reported in the
retrieval-manipulation block, wherein the manipulation was undertaken for both unstudied
and studied items. In contrast, for hits, performance could be reported as a function of the
memory stage, because for studied items, the fluency manipulation was carried both in the
retrieval-manipulation block and in the encoding-manipulation block. Examination of the data revealed that for recollection, the FAs in the fluent condition
(mean = 0.04, SE = 0.01) were only slightly higher than the FAs in the non-fluent condition
(mean = 0.03, SE = 0.01). Due to their small size, these responses were not submitted to statisti-
cal analysis. As for familiarity, the FAs in the fluent condition (mean = 0.25, SE = 0.02) were
higher than the FAs in the non-fluent condition (mean = 0.18, SE = 0.02). A dependent sam-
ples t-test revealed this increase in familiarity as a function of fluency to be significant, t(47) =
3.84, p < .01, d = 0.51. Thus, for FAs, when manipulated at retrieval, the fluency manipulation
significantly affected familiarity. For hit rates, the mean estimates of recollection and familiarity are presented in Table 1. Encoding Influences Recollection-Based Retrieval Table 1. Experiment 1. Mean estimates (and SE) of Recollection and Familiarity hit rates, as a function of Fluency (Fluent, Non-fluent) and Memory stage
(Encoding, Retrieval). Fluency
Process
Memory stage
Fluent
Non-Fluent
Cohen's da
Recollection
Encoding
.65 (.03)
.58 (.03)
0.34
Retrieval
.39 (.03)
.42 (.03)
-0.14
Familiarity
Encoding
.54 (.05)
.58 (.04)
-0.13
Retrieval
.60 (.03)
.51 (.03)
0.43
Note. Recollection and Familiarity were estimated using Yonelinas and Jacoby's correction formulas [47]. a Cohen's d represents the effect size of the fluency manipulation (for details and interpretation, see [75]). doi:10 1371/journal pone 0130403 t001 (and SE) of Recollection and Familiarity hit rates, as a function of Fluency (Fluent, Non-fluent) and Memory stage Table 1. Experiment 1. Mean estimates (and SE) of Recollection and Familiarity hit rates, as a function of Fluency
(Encoding, Retrieval). Note. Recollection and Familiarity were estimated using Yonelinas and Jacoby's correction formulas [47]. a Cohen's d represents the effect size of the fluency manipulation (for details and interpretation, see [75]). Note. Recollection and Familiarity were estimated using Yonelinas and Jacoby's correction formulas [47]. a Cohen's d represents the effect size of the fluency manipulation (for details and interpretation, see [75]). doi:10.1371/journal.pone.0130403.t001 Second, and of primary interest to our present concerns, the results of this experiment pro-
vide evidence for our notion that recollection is dependent on the processing that information
undergoes at encoding. Thus, at encoding, fluency affected recollection but not familiarity,
with higher recollection in the fluent condition. These results suggest that sensitivity to fluency
is not unique to familiarity. Rather, the answer to the question of whether fluency affects recol-
lection or familiarity depends on the memory stage at which the manipulation is carried out. When carried out at retrieval, fluency indeed affects familiarity. Critically, however, when car-
ried out at encoding, it affects recollection. This supports our thesis that the question of how
the fluency manipulation affects recollection may be ill conceived and may be tangential to the
critical element of the intimate relationship between recollection and encoding. Results and Discussion Though Whittlesea [9] did not examine the separate influence of the fluency manipulation to
recollection and familiarity, the theoretical analysis that he ascribed to this manipulation corre-
sponds to the finding of an influence on familiarity but not recollection (cf., [22]). PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 7 / 22 Encoding Influences Recollection-Based Retrieval particular, an attention-manipulation entails the exact same process—that of reducing or ele-
vating the amount of available resources—whether carried out at encoding or at retrieval
(though, possibly, because encoding and retrieval are different operations, they may still differ
in their resource-dependency). Because the manipulation of attention is based on a well-
defined theoretical structure, it would be more difficult to defend the idea that the characteris-
tics of this manipulation varies as a function of the memory stage. In a recent study, Knott and Dewhurst manipulated participants' attention both during
encoding and retrieval [41]. These authors measured R and K responses for memory of recog-
nized words. At encoding, Knott and Dewhurst replicated the typical (e.g., [7] [19]) pattern of
divided attention (DA) at encoding, with R responses affected to a greater extent by divided
attention than K responses [42]. However, at retrieval, DA affected K responses to a greater
extent than it affected R responses [40] [41] [42]. Thus, DA has been demonstrated to produce
different effect on R and K responses at encoding as compared to retrieval [41], in a manner
analogous to that which was found in Experiment 1, using the fluency manipulation. Critically, it is difficult to evaluate the effect of memory stage on R/F judgments across the
two manipulations (fluency, DA), in that the two manipulations were confounded by different
labs, materials, designs and procedures. Experiment 2 was thus designed to replicate Knott and
Dewhurst's findings [41], using the same materials, design and procedure as those used in
Experiment 1. We hypothesized that recollection-based retrieval of words will be reduced
when attention is divided at encoding, but not at retrieval. PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 Experiment 2 In Experiment 1, we demonstrated that fluency—a retrieval manipulation which has heretofore
not been found to affect recollection—produced an effect on recollection when manipulated at
encoding. We interpreted these findings as supporting our thesis that manipulations under-
taken at encoding create variations in the context in which the information is stored. Therefore,
because the process of recollection entails retrieving information that includes contextual
detail, manipulations carried out at encoding—at the time when context is bound with the
item—should affect recollection. In contrast to this interpretation, one could argue that encoding is not the critical factor
determining the presence of recollection, but rather, the characteristics of the manipulation is
possibly affected when shifting from retrieval to encoding (cf., [13]). Specifically, when fluency
was manipulated at retrieval, it affected processing speed or fluency-of-processing. However,
when this manipulation was shifted from retrieval to encoding, it no longer affected processing
speed or fluency-of-processing. Rather, it now affected the level of attention directed to stimuli
(or the amount of distinctive information which was extracted). This change in the nature of
the manipulation enabled recollection to emerge. Accordingly, the conclusions of our demon-
strations may more correctly be interpreted in terms of the characteristics of the manipulations
—which could be argued to change when moving from retrieval to encoding—rather than in
terms of the memory stage during which they were carried out. The argument against a memory-stage interpretation of our findings, targets the construct
validity (e.g., [49]) of the experimental manipulation, suggesting that the experimental manip-
ulation carried out at encoding does not represent the same theoretical variable as the exact
same manipulation carried out at retrieval. In Experiment 2, we wish to provide a rebuttal to
this argument by examining an additional manipulation, designed to affect attention. In PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 8 / 22 Method Participants. In exchange of course credit, 68 first-year psychology students at Tel-Aviv
University participated in this study (mean age = 23.55, SD = 3.72; 50 females). The study was
approved by the university ethics committee, with written consent of willingness to participate
in the study, provided by participants. Design, Materials, and Procedure. Attention (full, divided at encoding, divided at
retrieval) was manipulated within subject, in three separate blocks. In the first block, partici-
pants performed the memory task with full attention at both encoding and retrieval. The two
remaining blocks were the encoding-manipulation block—in which attention was divided only
at encoding—and the retrieval-manipulation block—in which attention was divided only at
retrieval). Presentation order of the encoding- and the retrieval-manipulation blocks was coun-
terbalanced across participants. Memory was assessed using the same materials as in Experiment 1. Specifically, 120 Hebrew
nouns served as target stimuli and were randomly assigned to three blocks of 40 words. Six
additional words served as buffers, with two words presented at the beginning of each of the
three test blocks. All words were presented in black Arial font (bold), size 60. In each of the blocks, the study list comprised 20 words, presented randomly at a rate of
1000 ms per word. Each word was preceded with a '+' sign presented for 250 ms, followed by a
blank screen for 100 ms. The offset of the target word was followed by a blank screen for 100
ms. At test, 40 words were presented randomly, of which half were studied and half were new. Participants were asked to determine for each test word whether it was studied or not studied,
by pressing designated buttons on a computer keyboard, marked with red (the L key; "not stud-
ied") and blue (the A key; "studied") stickers. If a word was judged as studied, participants were
prompted to perform a RKG judgment, as in described Experiment 1. The test was self-paced. The three 40-words sets were counterbalanced across the three attention conditions, such that
across participants, each set appeared an equal number of times under the full, divided-at-
encoding, and divided-at-retrieval conditions. PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 9 / 22 Encoding Influences Recollection-Based Retrieval Attention was divided using a secondary task of tone classification [7]. This task comprised
three tones of 220Hz (low), 440Hz (medium), and 880Hz (high). Results and Discussion As a manipulation check, we first turned to examine the effect of the attention manipulation
on overall recognition scores. We first observed that overall recognition was highest under full
attention (mean = 0.88, SE = 0.02), lower for DA-at-retrieval (mean = 0.76, SE = 0.02), and was
lowest for the DA-at-encoding condition (mean = 0.64, SE = 0.02). We applied the data to a
one-way repeated-measures analysis of variance (ANOVA), and found significant differences,
F(2,134) = 75.04, p < .001. A series of three dependent samples t-tests was employed as a post-
hoc analysis, with the significance level reduced to .017, according to Bonferroni's correction
for multiple comparisons. The analyses revealed that when attention was divided at encoding,
recognition scores for studied items were lower as compared to performance under full atten-
tion, t(67) = 12.79, p < .001, d = 3.13. When attention was divided at retrieval, recognition
scores for studied items were all lower as compared to performance under full attention, t(67)
= 6.58, p < .001, d = 1.61. Finally, when attention was divided at retrieval, recognition scores
for studied items were higher than when attention was divided at encoding, t(67) = 5.49, p <
.001, d = 1.34. As for unstudied items, a one-way repeated-measures analysis of variance (ANOVA) found
significant differences between the three attention conditions, F(2,134) = 11.35, p < .001. A
series of dependent samples t-tests revealed that when attention was divided at encoding, false-
alarm rate was higher (mean = 0.27, SE = 0.02) as compared to false-alarms under full attention
(mean = 0.21, SE = 0.02), t(67) = 2.66, p = .01, d = 0.65. When attention was divided at retrieval,
false-alarm rates were lower (mean = 0.16, SE = 0.02) as compared to false-alarms under full
attention, t(67) = 2.01, p = .05, d = 2.01. Finally, when attention was divided at retrieval, false-
alarm rates were lower than when attention was divided at encoding, t(67) = 5.04, p < .001,
d = 1.23. The effects of the attentional manipulation on total recognition scores replicates the
asymmetry between encoding and retrieval processes reported in the literature (e.g., [50]), such
that manipulating attention at encoding hurt memory performance for studied items more so
than did manipulating attention at retrieval. Next, we turned to examine the effects of the attention manipulation on recollection and
familiarity. Method The tones were generated by
professional audio software, and were 550 ms long. In the secondary task, participants were to
classify tones as “Low”, “Medium”, or “High”, by pressing designated keys on a numeric key-
pad connected to a computer. The tones were presented randomly, with a 300ms inter-stimu-
lus interval (ISI). If a given tone was not classified within the 850 ms interval (tone on-set plus
ISI), a new tone was presented. In each of the three blocks, participants performed the tone-classification task for 30 sec-
onds between the study and test stages, thus serving as a distractor task. More importantly, the
task was performed during the presentation of the study list in the encoding-manipulation
block, and during the recognition test in the retrieval-manipulation block. Participants were
informed that accuracy was very important for both the memory and the tone-classification
tasks, and that they should perform as fast as they could in the tone-classification task. Participants were given detailed instructions of the RKG recognition task and of the tone-
classification task at the beginning of the experiment. In addition, each block began with a
reminder of the instruction for the forthcoming task, as well with a short practice that simu-
lated the forthcoming task in that block. Encoding Influences Recollection-Based Retrieval and familiarity. Mean estimates of Recollection and Familiarity hits are presented in Table 2. Raw proportions of R and K responses are presented in S2 Table. For recollection, a one-way repeated measures ANOVA revealed a significant difference
between the three attention conditions, F(2,134) = 67.02, p < .001. Three dependent-samples t-
tests (with significance level of .017) revealed that recollection hits were lower when attention
was divided at encoding, as compared to full attention, t(67) = 10.22, p < .001, d = 2.50. Recol-
lection hits were slightly lower when attention was divided at retrieval as compared to full
attention, yet this difference did not reach significance, t(67) = 2.37, p = .02, d = 0.58. Finally,
recollection hits were lower when attention was divided at encoding, than when it was divided
at retrieval, t(67) = 8.37, p < .001, d = 2.05. For familiarity, a one-way repeated measures ANOVA revealed a significant difference
between the three attention conditions, F(2,134) = 21.76, p < .001. Three dependent-samples t-
tests (with significance level of .017) revealed that familiarity hits were lower when attention
was divided at encoding, as compared to full attention, t(67) = 5.51, p < .001, d = 1.35. Famil-
iarity hits were also lower when attention was divided at retrieval, as compared to full attention,
t(67) = 5.33, p < .001, d = 1.30. Finally, there was no difference between familiarity hits when
attention was divided at encoding, as compared to divided at retrieval, t(67) = 1.76, p = .08,
d = 0.43. Next, we examined the effect of the attention manipulation on familiarity false-alarms
(FAs). As in Experiment 1, we do not report or analyze recollection FAs due to their small sizes
(less than 3%). A one-way repeated measures ANOVA revealed that familiarity FAs were
affected by the attention manipulation, F(1,134) = 25.20, p < .001. Thus, FAs when attention
was divided at encoding (mean = 0.17, SE = 0.02) were higher than under full attention
(mean = 0.06, SE = 0.01), t(67) = 6.99, p < .001, d = 1.71. FAs when attention was divided at
retrieval (mean = 0.09, SE = 0.01) did not differ from FAs under full attention, t(67) = 1.53, p =
.13, d = 0.37. Results and Discussion As in Experiments 1, we present the data corrected for the estimates of recollection PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 10 / 22 PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 Experiment 3 Thus far, we have demonstrated that recollection is more likely to be affected by manipulations
carried at encoding, rather than manipulations carried at retrieval. However, a leading account
of the processes underlying R judgments is that it is the analysis of the distinctive or salient
attributes of the information creates memories that are later accompanied by Remember
responses [13]. Therefore, we next wished to generalize our findings to stimuli which were
highly distinctive, unlike the word-stimuli used in Experiment 2. Specifically, if R responses are
indeed influenced by increased processing of distinctive information at encoding, as suggested
by Rajaram, then using stimuli which are already highly distinctive, should eliminate, or at
least mitigate, any possible effect on R responses. If, however, R responses are mediated by pro-
cessing of information at encoding per se, then the distinctive nature of the stimuli should not
matter, and an influence on R response should be observed for distinctive stimuli, as it was
observed for word stimuli. To this end, in Experiment 3, we examined unfamiliar faces, which comprise a class of sti-
muli which are highly distinctive. Indeed, we chose facial stimuli which had particularly dis-
tinctive features, such as a long nose or crooked eyes. If an effect would be observed even for
these unique stimuli, then the notion that an analysis of distinct features is insufficient to
account for RK performance would be bolstered. Consistent with our hypothesis, we predicted that at encoding, the attentional manipulation
would affect recollection but that recollection should not be affected by the DA manipulation
during retrieval. Thus, an interaction was expected between the stage in which the manipulation
is carried-out (Encoding, Retrieval), and the amount of attentional-load (Low-load, High-load). Encoding Influences Recollection-Based Retrieval researches suggested that although encoding and retrieval are attention-demanding processes,
retrieval is more resilient to divided attention. Other researchers too ([40] [51]) have reported that recollection, but not familiarity, is sen-
sitive to changes in attention during encoding but not to changes during retrieval. Importantly,
however, their findings were not interpreted, as we propose here, in terms of the different
effects of memory stage. To reiterate, we wish to argue that manipulations undertaken at
encoding create variations in the context in which the information is stored. Therefore, because
the process of recollection entails retrieving information that includes contextual detail, only
manipulations carried out at encoding—at the time when context is bound with the item—
should affect recollection. Rather than interpreting the different influence of encoding and retrieval on R in terms of
memory stage, Knott and Dewhurst ([40]; see also [51]) interpreted this difference in terms of
Rajaram's fluency-distinctiveness model [13]. This fluency-distinctiveness model suggests that
changes in attention during encoding alter item's distinctiveness, thus affecting recollection. Changes in attention during retrieval may not alter item's distinctiveness, thus recollection was
not affected. Critically, Rajaram's fluency-distinctiveness model can just as easily apply to the findings
presented in our Experiment 2. In particular, one could argue that the attention-demanding
tone-classification task interfered with participants' ability to encode study words in a distinc-
tive manner, thus only a few of these words "achieved" distinctiveness. This manifested in low
recollection rate. We address this possible explanation in Experiment 3. Participants.
Twenty-four Tel-Aviv University students (mean age = 23.47 years,
SD = 2.15 years) participated in the experiment in exchange for course credit. This study was Finally, FAs when attention was divided at encoding were higher than when
attention was divided at retrieval, t(67) = 4.87, p < .001, d = 1.19. Taken together, the findings presented here support our notion that recollection will be
more affected by manipulations carried at encoding, but not when carried at retrieval. In this
experiment, recollection-based retrieval was dramatically decreased when attention was
divided at encoding, yet was not affected when attention was divided at retrieval. While com-
parable evidence was already presented in Experiments 1, here it is more difficult to provide
an alternate interpretation of the results in terms of differences in underlying theoretical
constructs. Put differently, our findings indicate an asymmetry between encoding and retrieval in
regard to the effects of divided-attention manipulation on memory performance. Such an
asymmetry is well documented in free-recall and in total recognition scores (e.g., [50]). These Table 2. Experiment 2. Mean estimates (and SE) of Recollection and Familiarity hit rates, as a function of
Attentional: Full, divided at encoding, and divided at retrieval. Attention manipulation
Process
Full
Divided at encoding
Divided at retrieval
Recollection
.58 (.03)
.22 (.02)
.51 (.03)
Familiarity
.67 (.04)
.49 (.02)
.42 (.04)
Note. Recollection and Familiarity were estimated using Yonelinas and Jacoby's correction formulas [47]. doi:10.1371/journal.pone.0130403.t002 Table 2. Experiment 2. Mean estimates (and SE) of Recollection and Familiarity hit rates, as a function of
Attentional: Full, divided at encoding, and divided at retrieval. Table 2. Experiment 2. Mean estimates (and SE) of Recollection and Familiarity hit rates, as a function o
Attentional: Full, divided at encoding, and divided at retrieval. PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 11 / 22 Encoding Influences Recollection-Based Retrieval approved by the ethics committee for studies in psychology at Tel-Aviv University. Also, all
participants gave their consent to participate in the experiments by pressing a key when they
were prompt to declare that they agree to participate. They were informed that they can termi-
nate their participation at anytime by pressing the "escape" key on the keyboard. Participants'
consent was recorded in the data file of each experiment. Design. Attentional-load (Low-load, High-load) was manipulated within subject in two
separate blocks—which were also manipulated within subject. The two blocks—corresponding
to memory stage—were the encoding-manipulation and the retrieval-manipulation blocks,
with attentional load manipulated at encoding and retrieval, respectively. Apparatus and Materials. A total of 128 photos of human faces were used in this experi-
ment. Photos were taken from the Karolinska Directed Emotional Faces database (KDEF; [52])
and downloaded from the Internet, with the constraint that each photo should bare a unique—
exaggerated—extreme facial expression, hairstyle, color, gender, age, and position in the pic-
ture frame. All photos were of high quality and standardized to 225 pixels width and 305 pixels
height. The experiment consisted of two experimental blocks. In the encoding-manipulation block,
attentional load was manipulated at study (as described below), with 15 photos presented in
the high-load and 15 photos in the low-load condition. At test, 30 old and 30 new photos were
presented without an attentional manipulation. In the retrieval-manipulation block, at study,
participants studied 30 photos, without an attentional manipulation. At test, 30 old and 30 new
photos were presented with each type equally divided between the high-load and the low-load
conditions. Because pictures are spatial stimuli, attention was not manipulated using the tone classifica-
tion task (a spatial task), but rather with a verbal task. Specifically, to manipulate attention,
each photo was randomly paired with either a common 6-letter Hebrew word, taken from a
contemporary Hebrew-Hebrew dictionary (e.g., SCANNER) or with a 6-letter string of an
identical letter (e.g., AAAAAA). Across trials, each word was paired an equal number of times
with a word and with an identical-letter string. To manipulate attention, the 6-letter string or
word was presented and participants had to recite the letters backwards. After 750 ms., the
word or letter-string were replaced by the photo for 2000 ms. Method Participants. Twenty-four Tel-Aviv University students (mean age = 23.47 years, Participants. Twenty-four Tel-Aviv University students (mean age = 23.47 years,
SD = 2.15 years) participated in the experiment in exchange for course credit. This study was 12 / 22 PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 Results and Discussion As in Experiments 1 and 2, we present the data, corrected for the estimates of recollection and
familiarity. Raw proportions of R and K responses are presented in S3 Table. As in Experiments 1 and 2, we present the data, corrected for the estimates of recollection and
familiarity. Raw proportions of R and K responses are presented in S3 Table. As a manipulation check, we first turned to examine the effect of the attentional-load
manipulation on overall recognition scores. When the manipulation was carried out at encod-
ing, recognition scores for items that were studied under low-load (mean = 0.84, SE = 0.03)
were higher than those under high-load (mean = 0.57, SE = 0.04). This difference was signifi-
cant, t(23) = 8.86, p < .01, d = 1.57. When the manipulation was carried out at retrieval, recog-
nition scores for studied items that were retrieved under low-load (mean = 0.91, SE = 0.02)
were slightly higher than those retrieved under high-load (mean = 0.88, SE = 0.03). This differ-
ence was not significant, t(23) = 1.20, p > .05, d = 0.24. As for unstudied items, false alarms for
items that were retrieved under low-load (mean = 0.10, SE = 0.02) were lower than those
retrieved under high-load (mean = 0.21, SE = 0.03). This difference was significant, t(23) =
-5.00, p < .01, d = -0.9. The effects of the attentional-load manipulation on total recognition
scores replicated the asymmetry between encoding and retrieval processes found in Experi-
ment 2. Next we asked whether a similar asymmetry can be found on the effects of attention on the
estimates of recollection of studied items. Mean estimates of Recollection and Familiarity hits
are presented in Table 3. Examination of Table 3 revealed two interactions. First—as predicted—for recollection, an
attentional-load effect was found when the manipulation was carried out at encoding, but not
at retrieval. Second, for familiarity, a similar interaction was found, with attention affecting
familiarity when the manipulation was carried out at encoding and less so, when at retrieval. We submitted each of the interactions separately to an analysis of variance (ANOVA), with
both memory stage (Encoding, Retrieval) and attentional-load (Low, High) as within-subject
variables. For recollection, the attentional-load X memory stage interaction was significant, F
(1, 23) = 13.97, MSE = 0.02, p < .01. During the presentation of the
photo, participants were required to continue reciting the letters from back to front from mem-
ory. For identical-letter strings, this was a relatively easy task—constituting the low-load condi-
tion. However, for words, this entailed maintaining the word in memory and constantly
retrieving its spelling—thereby constituting the high-load condition. All words and identical-
letter strings were presented in black Tahoma font, size 40 and only appeared when attention
was manipulated. Eight photos served as buffers, two photos in each study or test stage. In addition, 10 extra
photos and 10 extra words and identical-letter strings were used for a short practice. Procedure. As in Experiments 1 and 2, individually tested participants were given printed
instructions for the experiment, in which they were acquainted with the two experimental
blocks and with the RKG procedure. In a short practice session, participants were acquainted with the memory task and with the
high- and low-load tasks. They were also encouraged to deeply encode the photos by generat-
ing, for each photo, a corresponding association. Following the practice session, participants
were prompted to declare whether they agree or not agree to participate in the study, with the
experiment then beginning. Each block consisted of the presentation of 30 photos. Each trial started with a '+' sign pre-
sented at the middle of the screen for 1000 ms., followed by a blank screen for 500 ms. Subse-
quently, the target photo was presented for 2000 ms., after which it was masked with a black PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 13 / 22 Encoding Influences Recollection-Based Retrieval rectangle for 250 ms. As described above, for the attention manipulation (at either study or
test)—prior to the presentation of the target photo—a word (high-load condition) or the letter-
string (low-load condition) was presented for 750 ms, and was replaced by the photo. At test, a message appeared on the screen informing participants about the upcoming recog-
nition test. As described above, test trials had the same structure as study trials, except the off-
set of each target photo was followed by an “old/new” judgment notification on the screen. Participants performed the judgment by pressing a designated key on the keyboard for the dif-
ferent judgments. When the word was judged as old, an RKG judgment notification appeared
on the bottom of the screen, and participants pressed the designated keys according to their
judgment. Immediately after making the RKG judgment, a new trial began. Process Note. Recollection and Familiarity were estimated using Yonelinas and Jacoby's correction formulas [47]. a Cohen's d represents the effect size of the attentional-load manipulation (for details and interpretation, see [75]). Note. Recollection and Familiarity were estimated using Yonelinas and Jacoby's correction formulas [47]. a Cohen's d represents the effect size of the attentional-load manipulation (for details and interpretation see [75 doi:10.1371/journal.pone.0130403.t003 SE = 0.02) were lower than the FAs in the high-load condition (mean = 0.15, SE = 0.03). This
effect was significant, F(1, 23) = 21.12, MSE = 0.003, p < .01. SE = 0.02) were lower than the FAs in the high-load condition (mean = 0.15, SE = 0.03). This
effect was significant, F(1, 23) = 21.12, MSE = 0.003, p < .01. The results of Experiment 3 lend further support to our notion that the stage in which a
manipulation is carried out—rather than the type of manipulation per se’—best determines
whether recollective processes would be affected. Specifically, when employed at retrieval, the
attentional-load manipulation did not affect recollection or familiarity hits. Critically, in accor-
dance with our prediction, when employed at encoding, this same manipulation did affect
recollection. Opposed to Rajaram's fluency-distinctiveness model [13], our study eliminated the ability
to rely on item's distinctiveness during retrieval, because all items, both old and new, were
highly distinctive. Thus, the fact that recollection was affected by changes in attention during
encoding but not during retrieval could not be easily attributed to changes in item's distinc-
tiveness, but rather to the stage in which the manipulation was carried. Hence, recollection
was more likely to be affected when variations in processing took place at encoding, than at
retrieval. Still, a few studies have reported an affect of divided attention on recollection at retrieval
[53] [54]. Note, however, that in these studies, the effects of DA at retrieval on recollection
were obtained by manipulating attention along with several other manipulations within a sin-
gle design [53]. The inclusion of several variables makes it difficult, if not impossible, to isolate
the effects of the DA manipulation. A different difficulty in interpretation is found in a study by Gruppuso et al. [54]. In their
study, attention was divided using a secondary task which had similar semantic structure to the
primary-memory task. Results and Discussion The simple effects revealed that at encoding, the higher
attentional load decreased recollection, F(1, 23) = 43.40, MSE = 0.01, p < .01, while at retrieval
it did not, F(1, 23) = 1.40, MSE = 0.01, p > .05. For familiarity, examination of the estimates revealed that the attentional-load X memory
stage interaction was also significant, F(1, 23) = 5.81, MSE = 0.04, p < .05. At encoding, the
simple effects revealed that high attentional-load decreased familiarity, F(1, 23) = 22.27,
MSE = 0.02, p < .01, while at retrieval there was no effect on familiarity, F(1, 23) < 1. Examination of the estimates of the false-alarms (FA) revealed that the attentional-load
manipulation affected familiarity. As in Experiments 1 and 2, these data can only be reported
for the retrieval-manipulation block, in which the manipulation was undertaken for unstudied
as well as for studied words. For familiarity, the FAs in the low-load condition (mean = 0.08, PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 14 / 22 PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 Encoding Influences Recollection-Based Retrieval Table 3. Experiment 3. Mean estimates (and SE) of Recollection and Familiarity hit rates, as a function of Attentional-load (High, Low) and Memory stage
(Encoding, Retrieval). Attentional-load
Process
Memory stage
Low
High
Cohen's da
Recollection
Encoding
.43 (.05)
.21 (.03)
1.12
Retrieval
.53 (.05)
.49 (.06)
0.15
Familiarity
Encoding
.68 (.05)
.46 (.04)
1.00
Retrieval
.77 (.06)
.75 (.05)
0.07
Note. Recollection and Familiarity were estimated using Yonelinas and Jacoby's correction formulas [47]. a Cohen's d represents the effect size of the attentional-load manipulation (for details and interpretation, see [75]). (and SE) of Recollection and Familiarity hit rates, as a function of Attentional-load (High, Low) and Memory stage Table 3. Experiment 3. Mean estimates (and SE) of Recollection and Familiarity hit rates, as a function of Attentio
(Encoding, Retrieval). General Discussion Our goal was to investigate whether the memory stage (encoding, retrieval) in which a manipu-
lation is carried out can help predict whether recollection is affected. Our results suggest that
when the manipulation is carried out at encoding, recollection is likely to be affected. In con-
trast, when the manipulation is carried out at retrieval, recollection is likely to not be affected. Therefore, an encoding-retrieval asymmetry was found for the effects of manipulations on
recollection. In Experiment 1, we demonstrated that a manipulation designed specifically to affect famil-
iarity—the fluency manipulation—affected recollection when carried out at encoding while not
affect recollection—but affecting familiarity—when carried out at retrieval. In Experiments 2
and 3, we demonstrated that a manipulation known to affect recollection—the divided atten-
tion (DA) manipulation—indeed affected recollection (as well as familiarity; see discussion
below on non-criterial recollection), but only when carried out at encoding. When attention
was divided at retrieval, neither recollection nor familiarity was affected. Put altogether, these findings suggest that the characteristics of recollection are better
understood when considering the stage in which the manipulation is carried out. This finding
is unintuitive in that both recollection and familiarity are retrieval processes and, as such,
might both be expected to recover information encoded at study. However, our findings sug-
gest that because recollection is a contextual-based retrieval process, it—but not necessarily
familiarity—is influenced almost exclusively by the manner in which information was pro-
cessed at encoding. One caveat to this suggestion is that like recollection, familiarity too may at times be sensi-
tive to manipulations carried out at encoding. Indeed, in Experiment 3, familiarity was also
affected by the attentional-load manipulation that was carried out at encoding. Interestingly,
though this pattern was revealed in the corrected scores, it was not found in the raw K
responses reported in S3 Table. Either way, to the extent that an effect on familiarity was found
from an encoding manipulation, it seems to devizate from our notion that only recollection is a
context-based process, and therefore it alone should be influenced by study manipulations. y
y
Still, this finding does not challenge our thesis [19] [41] [55]. Our thesis only focuses on the
intimate relationship between encoding and recollection. The finding that encoding manipula-
tions can affect familiarity has been interpreted as non-criterial recollection [56]. Process Therefore, in their study, the effect of DA at retrieval on task perfor-
mance may have stemmed from the competition for semantic-resources rather than from a
competition for attentional-resources. In contrast, in our study, we intentionally used primary and secondary tasks from separate
domains, face processing and word reading, respectively. Because both the primary task and
the secondary task—reading the word or the identical-letter string—relied on memory, both
tasks competed directly for attentional-resources required for memory performance. By doing
so, we replicated the finding of a decrease in recollection as a function of DA at encoding. More important, we were able to demonstrate that recollection is not affected by the same
manipulation carried out at retrieval as when it is carried out at encoding. When manipulated at encoding, our manipulation also affected familiarity. Although this
effect was not predicted, it did not contradict any of our predictions. This effect was probably
due to non-criterial recollection, which is a form of recollection that is manifested in the esti-
mates of familiarity. In the General Discussion we describe the idea of non-criterial recollection PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 15 / 22 Encoding Influences Recollection-Based Retrieval and discuss possible reasons for its appearance in this experiment and in Experiment 2, but not
in Experiments 1. and discuss possible reasons for its appearance in this experiment and in Experiment 2, but not
in Experiments 1. PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 Encoding Influences Recollection-Based Retrieval several contextual cues could fuel the process of recollection. We speculate that under such cir-
cumstances—where an abundance of non-diagnostic contextual-information is available—an
“indirect” influence of the contextual information can affect familiarity in the guise of non-cri-
terial recollection. Nevertheless, this explanation is well accommodated with the effects found
on familiarity in Experiment 3, but not in Experiment 2. Yet, one should note that non-criterial
recollection is typically observed for verbal stimuli [57] [59]; thus, it is possible that in Experi-
ment 2 as well, the effect of attention on familiarity may be a manifestation of non-diagnostic
contextual-information. A second caveat to our thesis regarding the importance of the stage in which the manipula-
tion is carried out, is that this suggestion can only apply to a limited—albeit large—set of
manipulations that involve only a single memory stage. In this article, we demonstrated that
fluency and attentional-load manipulations had a differential effect on recollection as a func-
tion of the stage in which the manipulation was carried out. Nevertheless, there are many
manipulations for which we do not make any predictions, in that there is no clear stage at
which they can be said to be carried out. Such manipulations include aging (e.g., [50]), amnesia
(e.g., [34]), delay between encoding and retrieval (e.g., [60]) and word frequency (e.g., [61]). More research is needed to better understand when these manipulations influence recollection,
when they influence familiarity and when they influence both (cf., [35]). Likewise, some
manipulations (e.g., massed versus spaced presentation, e.g., [62]) can only be carried out at
encoding, and so our thesis cannot be empirically tested for these manipulations (though, as
predicted by our suggestion, they do show a consistent effect on recollection). A final, third caveat has to do with an effect on recollection found when response-deadline
is manipulated, in which the time allotted to retrieval is limited to less than 1000 milliseconds
(i.e., the "fast" condition in [63]) or to more than 1000 milliseconds (i.e., the "slow" condition). The response-deadline manipulation can only be carried out at retrieval, and has typically been
found to affect recollection. We argue that our framework addresses manipulations that modulate the magnitude of rec-
ollection-based retrieval, reflected by different levels of recollection-based retrieval in perfor-
mance. The response deadline procedure, in contrast, is designed to completely eliminate
recollection-based retrieval—which it succeeds in doing. General Discussion Non-criterial
recollection was suggested to be the manifestation of recollection in estimates of familiarity,
under conditions where retrieval is mainly recollection-based [57] [58]. Thus, the most accurate
prediction we can make is that to the extent that an encoding manipulation affects recognition
performance, it should always influence recollection and may at times also affect familiarity in
the same direction as it affected recollection. In those cases, the effects of the manipulation on
familiarity would be the product of non-criterial recollection. Still, this finding does not challenge our thesis [19] [41] [55]. Our thesis only focuses on the
intimate relationship between encoding and recollection. The finding that encoding manipula-
tions can affect familiarity has been interpreted as non-criterial recollection [56]. Non-criterial
recollection was suggested to be the manifestation of recollection in estimates of familiarity, Unfortunately, current understanding of non-criterial recollection is still limited and the
conditions under which it appears can only be explained on a post-hoc manner. Though not
predicted by us, the effects of divided attention at encoding on familiarity in Experiments 2
and 3, may suggest that perceptual distinctiveness may mediate the appearance of non-criterial
recollection. According to this suggestion, in Experiment 3, not only standard facial-features,
but also expression, hairstyle and color were encoded as contextual information. This makes
the attribute of “uniqueness”, one which is not diagnostic with regard to its appearance in the
study phase. Instead, only the more standard facial features or the retrieval of a combination of PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 16 / 22 Encoding Influences Recollection-Based Retrieval intimately related to the encoding stage, using different estimation methods of recollection and
familiarity [70] [71] [72]. Critically, we do not rule out the notion that recollection may be affected by manipulations
carried at retrieval; we argue that recollection is prone to be affected when some variability in
the processing of to-be-remembered information can be attributed to the encoding stage (see
also [54] [73]). A study also highly relevant to our current work is McCabe and Balota's study
[69], in which medium-frequency target words were paired with low or high frequency unre-
lated prime words. When carried at encoding, high frequency primes increased estimates of
recollection (but not of familiarity), as compared to low frequency primes. When carried at
retrieval, this manipulation again increased estimates of recollection, but not of familiarity. Critically, we do not rule out the notion that recollection may be affected by manipulations
carried at retrieval; we argue that recollection is prone to be affected when some variability in
the processing of to-be-remembered information can be attributed to the encoding stage (see
also [54] [73]). A study also highly relevant to our current work is McCabe and Balota's study
[69], in which medium-frequency target words were paired with low or high frequency unre-
lated prime words. When carried at encoding, high frequency primes increased estimates of
recollection (but not of familiarity), as compared to low frequency primes. When carried at
retrieval, this manipulation again increased estimates of recollection, but not of familiarity. McCabe and Balota explained their results within the expectancy-heuristic framework (see also
[9]). According to this framework, participants expect an item to have a certain level of activa-
tion. If there is a discrepancy between the actual and the expected levels of activation, then this
discrepancy is attributed to past occurrence of the item. Whereas Whittlesea [9] suggested that
expectancy discrepancy is attributed to item's familiarity, McCabe and Balota [69] suggested
that if an item reaches a certain amount of activation, expectancy discrepancy may be attrib-
uted to recollection. Therefore, recollection can be affected by manipulation carried at retrieval
as well as at encoding. The findings presented in this article may be interpreted within the
expectancy-discrepancy framework; we discuss it later on from a single process perspective. An additional issue that needs to be addressed is the effect of encoding-retrieval compatibil-
ity on recollection and familiarity. Dewhurst and Brandt manipulated the depth to which infor-
mation was processed (specifically, Read versus Anagram) at both encoding and retrieval [74]. These authors found that remember—but not know—responses were increased when informa-
tion was presented in the same format (read, anagram) at encoding and retrieval (see also [65]
and [13], for similar results using size congruency). However, in contrast to these findings, our
findings suggest that encoding-retrieval compatibility cannot completely explain the contribu-
tion of recollection to memory performance. Specifically, in our study, we deliberately manipulated the stage in which the manipulation
was carried out in two separate blocks, thereby enabling us to examine the pure effects of the
manipulation at encoding or at retrieval. For instance, in the encoding-manipulation block of
Experiment 1, participants studied words in sentences that evoked a fluent or a non-fluent pro-
cessing of the target word. At test, words were presented individually, without a sentence. Hence, there was no perceptual compatibility between encoding and retrieval presentation and
yet recollection was higher at the fluent as compared to the non-fluent processing. Thus, although we do not dispute the notion that recollection-based retrieval may benefit
from encoding-retrieval compatibility more than familiarity-based retrieval (but see [63], we
argue that the extent to which each process would be affected by the manipulation is better
determined by the stage in which the manipulation is carried. Our thesis is that the effect on
recollection is, for the most part, unrelated to the nature of the manipulation. Rather, it is the
stage—encoding versus retrieval—in which the variability of processing the information takes
place that primary determines which process would be affected. We propose that given that the
fluency manipulation—or for that matter any manipulation at all—was carried out at encoding,
it would affect recollection. Specifically, recollection-based
retrieval is typically initiated between 500–800 milliseconds after stimulus onset, whereas
familiarity-based retrieval is typically initiated between 300–500 milliseconds (e.g., [64]). Thus,
in the fast response-deadline condition, where response time is usually limited to 700 millisec-
onds (e.g., [65]) recollection-based retrieval has little chance of being initiated, and indeed,
does not show up. Therefore, although this manipulation is carried out at retrieval, it affects
recollection by eliminating it (in the fast condition) rather than by modulating its contribution
to performance. After delineating the limits of our thesis, we would like to focus of the big picture. The
notion that the stage in which the manipulation is carried out predicts the processes to be
affected can explain the majority of the data reported in the literature. For example, modality
change affects recollection only when carried out at encoding but not at retrieval [66]. Likewise,
levels of processing (e.g., [15]) and study duration (e.g., [62]) mainly affect recollection because
these manipulations, too, are manipulated at encoding. Finally, semantic priming does not
affect recollection (e.g., [21]) when manipulated at retrieval. In contrast to our thesis, one could argue that there are several studies that demonstrated an
effect on recollection by manipulations carried out at retrieval [22] [67] [68] [69]. However,
most of these studies either did not estimate the independent contribution of recollection and
familiarity (e.g., [68]) or did so by measuring changes in response-confidence levels (e.g., [22]
[67]). Therefore, further work is needed in order to examine our notion that recollection is PLOS ONE | DOI:10.1371/journal.pone.0130403
July 2, 2015 17 / 22 S2 Table. Mean estimates (and SE) of the proportion of Remember and Know responses hit
rates, as a function of Attentional: Full, divided at encoding, and divided at retrieval.
(DOCX) S3 Table. Mean estimates (and SE) of the proportion of Remember and Know responses hit
rates, as a function of Attentional-load (High, Low) and Memory stage (Encoding,
Retrieval). Author Contributions Conceived and designed the experiments: ER YGG. Performed the experiments: ER YGG. Ana-
lyzed the data: ER YGG. Contributed reagents/materials/analysis tools: ER YGG. Wrote the
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July 2, 2015 Supporting Information S1 Table. Mean estimates (and SE) of the proportion of Remember and Know responses hit
rates, as a function of Fluency (Fluent, Non-fluent) and Memory stage (Encoding, Retrieval). (DOCX) 18 / 22 PLOS ONE | DOI:10.1371/journal.pone.0130403
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primer for t-tests and ANOVAs. Front Psychol 4:863. doi: 10.3389/fpsyg.2013.00863 PMID: 24324449 22 / 22
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Modelling of a CFD Microscale Model and Its Application in Wind Energy Resource Assessment
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MATEC web of conferences
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1 Introduction Energy is one of the most valuable consumables of
human society. With energy shortage becoming obvious,
the demand for renewable energy is rapidly increasing. Among these available new energy resources is wind
power which has widely been used during recent years. However, drastic fluctuations of wind power generation
being caused by random wind variation will result in a
serious strike on the power grid [1]. The reliable and
accurate prediction of wind farm near the wind turbines
can effectively improve the safety and economy for wind
power generation. Currently, the most general method for
wind farm prediction is using the mesoscale weather
forecast models, e.g. WRF, MM5 [2]. The demand for a
more precise prediction of atmospheric processes calls
for the improvement of the resolution of model and grid. But it will cause extreme increase of computation and
time expense if the fine mesh model is used with higher
resolution in the whole prediction area and consequently
making the model unable to meet the requirements of fast
prediction. Therefore, the solution is applying the
microscale model in the concerned local region on the
background of mesoscale meteorological model. This
method is called the coupling model method. Models
developed on computational fluid dynamics (CFD)
method can be used to simulate the local wind field on a
fine grid. Several meteorological CFD models have been The coupling method is derived from the idea of the
regional model developed by Richardson [5]. The
advantage of the coupling method is that the two models
that are based on the coarse grid and the fine grid
respectively can be independently programmed and
launched. Between the two models it only needs to build
a reasonable coupling scheme to achieve the exchange of
information, thus helping to improve the efficiency and
accuracy of prediction. In recent years, along with the air
pollution problem becoming the international issue, the
method coupling the mesoscale model and microscale
CFD model has widely been applied in the simulation of
urban atmospheric flow and environmental pollution flow. M. Tewari [6] calculated the wind speed and direction
above the Salt Lake City via coupling WRF and CFD-
Urban method. Wyszogrodzki [7] simulated the pollutant
dispersion of Oklahoma City by coupling WRF model
with a CFD-LES solver. Kwak [8] did the air quality
simulation in a high-raise building area of Seoul by
coupling a CFD model with mesoscale meteorological
and chemistry-transport model. Modelling of a CFD Microscale Model and Its Application in Wind
Energy Resource Assessment Jie-shun Yue1,a, Song-ping Wu1,2 and Fei-shi Xu3 1School of Aeronautical Science and Engineering, Beihang University, 100083, Beijing, P. R. China
2National computational fluid dynamics laboratory of China, 100083, Beijing, P. R. China
3Sino-French Engineering School, Beihang University, 100083, Beijing, P. R. China 1School of Aeronautical Science and Engineering, Beihang University, 100083, Beijing, P. R. China
2National computational fluid dynamics laboratory of China, 100083, Beijing, P. R. China
3Sino-French Engineering School, Beihang University, 100083, Beijing, P. R. China Abstract. The prediction of a wind farm near the wind turbines has a significant effect on the safety as well as
economy of wind power generation. To assess the wind resource distribution within a complex terrain, a
computational fluid dynamics (CFD) based wind farm forecast microscale model is developed. The model uses
the Reynolds Averaged Navier-Stokes (RANS) model to characterize the turbulence. By using the results of
Weather Research and Forecasting (WRF) mesoscale weather forecast model as the input of the CFD model, a
coupled model of CFD-WRF is established. A special method is used for the treatment of the information
interchange on the lateral boundary between two models. This established coupled model is applied in
predicting the wind farm near a wind turbine in Hong Gang-zi, Jilin, China. The results from this simulation
are compared to real measured data. On this basis, the accuracy and efficiency of turbulence characterization
schemes are discussed. It indicates that this coupling system is easy to implement and can make these two
separate models work in parallel. The CFD model coupled with WRF has the advantage of high accuracy and
fast speed, which makes it valid for the wind power generation. founded, such as WindSim of Norway and Meteodyn WT
of France [3][4]. Co espo d g au o : buaayjs@buaa.edu.c
© 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/). a Corresponding author: buaayjs@buaa.edu.cn
© 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/). lished by EDP Sciences. This is an open access article distributed under the terms of the Creative
Commons Attribution
reativecommons.org/licenses/by/4.0/). © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creativ
License 4.0 (http://creativecommons.org/licenses/by/4.0/). ,
MATEC Web of Conferences 7
ICMIT 2016
0 12004 (2016) ,
MATEC Web of Conferences 7
ICMIT 2016
0 12004 (2016) DOI: 10.1051/matecconf/20167012004 2.1 Dynamics equation WRF model uses the dynamics equations built in the p-σ
coordinates, the output results will be transformed into
spherical coordinates of the earth. Due to the small
computational area of the regional wind field, the
curvature of the earth can be neglected, and thus the CFD
model can be established in local Cartesian coordinate. Considering the external force, the dynamics equations of
the compressible NS equations is as follows: where the preconditioning parameter is
2
r
M 2
r
M
,
0
min 1, max
,
r
M
M M
min 1
, while M and
0
M
are
the local Mach number and reference Mach number,
respectively. where the preconditioning parameter is
2
r
M 2
r
M
,
0
min 1, max
,
r
M
M M
min 1
, while M and
0
M
are
the local Mach number and reference Mach number,
respectively. v
t
v
t
q
F
F
s (1) (1) The control equation after preconditioning is: where
,
,
,
,
T
u
v
w E
q
is the conserved variables,
v
t
v
t
Q
q
F
F
s (4) (4)
0,
,-
,-
,0
T
f v f u
g
0, f
f
s
is
the
source
term,
2 sin
f
2 sin
is the Coriolis force and g is gravitational
acceleration.
0,
,-
,-
,0
T
f v f u
g
0, f
f
s
is
the
source
term,
0,
,-
,-
,0
T
f v f u
g
0, f
f
s
is
the
source
term,
2 sin
f
2 sin
is the Coriolis force and g is gravitational
acceleration. During the numerical solution of the above equations,
LUSGS algorithm is used for the inner iteration along the
pseudo time. The backward Euler implicit scheme is used
for the outer iteration of physical time, where the
convection term using implicit processing and the viscous
term and the source terms using explicit processing. Inviscid flux is discretized by the Roe scheme. If a reference pressure
0
p is specified, the difference
between the pressure of a certain point and the reference
pressure, is called the perturbation pressure, denoted as
0
p
p
p0
p
p . ,
MATEC Web of Conferences 7
ICMIT 2016
0 12004 (2016) DOI: 10.1051/matecconf/20167012004
0
p
p
p
RT
p
RT
0
p
0 the flow structure and simulate the unsteady variation of
the vortex in the wind field. Emeis [11] suggested that
turbulence, thermal convection and surface-induced
secondary circulations could be the three main challenges
to mesoscale–microscale models. (2) Among dozens of parameterize schemes of turbulence,
two Reynolds-averaged Navier–Stokes turbulence models
are considered. One is B-L model. This model is an
algebra model, which does not need to introduce extra
control equations as well as costs small amount of
calculation. It especially suits for the simulation of near
wall turbulent flow [15]. The other approach to
parameterize the turbulence is using the k-ω SST model. This model solves two dynamic equations, one is the
equation for turbulence kinetic energy, k, the other is for
turbulence dissipation rate, ω. This model is more
complex than the B-L model, but it is suitable for all
kinds of turbulence including near wall flow and shear
flow [16]. The speed of wind is naturally much lower than the
speed of sound. Assuming that the wind velocity does not
influence the density of flow, the simulation will adopt
the incompressible flow control equations, so that the
pressure and velocity can be solved separately [12]. For
the wind field prediction, it’s more suitable to use the
compressible flow control equations due to the severe
changes in air density. Yet in order to avoid the apparent
decrease of convergence rate and accuracy, i.e. ‘stiffness’
problems, while solving the compressible equations, the
preconditioning
for
equations
is
necessary. The
preconditioning method has been well developed and
been widely used in solving a variety of low speed flow
problems [13][14]. 2.2 CFD discretization scheme based on the
preconditioning method According to the preconditioning algorithms, the
prime
variable
is
T
p u v w T
Q
and
the
preconditioning matrix is [17]:
1
0
0
0
0
0
0
0
0
0
1
p
RT
T
u
u
RT
T
v
v
RT
T
w
w
RT
T
H
H
u
v
w
c
RT
T
0
0
0
1
T
1
RT
RT
RT
RT
u
u
T
u
RT
RT
RT
RT
v
v
T
v
RT
RT
RT
RT
RT
w
w
T
w
RT
RT
RT
RT
RT
H
1
H
1
H
T
1
pc
T
p
RT
RT
(3)
1
0
0
0
0
0
0
0
0
0
1
p
RT
T
u
u
RT
T
v
v
RT
T
w
w
RT
T
H
H
u
v
w
c
RT
T
0
0
0
1
T
1
RT
RT
RT
RT
u
u
T
u
RT
RT
RT
RT
v
v
T
v
RT
RT
RT
RT
RT
w
w
T
w
RT
RT
RT
RT
RT
H
1
H
1
H
T
1
pc
T
p
RT
RT
(3) 2 Method: Forecast model and coupling
process (3) 2.2 CFD discretization scheme based on the
preconditioning method Taking the advantage of CFD’s ability of accurately
simulating the flow details and using the preconditioning
method for the compressible Navier-Stokes equation of
the low speed flow, this paper developed a fine mesh
method for local wind field prediction. A coupling
process of the CFD model and the mesoscale weather
forecast model WRF is established. The coupled model
carries out a refinement on the coarse mesh in WRF. A
special method for information exchange on the lateral
boundary of two models is adopted. The wind speed near
Hong Gang-zi in Jilin, China wind power station is
calculated as an application of this coupled model. The
results are compared with the results generated by WRF
and the measured data of wind towers. In the case of low speed flow, the NS equations will
appear the ‘stiffness’ problem of slow convergence rate
and poor accuracy. In order to overcome this problem,
the preconditioning for the equations is necessary. For the
change of wind speed within the 24h-72h, it needs to
adopt a dual time step algorithm for calculating the
unsteady time integration. A pseudo time is introduced
and the preconditioning only plays role on the pseudo
time term. 1 Introduction In the field of wind
energy, Katurji [9] simulated the turbulent flow of
complex terrain by coupling the WRF model and
WindSim model. Gopalan [10] compared several
different CFD models by coupling them with the WRF
model. The results showed that the CFD model can refine ,
MATEC Web of Conferences 7
ICMIT 2016
0 12004 (2016) 3 Numerical forecast design The wind field near a wind tower in Hong Gangzi of Jilin,
China is selected for the wind velocity prediction. The
average altitude of this area is about 130m. The
horizontal resolution of WRF is 5km. 4 coarse grids
around the wind tower are chosen with the total area for
CFD model becoming 10km×10km. As is seen from the
computational area for the coupling model in Figure 2,
the red dot is the wind tower location, while the selected
grid for refinement is within the dashed-line box. Each
coarse grid is divided into 20 × 20 fine mesh, namely a
horizontal resolution of 250m for CFD model. In the
vertical direction, the upper boundary is taken as 1km and
the height of the first layer from the ground is 2 meters. The coordinates of the wind tower are 123.892 east
longitude and 45.549 north latitude, in which 4 measuring
points arranged at the height of 10m, 30m, 50m and 70m,
respectively. Figure 2. Computational area for the coupling model. The whole period for a prediction is 72h, i.e. 3 days. WRF provides a data every 15 minutes. Hence the
boundary values need to be updated every 15 minutes. A
new forecast starts every 24h according to the data
sequence of WRF. 12:00 is chosen as the starting time for
a 72h forecast. The prediction process lasts from July
24th to July 29th, 2012, with a total of 6 time series
(Table 1). Figure 2. Computational area for the coupling model. Figure 1. Working process of the coupling model. The horizontal grid scale in the WRF model is
thousands of meters, while vertical hundreds of meters. The coupling model chooses several pieces of coarse grid
corresponding to the concerned local wind field to be the
refining region. The refinement decreases the mesh size
by one order of magnitude. Physical quantities on fine
mesh point are obtained by interpolating the values on
coarse mesh, of which the interpolated result in the initial
time is regarded as the initial flow field for CFD
simulation. The interpolated results in the follow-up
moment are used as boundary conditions, to achieve the
information exchange between the two models. There are two methods to deal with boundary
conditions. One is to directly use interpolated data to
update the physical quantities on the boundary of the
CFD calculation, which is called the fixed boundary
condition. 2.1 Dynamics equation At this time, the state equation can be
written as: 2 ,
MATEC Web of Conferences 70 12004 (2016) ,
MATEC Web of Conferences 7
ICMIT 2016
0 12004 (2016) DOI: 10.1051/matecconf/20167012004 2.3 The coupling of coarse mesh and fine mesh
model To reduce the horizontal gradient of variable near the
boundary, another method applied is called the transition
zone method [19]. In this method, several layers of grid,
i.e. the transition zone, should be added outside the
computational area. In the outermost lateral of the
transition zone, the fixed boundary condition is applied,
while in the innermost lateral the result from CFD
calculation is preserved. For the transition zone, a
transition function is used for linking these two parts. The output file format of mesoscale prediction model
WRF is NetCDF, which contains geographic coordinates
of the coarse grid and physical quantities such as velocity,
pressure and humidity. In this coupling model, the data of
time, geographic coordinates, velocity, pressure and
temperature are extracted as the required information for
CFD computation. All the extracted data is stored in
single files in time series. Along with the time advance,
the program regularly read the file of each time, as the
input to update the computational boundary condition. The working process of the coupling model is shown in
Fig. 1: The time step of CFD is smaller than the time interval
of WRF data. Therefore, during the calculation of the
CFD model, at each physical time step, it needs to do the
interpolation for WRF data between two adjacent
moments. g
Figure 1. Working process of the coupling model. ,
MATEC Web of Conferences 7
ICMIT 2016
0 12004 (2016) results provided by WRF and CFD model have almost
the same trend. For the time integration scheme, the dual-time step
method is adopted. The iterative process includes the
inner iteration of pseudo time and the outer iteration of
real physical time marching. In order to improve the
efficiency and ensure the high time accuracy, the time
step of outer iteration is 20s, and the maximum step of
inner iteration is 20. time (min.)
velocity(m/s)
0
1000
2000
3000
4000
0
2
4
6
8
10
12
14
16
CFD(70m)
WRF
EXP. time (min.)
velocity(m/s)
0
1000
2000
3000
4000
0
2
4
6
8
10
12
14
16
CFD(70m)
WRF
EXP. Figure 5. Wind velocity curve of 7/26 12:00 at 70m. 70m
time (min.)
0
30
60
90
120
150
180
210
240
270
300
330
0
1000
2000
3000
4000
CFD
WRF
EXP. Figure 6. Wind direction curve of 7/26 12:00 at 70m. velocity(m/s) Table 1. Time series. Serial
number
Starting time
Serial
number
Starting time
1
7/24 12:00
2
7/25 12:00
3
7/26 12:00
4
7/27 12:00
5
7/28 12:00
6
7/29 12:00 Table 1. Time series. Figure 5. Wind velocity curve of 7/26 12:00 at 70m. Figure 5. Wind velocity curve of 7/26 12:00 at 70m. 70m
time (min.)
0
30
60
90
120
150
180
210
240
270
300
330
0
1000
2000
3000
4000
CFD
WRF
EXP. Figure 6. Wind direction curve of 7/26 12:00 at 70m. 4.1 Results Fig. 3 and Fig. 4 show the instantaneous distribution of
wind velocity component u in extracted fine mesh area at
the height of about 50 meter. Comparing the WRF and
the numerical results of this coupled model, it reveals that,
because of the refinement of the grids and improvement
of the model’s accuracy, CFD model can be able to
describe the flow details and characterize the gradient of
the wind field variable. This feature makes it particularly
suitable for processing in a region with a plurality of
wind towers or wind turbines. Figure 3. Counter of velocity component u of WRF model. Figure 6. Wind direction curve of 7/26 12:00 at 70m. Due to the results of WRF are used as the initial field
and the boundary condition for CFD calculations. The
results of CFD and WRF have strong similarity. It can be
named as ‘the following property’ of the fine mesh model
for the coarse mesh model. Because the WRF model is
the only input of the CFD model, the wind velocity of
two models increases or decreases similarly with the
inflection point appearing in different positions. In fact,
the CFD model corrects the position of the inflection
point, so that the shape of the wind speed curve is closer
to the measured data. Figure 3. Counter of velocity component u of WRF model. CFD simulation has a certain time lag effect relative
to WRF data, namely ‘the lag property’ of fine mesh
model to the coarse mesh model. That is because in each
time step, the data of WRF is just input as the boundary
condition for CFD calculation, which means the changes
of the flow field will take time to spread from the
boundary to interior of the wind field. Figure 4. Counter of velocity component u of CFD result. Figure 4. Counter of velocity component u of CFD result. The CFD simulation has a certain ‘smoothing’ effect
on the wind velocity curve, making the temporal
variation of wind velocity not so intense. Results of WRF
vibrate acutely at some point, seriously deviating from
the measured data. CFD model can avoid wind velocity
to appear large fluctuation, which improves the
calculation results of WRF. By modifying the calculation The result of a set of time series (July 26th 12:00) at
the height of 70m is presented in Figure 5 and Figure 6. 3 Numerical forecast design In specific, if the boundary is the flow entrance,
by given the velocity and density, the temperature is
obtained from interpolation of the inner field, while
pressure is determined by the state equation; if the
boundary is outlet, by given the outlet pressure (result
from the interpolation), velocity and temperature are
obtained from the interpolation of the inner field [18],
while density is determined by the state equation. Figure 2. Computational area for the coupling model. The whole period for a prediction is 72h, i.e. 3 days. WRF provides a data every 15 minutes. Hence the
boundary values need to be updated every 15 minutes. A
new forecast starts every 24h according to the data
sequence of WRF. 12:00 is chosen as the starting time for
a 72h forecast. The prediction process lasts from July
24th to July 29th, 2012, with a total of 6 time series
(Table 1). 3 ,
MATEC Web of Conferences 7
ICMIT 2016
0 12004 (2016) DOI: 10.1051/matecconf/20167012004 ,
MATEC Web of Conferences 7
ICMIT 2016
0 12004 (2016) 4.2 Parallel programming and time consumption Accuracy of CFD simulation requires a big amount of
computation time. In order to increase the efficiency of
the coupling model and meet the requirement of fast
forecast, parallel programming is necessary. The
OpenMP multi-thread parallel programming is adopted. By optimizing the loop calculation in the CFD simulation,
this method is easy to program and can provide a
considerable improvement in efficiency. (b) (a) Figure 8. Correlation coefficient (a) 10m (b) 50m. From Fig. 7 it can be seen that, the coupled CFD
model has a larger mean value than the WRF model,
whereas the values are minus. It shows that in general,
the wind velocity calculated by CFD is smaller than that
by WRF. The near wall boundary layer effect decreases
the wind speed magnitude. Therefore, the extremely high
wind speed deriving from WRF can be avoided. Figure 8
shows that the coupled CFD model has lager correlation
coefficient than the WRF model, which means the curve
computed by the CFD model is closer to the measured
data. Overall, the result of coupling model is more
sophisticated, closer to the measured wind velocity and
has optimized the forecast results of WRF. Table 2. Computation time of the coupling model. The parallel model is running with 6 threads. Tab. 2
reveals the time consumption before and after the parallel
is adopted. The parallel gives the program an acceleration
of more than 20%. 4.1 Results It
can be roughly seen from the figures that, through
enhancing the accuracy of the WRF model, the result of
the coupling model is more close to the measured curve
of wind velocity. On the other hand, the wind direction 4 DOI: 10.1051/matecconf/20167012004 ,
MATEC Web of Conferences 7
ICMIT 2016
0 12004 (2016) time step, the ‘smoothing’ effect can be increased or
decreased, to achieve the resolution adjustment according
to the actual situation and the requirements. serial number
correlation
0
1
2
3
4
5
6
7
0
0.2
0.4
0.6
0.8
1
CFD
WRF
serial number
correlation
0
1
2
3
4
5
6
7
0
0.2
0.4
0.6
0.8
1
CFD
WRF
(a) (b)
Figure 8. Correlation coefficient (a) 10m (b) 50m. serial number
correlation
0
1
2
3
4
5
6
7
0
0.2
0.4
0.6
0.8
1
CFD
WRF
(b) serial number
correlation
0
1
2
3
4
5
6
7
0
0.2
0.4
0.6
0.8
1
CFD
WRF
(a) 4.4 Comparison of turbulence models Two models are chosen for the parameterization of
turbulence, one the algebraic B-L model, the other is two-
equation k-ω SST model. Theoretically, the algebraic
operation of B-L model is efficient and fast, and it is
suitable for near wall flow. However, it is not suitable for
the wake flow, the shear flow, etc. SST model is a
relatively well-developed two-equation model, which
more accurately calculates the adverse pressure gradient
and separated flow. Yet solving two extra equations,
computational efficiency of this model is lower than the
algebraic model. The model has to take into account the
efficiency and accuracy of wind prediction. Thus the
evaluation of these two models is very important. Table 3
is the comparison of the two models in the aspect of
calculation time. 4.3 Error analysis In order to evaluate the optimization effect of CFD model,
the average error and the correlation coefficient between
the WRF and CFD results are compared with the
measured data of each time series. The average error
indicates the mean value of the error between
computational results and measured data, while the
correlation coefficient indicates the degree of the linear
correlation between computational results and measured
data. Computing the 6 sets of time series shown in Table
1 with B-L model, the error analysis at the height of 10m
and 50m is carried out and the results are shown in Fig. 7
and Fig. 8, respectively. Table 3. Computation time of two turbulent models. Table 3. Computation time of two turbulent models. Table 3. Computation time of two turbulent models. Time(s)
B-L
15163
k-ω SST
16386 serial number
mean error
0
1
2
3
4
5
6
7
-5
-4
-3
-2
-1
0
1
2
CFD
WRF
serial number
mean error
0
1
2
3
4
5
6
7
-5
-4
-3
-2
-1
0
1
2
CFD
WRF
(a) (b)
Figure 7. Mean error (a) 10m (b) 50m. serial number
mean error
0
1
2
3
4
5
6
7
-5
-4
-3
-2
-1
0
1
2
CFD
WRF
(b) serial number
mean error
0
1
2
3
4
5
6
7
-5
-4
-3
-2
-1
0
1
2
CFD
WRF
(a) Time(s)
B-L
15163
k-ω SST
16386 The results show that for short-term forecasting, both
models can achieve completing 72 hours’ forecasts
within 5 hours. Since the turbulence equations are not a
major part of the calculation, and the parallel
programming methods also have a significant impact on
the calculation speed, B-L mode computing speed is not
much faster than the k-ω SST model. From this point of (b) (a) Figure 7. Mean error (a) 10m (b) 50m. 5 ,
MATEC Web of Conferences 7
ICMIT 2016
0 12004 (2016) DOI: 10.1051/matecconf/20167012004 view, by introducing of parallel computing, slow
shortcomings
of
two-equation
k-ω
SST
model
calculations can be made up. According to the results of a series of time sequence, as
the fine grid model, the CFD model can effectively
augment the prediction precision of the WRF model. The
wind velocity curve of the coupled model is overall close
to that of WRF, presenting ‘the follow property’, ‘the lag
property’ and ‘smoothing’ effect. These characteristics of
CFD model modify the inflection point and smoothness
of WRF model curve, thus make it closer to the measured
data. In addition, as can be seen from the error analysis,
CFD model can significantly reduce the calculation error
of the WRF model. Figure 9. Mean error of different turbulent models. Figure 10. Correlation coefficient of different turbulent models. The established CFD and WRF coupling model can
undertake delicate simulation for the wind field above
local complex terrains and can be used for short-term
wind velocity forecast. References 1. M. Jannatia, S. H. Hosseiniana, B. Vahidi,
Renew. Sust. Energ. Rev. 29 158-127( 2014) 2. A. Gsella, A. de Meij, A. Kerschbaumer et al,
Atmos. Environ. 89 797-806( 2014) Figure 10. Correlation coefficient of different turbulent models. 3. S. Milashuk, W. Crane, Environ. Mod. Soft. 26
429-433(2011) Fig. 9 and Fig. 10 further compare the average error
and the correlation coefficient of B-L model and k- ω
SST model. It can be seen from the figures that,
compared to the k-ω SST model, the B-L model is more
accurate. B-L model is an algebraic model, so in each
calculation step it directly gives the exact value of the
turbulent viscosity coefficient. While in k-ω SST model
the accurate obtaining of turbulent viscosity coefficient
requires a lot of iteration, and the excessive number of
iteration will reduce the efficiency. Thus, considering the
computational efficiency and accuracy, for this coupling
mode, B-L model is a better choice. However, the terrain
of this forecast is relatively flat. Whether k-ω SST model
will perform better in the undulating terrain with
mountains still needs to be verified. (
)
4. J. Manning, P. Hancock, R. Whiting, Wind Eng. 5 477-500(2010) 5. L. Richardson, Weather Prediction by Numerical
Process(London: Cambridge University Press,
1922) 6. M. Tewari, H. Kusaka, F. Chen et al, Atmos. Res. 96 656-664(2011) 7. A. Wyszogrodzki, S. Miao, F. Chen. Atmos. Res. 118 324-345(2012) 8. K. Kwak, J. Baik, Y. Ryu et al, Atmos. Environ. 100 167-177(2015) (
)
9. M. Katurji, (Ph. D. thesis, University of
Canterbury, (2011) 10. H. Gopalan, C. Gundling, K. Brown, J. Wind
Eng. Ind. Aerod. 132 13-26 (2014) Table 3. Computation time of two turbulent models. In order to improve the accuracy
of CFD model and more accurately describe the velocity
fluctuations, the next research direction will be to reduce
the coupling degree, optimize the iterative scheme of
time
integration,
to
optimize
the
turbulence
parameterization schemes, and to consider the land
surface process. Figure 9. Mean error of different turbulent models. Figure 9. Mean error of different turbulent models. 5 Conclusion 11. S. Emeis, J. Wind Eng. and Ind. Aerod. 144
24-30(2015) The author has established a CFD model suitable for
microscale wind field simulation. This model uses the
preconditioning technique to solve the compressible NS
equations,
parameterizes
the
turbulent
viscosity
coefficient with the BL model and k-ω SST model. On
this basis, by coupling the CFD model and WRF model, a
nested model which can realize the fine forecast for local
wind field is developed. The coupled model is
successfully applied in the wind field prediction near a
wind tower in Hong Gang-zi, Jilin, China. The prediction
results are compared with that of the WRF model and the
measured data. 12. C. Bruneau, M. Saad, Comput. Fluids, 35 326–
348(2006) 3. E. Turkel, Appl. Numer. Math. 12 257-284(1993 14. Y. Colin, H. Deniau, J. Boussuge, Comput. Fluids,
47 1-15(2011) 15. B. Baldwin, H. Lomax, Aerospace Sciences
Meeting, 16th(1978) 16. F. Menter, I. Rumser, 25th AIAA Fluid Dynamics
Conference, Colorado(1994) 17. C. Merkle, P. Buelow, J. Sullivan et al, AIAA J. 36 515–521(1998) 18. E. Turkel, Ann. Rev. Fl. Mech. 31 385-416(1999) The CFD model can be quickly coupled with the
WRF model, and can carry on the delicate simulation of
the local wind field, to describe the wind field details. 19. A. McDoland, PINSA, 65 91-105(1999) 6
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https://openalex.org/W4378746575
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https://www.frontiersin.org/articles/10.3389/fneur.2023.1152730/pdf
|
English
| null |
CT-based thrombus radiomics nomogram for predicting secondary embolization during mechanical thrombectomy for large vessel occlusion
|
Frontiers in neurology
| 2,023
|
cc-by
| 7,517
|
TYPE Original Research
PUBLISHED 12 May 2023
DOI 10.3389/fneur.2023.1152730 TYPE Original Research
PUBLISHED 12 May 2023
DOI 10.3389/fneur.2023.1152730 TYPE Original Research
PUBLISHED 12 May 2023
DOI 10.3389/fneur.2023.1152730 TYPE Original Research
PUBLISHED 12 May 2023
DOI 10.3389/fneur.2023.1152730 stroke, mechanical thrombectomy, thrombus, secondary embolization, radiomics,
nomogram, large vessel occlusion CT-based thrombus radiomics
nomogram for predicting
secondary embolization during
mechanical thrombectomy for
large vessel occlusion OPEN ACCESS
EDITED BY
Xin Zhang,
Southern Medical University, China
REVIEWED BY
Chubin Ou,
Macquarie University, Australia
Xin Feng,
Southern Medical University, China
*CORRESPONDENCE
Peng Qi
qipeng2008@aliyun.com
Jun Lu
lujun3655@bjhmoh.cn
Daming Wang
dm.wang@bjhmoh.cn
RECEIVED 28 January 2023
ACCEPTED 14 April 2023
PUBLISHED 12 May 2023
CITATION
Yusuying S, Lu Y, Zhang S, Wang J, Chen J,
Wang D, Lu J and Qi P (2023) CT-based
thrombus radiomics nomogram for predicting
secondary embolization during mechanical
thrombectomy for large vessel occlusion. Front. Neurol. 14:1152730. doi: 10 3389/fneur 2023 1152730 OPEN ACCESS
EDITED BY
Xin Zhang,
Southern Medical University, China
REVIEWED BY
Chubin Ou,
Macquarie University, Australia
Xin Feng,
Southern Medical University, China
*CORRESPONDENCE
Peng Qi
qipeng2008@aliyun.com
Jun Lu
lujun3655@bjhmoh.cn
Daming Wang
dm.wang@bjhmoh.cn
RECEIVED 28 January 2023
ACCEPTED 14 April 2023
PUBLISHED 12 May 2023
CITATION
Yusuying S, Lu Y, Zhang S, Wang J, Chen J,
Wang D, Lu J and Qi P (2023) CT-based
thrombus radiomics nomogram for predicting
secondary embolization during mechanical
thrombectomy for large vessel occlusion. Front. Neurol. 14:1152730. d i 10 3389/f
2023 1152730 Shadamu Yusuying1,2, Yao Lu3,4, Shun Zhang1, Junjie Wang1,
Juan Chen4, Daming Wang1,2*, Jun Lu1,2* and Peng Qi1* Shadamu Yusuying1,2, Yao Lu3,4, Shun Zhang1, Junjie Wang1,
Juan Chen4, Daming Wang1,2*, Jun Lu1,2* and Peng Qi1* 1Department of Neurosurgery, Beijing Hospital, National Center of Gerontology, Institute of Geriatric
Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China,
2Graduate School of Peking Union Medical College, Beijing, China, 3Beijing Hospital, National Center of
Gerontology, Beijing Institute of Geriatrics, Beijing, China, 4Department of Radiology, Beijing Hospital,
National Center of Gerontology, Beijing, China Yusuying S, Lu Y, Zhang S, Wang J, Chen J,
Wang D, Lu J and Qi P (2023) CT-based
thrombus radiomics nomogram for predicting
secondary embolization during mechanical
thrombectomy for large vessel occlusion. Front. Neurol. 14:1152730. Background
and
aims:
Secondary
embolization
(SE)
during
mechanical
thrombectomy (MT) for cerebral large vessel occlusion (LVO) could reduce
the anterior blood flow and worsen clinical outcomes. The current SE prediction
tools have limited accuracy. In this study, we aimed to develop a nomogram
to predict SE following MT for LVO based on clinical features and radiomics
extracted from computed tomography (CT) images. doi: 10.3389/fneur.2023.1152730 KEYWORDS Introduction The baseline characteristics of the patients, including age,
gender, history of stroke, hypertension, hyperlipidemia, diabetes
mellitus, atrial fibrillation, current use of anticoagulants, and
smoking history; stroke subtype of Trial of Org 10172 in acute
stroke treatment (28); conventional thrombus imaging markers,
such as thrombus density and the vessel occlusion site, were
extracted from the patient’s medical records. In addition, the
pre-interventional parameters, such as the administration of
intravenous thrombolysis therapy, and interventional parameters,
including the use of aspiration, stent retriever, Solumbra, and
measurements, were also extracted. Figure 2 shows the detailed
process of model building. Mechanical thrombectomy (MT) has become a standard
treatment for large vessel occlusion (LVO) stroke (1), with
a successful recanalization rate ranging from 58.7% to 88%
(2, 3). Complete recanalization still cannot be obtained in a
small proportion of patients due to the formation of thrombus
fragmentation and secondary embolization (SE) during the
procedure. Thrombus fragmentation and SE can reduce the
anterior blood flow and often require more complex surgical
maneuvers to relieve the obstruction, increasing the risk of
hemorrhagic transformation (4). Generally, the stent retriever and contact aspiration remain
the first-line thrombectomy strategies in clinical practice (5). However, both techniques can cause thrombus fragmentation
(6, 7). These fragments must be curtailed to reduce the risk of
emboli and micro-emboli obstructing the downstream cerebral
vessels. Several new surgical devices and procedures, such as
the balloon guide catheter (8), EmboTrap device (9), Lazarus
funnel (10), stent retriever assisted vacuum-locked extraction
(SAVE) (11), continuous aspiration before intracranial vascular
embolectomy (CAPTIVE) (12), and balloon guide with large-
bore distal access catheter with dual aspiration with the stent
retriever (BADDASS) (13), can reduce the risk of developing
SE and improve the complete recanalization. Nevertheless, their
routine use has not been proven to be always necessary or cost-
effective. Therefore, there is a need to identify patients at risk
of developing SE to optimize the surgical approach for LVO
stroke patients. Clinical
and
radiological
features,
such
as
the
use
of
anticoagulant
therapy
(14),
pre-interventional
systemic
thrombolysis,
and
internal
carotid
artery
(ICA)
occlusion
(15), may play an important role in the development of SE. Furthermore,
previous
studies
have
also
identified
various
thrombus features, such as thrombus density and thrombus
length, that might be related to the development of SE (16–18). COPYRIGHT COPYRIGHT
© 2023 Yusuying, Lu, Zhang, Wang, Chen,
Wang, Lu and Qi. 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. Materials and methods: A total of 61 patients with LVO stroke treated by MT at
Beijing Hospital were included in this retrospective study, of whom 27 developed
SE during the MT procedure. The patients were randomly divided (7:3) into training
(n = 42) and testing (n = 19) cohorts. The thrombus radiomics features were
extracted from the pre-interventional thin-slice CT images, and the conventional
clinical and radiological indicators associated with SE were recorded. A support
vector machine (SVM) learning model with 5-fold cross-verification was used
to obtain the radiomics and clinical signatures. For both signatures, a prediction
nomogram for SE was constructed. The signatures were then combined using the
logistic regression analysis to construct a combined clinical radiomics nomogram. Results: In the training cohort, the area under the receiver operating characteristic
curve (AUC) of the nomograms was 0.963 for the combined model, 0.911 for
the radiomics, and 0.891 for the clinical model. Following validation, the AUCs
were 0.762 for the combined model, 0.714 for the radiomics model, and 0.637 for
the clinical model. The combined clinical and radiomics nomogram had the best
prediction accuracy in both the training and test cohort. Conclusion: This nomogram could be used to optimize the surgical MT procedure
for LVO based on the risk of developing SE. stroke, mechanical thrombectomy, thrombus, secondary embolization, radiomics,
nomogram, large vessel occlusion 01 Frontiers in Neurology frontiersin.org Yusuying et al. 10.3389/fneur.2023.1152730 10.3389/fneur.2023.1152730 Identification of SE The images of the multiphase CTA acquired on admission
and the digital cerebral angiography acquired during MT were
reviewed. SE was identified according to the criteria published in
our previous report (29). According to these criteria, patients are
diagnosed with SE if they have a patent intracranial artery on
the admission CTA, or pre-interventional angiography occluded
during or after the MT procedure with visible embolisms in either
the distal part of the primary occluded vessel or in a completely
new location. Radiomics can extract additional features from medical images
(19–21) and is increasingly being used to improve the diagnosis
and treatment of LVO (22–24). Previous studies used clot-
based radiomics features to predict the optimal thrombectomy
strategy for successful recanalization (25, 26) and the histological
composition of the clot (27). However, to the best of our knowledge,
no studies have been conducted investigating the use of CT-based
radiomics to predict the risk of SE before MT for patients with LVO
stroke. Therefore, in this study, we aimed to develop a prediction
nomogram for SE following MT for LVO stroke patients based on
clinical and CT-based radiomics features. Introduction However, these studies did not quantitatively analyze the wide
range of features of the whole thrombus that can increase the risk
of developing SE. Introduction in this retrospective study. The patients were included in the
study if they had an extracranial or intracranial LVO involving
either the anterior or posterior circulation; they underwent a
one-stop CT examination that contains computed tomography
angiogram (CTA) and computed tomography perfusion (CTP)
on admission using the same scanner; they were treated with
a thrombectomy strategy involving a stent retriever, contract
aspiration, or both (Solumbra); they were with good preservation
of the retrieved thrombi. Patients with incomplete CT or poor-
quality images that could not be segmented, immeasurable
thrombus location, and obvious vascular calcifications were
excluded (Figure 1). Subsequently, the patients were randomly
divided into training and testing cohorts, in a proportion of
7:3, and the overall distribution of the patient was maintained
consistent (the proportion of SE and without SE is the same). The baseline characteristics of the patients, including age,
gender, history of stroke, hypertension, hyperlipidemia, diabetes
mellitus, atrial fibrillation, current use of anticoagulants, and
smoking history; stroke subtype of Trial of Org 10172 in acute
stroke treatment (28); conventional thrombus imaging markers,
such as thrombus density and the vessel occlusion site, were
extracted from the patient’s medical records. In addition, the
pre-interventional parameters, such as the administration of
intravenous thrombolysis therapy, and interventional parameters,
including the use of aspiration, stent retriever, Solumbra, and
measurements, were also extracted. Figure 2 shows the detailed
process of model building. in this retrospective study. The patients were included in the
study if they had an extracranial or intracranial LVO involving
either the anterior or posterior circulation; they underwent a
one-stop CT examination that contains computed tomography
angiogram (CTA) and computed tomography perfusion (CTP)
on admission using the same scanner; they were treated with
a thrombectomy strategy involving a stent retriever, contract
aspiration, or both (Solumbra); they were with good preservation
of the retrieved thrombi. Patients with incomplete CT or poor-
quality images that could not be segmented, immeasurable
thrombus location, and obvious vascular calcifications were
excluded (Figure 1). Subsequently, the patients were randomly
divided into training and testing cohorts, in a proportion of
7:3, and the overall distribution of the patient was maintained
consistent (the proportion of SE and without SE is the same). CT data acquisition Whole brain dynamic volume CTA and CTP were obtained
from the Aquilion ONE, Canon Medical Systems CT scanner with
320 × 0.5 mm detector rows and 160 mm coverage. Iopamidol
(370 mg/ml iodine, Bracco, Italy) or iopromide (370 mg/ml iodine,
Bayer, Germany) were injected using a high-pressure syringe via
the elbow veins with a dose of 0.6 ml/kg, followed by a 30-ml
bolus injection of saline. The CTP parameters were 80 kV, 100 mA,
and 0.5-mm thickness reconstructed slices. The images were
reconstructed using a hybrid-iterative reconstruction algorithm
and 19 volume data packets, resulting in a total of 6,080 images for
each patient. Data acquisition The patients with LVO stroke treated by MT at a Beijing
Hospital between July 2017 and August 2022 were included 02 Frontiers in Neurology frontiersin.org Yusuying et al. 10.3389/fneur.2023.1152730 FIGURE 1
Flow chart for patient enrolment. FIGURE 2
Overall workflow of this study. Thrombus density measurements
The thrombus density was measured by positioning a region
of interest (ROI) at ∼2 mm behind the occlusion site, covering
approximately half to two-thirds of the vascular area, and another
ROI along with its corresponding position of the contralateral
artery as explained in our previously reported method (30). The mean Hounsfield Unit (HU) value was recorded on the
reconstructed NCCT images for the thrombus and contralateral
artery. The relative HU (rHU) (thrombus density/contralateral FIGURE 1
Flow chart for patient enrolment. Flow chart for patient enrolment. FIGURE 2
Overall workflow of this study. FIGURE 2
Overall workflow of this study. Frontiers in Neurology Radiomics features extraction Radiomics features were automatically extracted from the
segmented thrombus using Python’s Pyradiomics package (31)
(https://pypi.org/project/pyradiomics/). From the NCCT images,
107 radiomics features were extracted. The extracted features
were classified into first-order statistics, shape-based, gray-level co-
occurrence matrix (GLCM), gray-level size zone matrix (GLSZM),
gray-level run length matrix (GLRLM), neighboring gray tone
difference matrix (NGTDM), and gray-level dependence matrix
(GLDM). These radiomics features are described in detail on the
PyRadiomics documentation site (http://pyradiomics.readthedocs. io). All of the above-mentioned features were standardized using
the z-score. Development of the clinical radiomics
nomogram Logistic regression analysis was used to develop the clinical
radiomics nomogram. The diagnostic accuracy of the clinical
model, the radiomics model, and the clinical radiomics nomogram
was assessed by calculating the AUC in both the training and
testing cohorts. A decision curve analysis (DCA) was conducted
to evaluate the clinical effectiveness of the clinical radiomics
nomogram. The DCA involves calculating the net benefit of a
threshold probability range across the training and testing cohorts. Development of the clinical signature The process used to develop the radiomics signature was
applied to the development of the clinical signature. The collected
clinical features were included in a LASSO regression model to
select the most valuable features in the training set, and the features
with non-zero coefficients were retained. Then, the selected clinical
features were inputted into the same SVM machine learning model
to construct the risk model. The final clinical signatures were
obtained by 5-fold cross-verification. Development of the radiomics signature The feature dimensions were reduced to minimize radiomics
bias, and the course of dimensionality was as follows. The
features with a good inter- and intraobserver agreement defined as
having an ICC above 0.75 were selected. The Pearson correlation
coefficient was then calculated to identify the redundant features. Features with the largest mean absolute correlation or features that
had a correlation coefficient of 0.9 or greater were removed. Finally,
the least absolute shrinkage and selection operator (LASSO)
regression model was used to identify the most useful features
based on the training set. Depending on the regulation weight
λ, LASSO shrinks all regression coefficients toward zero and sets
the coefficients of many irrelevant features exactly to zero. The
optimal λ was determined by calculating the minimum cross-
validation error following a 10-fold cross-validation. An analysis
of retained features with non-zero coefficients was performed to
fit the regression models and construct the radiomics signatures. The radiomics score (Rad-score) was then calculated for each Thrombus density measurements ROI along with its corresponding position of the contralateral
artery as explained in our previously reported method (30). The mean Hounsfield Unit (HU) value was recorded on the
reconstructed NCCT images for the thrombus and contralateral
artery. The relative HU (rHU) (thrombus density/contralateral The thrombus density was measured by positioning a region
of interest (ROI) at ∼2 mm behind the occlusion site, covering
approximately half to two-thirds of the vascular area, and another 03 frontiersin.org 10.3389/fneur.2023.1152730 Yusuying et al. patient via a linear combination of the selected features weighted
by their respective LASSO coefficients. For selecting the optimal
radiomics model, different radiomics models were developed and
tested, respectively, to predict the risk of SE based on the following
eight machine learning classification algorithms: logistic regression
(LR), support vector machine (SVM), K nearest neighbor (KNN),
random forest (RF), extremely randomized trees (Extra-Trees),
eXtreme Gradient Boosting (XGBoost), light gradient boosting
machine (LightGBM), and multilayer perceptron (MLP). Then,
the SVM machine learning model was identified which has the
highest average area under the receiver operating characteristic
(ROC) curve (AUC) on the testing set (Supplementary Figure 1). Therefore, the final selected features were inputted into the
SVM machine learning models to construct the risk model. A 5-fold cross-verification was performed to obtain the final
radiomics signature. artery density) and 1HU (thrombus density-contralateral artery
density) were calculated. ROI segmentation All NCCT and CTA images of 0.5 mm thickness were loaded
into the 3D slicer software version 4.13.0 (https://www.slicer.org/;
3D Slicer image computing platform | 3D Slicer) and registered
with ELASTIX [https://elastix.lumc.nl/; elastix (lumc.nl)]. Using
the CTA images for guidance, two neuroradiologists (YL and
JC) manually segmented the thrombus on each NCCT image
(Figure 3). Intra-observer and inter-observer variability on the
segmentation of ROI are presented as intra- and inter-class
correlation coefficients (ICCs). The detailed process is as follows. In total, 30 cases were randomly selected, then YL and JC
independently segmented ROIs during the same period to assess
inter-observer agreement. After 1 month, YL repeated manually
segmented ROIs again based on the randomly selected 30 cases to
assess intra-observer agreement. Frontiers in Neurology frontiersin.org Screening and construction of the clinical
signatures After applying the LASSO feature screening, seven clinical
features were selected, including smoking history, intravenous
thrombolysis, internal carotid artery (ICA) occlusion, stent
retriever, Solumbra, rHU, and cardioembolism. These features were
used to establish the clinical signature. Establishment of the clinical radiomics
nomogram The combined clinical radiomics nomogram used to calculate
the SE risk is illustrated in Figure 5. The risk of developing SE was
calculated as follows. A score is first assigned to each influencing
factor. Subsequently, all scores are summed up to calculate the total
value. A line is then drawn from the total score to the risk axis to
calculate the total SE risk. A higher total score is associated with
greater SE risk. Performance of the clinical, radiomics, and
combined nomograms A summary of the diagnostic performance of the clinical,
radiomics, and combined nomograms is provided in Table 2. In the training cohort, the AUCs of the nomograms were
0.963
for
the
combined
model,
0.911
for
the
radiomics,
and
0.891
for
the
clinical
model
(Figure 6A). Following
validation, the AUCs were 0.762 for the combined model,
0.637 for the clinical model, and 0.714 for the radiomics model
(Figure 6B). Patient characteristics A total of 61 patients were eligible for the study. The training
cohort consisted of 42 patients, and the testing cohorts consisted
of 19 patients. The clinical and radiological features of all patients
included in the study are summarized in Table 1. SE occurred
in 27 of the 61 patients. Only large-artery atherosclerosis (p =
0.029) and stroke of other determined etiology (p = 0.045) differed
significantly between the training and testing cohorts. Frontiers in Neurology Statistics analysis Statistical analysis was performed using the IBM statistical
package for social sciences software (SPSS) version 26.0. The
Kolmogorov–Smirnov and Shapiro–Wilk tests were used to assess
the normality of the evaluated variables. The continuous variables
were reported as means ± standard deviations, and the categorical
variables were reported as frequency counts and percentages. The chi-square or Fisher’s exact tests were used to compare
the categorical variables, while the Mann–Whitney U-tests or
independent t-tests were used for the continuous variables. The
Python 3.11.1 software (https://www.python.org) was used for
feature extraction and screening and to build the models. The
package “regression modeling strategies (rms [R])” was used 04 frontiersin.org Yusuying et al. 10.3389/fneur.2023.1152730 FIGURE 3
Delineation of the thrombus using the 3D-slicer software. The thrombus on the non-contrast computed tomography (A) was manually segmented
using the computed tomography angiography image (B) as guidance. The red arrow indicates the location of the thrombus ROI. FIGURE 3
Delineation of the thrombus using the 3D-slicer software. The thrombus on the non-contrast computed tomography (A) was manually segmented
using the computed tomography angiography image (B) as guidance. The red arrow indicates the location of the thrombus ROI. relevant features were identified by LASSO analysis and used
to construct the radiomics signature. The best regularization
parameter was 0.059636 (Figures 4A, B). Figure 4C shows the
selected features and weights. Based on these features, the Rad-
score was calculated as follows: to develop the nomogram. The metrics used to evaluate the
performance of the three nomograms were AUC and 95%
confidence interval (95%CI), accuracy, sensitivity, and specificity. The Delong test was used to estimate the differences in the AUC
values between the three nomograms. For all statistical tests, a
p-value below 0.05 was considered to be statistically significant. Rad-score
=
0.3736422217481793
+
0.038825
×
original_glcm_InverseVariance
+0.085880
×
original_glrlm_LongRunHighGrayLevelEmphasis
+
0.046497
× original_glrlm_ShortRunHighGrayLevelEmphasis – 0.143520
× original_shape_Maximum2DDiameterSlice. Rad-score
=
0.3736422217481793
+
0.038825
×
original_glcm_InverseVariance
+0.085880
×
original_glrlm_LongRunHighGrayLevelEmphasis
+
0.046497
× original_glrlm_ShortRunHighGrayLevelEmphasis – 0.143520
× original_shape_Maximum2DDiameterSlice. Extraction, selection, and construction of
the radiomics signatures In total, 107 radiomics features were extracted from the
reconstructed NCCT images, of which 64 features (ICC >0.75)
were found to have satisfactory intra-observer and inter-observer
reproducibility. Feature pairs with high correlations were omitted,
leaving 27 features per patient for further selection. Finally, four The
Delong
test
results
of
the
training
set
showed
that no significant difference was noted between the AUC
values of the nomogram and the clinical model (p-value 05 frontiersin.org 10.3389/fneur.2023.1152730 Yusuying et al. TABLE 1 Summary of the patients’ characteristics in the training and test cohorts. TABLE 1 Summary of the patients’ characteristics in the training and test cohorts. Extraction, selection, and construction of
the radiomics signatures Variable
Training cohort (n = 42)
Testing cohort (n = 19)
p-value
Gender, n (%)
0.539
Male
23 (54.8)
12 (63.2)
Female
19 (45.2)
7 (36.8)
Age, y (mean)
74.52 ± 11.351
71.47 ± 14.946
0.435
Stroke history, n (%)
9 (21.4)
6 (31.3)
0.595
Hypertension, n (%)
27 (64.3)
10(52.6)
0.388
Hyperlipemia, n (%)
9 (21.4)
4 (21.1)
1
Diabetes mellitus, n (%)
18 (42.9)
6 (31.6)
0.404
Atrial fibrillation, n (%)
19 (45.2)
7 (36.8)
0.539
Anticoagulation, n (%)
7 (16.7)
5 (26.3)
0.596
Smoking history, n (%)
15 (35.7)
6 (31.6)
0.753
Intravenous thrombolysis, n (%)
13 (31.0)
3 (15.8)
0.351
Large-artery atherosclerosis, n (%)
28 (66.7)
7 (36.8)
0.029
Cardioembolism, n (%)
11 (26.2)
7 (36.8)
0.398
Stroke of other determined etiology, n (%)
0 (0)
3 (15.8)
0.045
Stroke of undetermined etiology, n (%)
3 (7.1)
2 (10.5)
1
Internal carotid artery occlusion, n (%)
18 (42.9)
8 (42.1)
0.956
Middle cerebral artery occlusion, n (%)
17 (40.5)
8 (42.1)
0.905
Anterior cerebral artery occlusion, n (%)
2 (4.8)
2 (10.5)
0.777
Basilar artery occlusion, n (%)
5 (11.9)
1 (5.3)
0.732
Aspiration, n (%)
20 (47.6)
13 (68.4)
0.131
Stent Retriever, n (%)
12 (28.6)
2 (10.5)
0.221
Solumbra, n (%)
10 (23.8)
4 (21.1)
1
rHU (mean)
1.08 ± 1.45
1.17 ± 0.43
0.789
1HU (mean)
6.67 ± 26.34
5.97 ± 18.73
0.918 Variable
Training cohort (n = 42)
Testing cohort (n = 19)
p-value
Gender, n (%)
0.539
Male
23 (54.8)
12 (63.2)
Female
19 (45.2)
7 (36.8)
Age, y (mean)
74.52 ± 11.351
71.47 ± 14.946
0.435
Stroke history, n (%)
9 (21.4)
6 (31.3)
0.595
Hypertension, n (%)
27 (64.3)
10(52.6)
0.388
Hyperlipemia, n (%)
9 (21.4)
4 (21.1)
1
Diabetes mellitus, n (%)
18 (42.9)
6 (31.6)
0.404
Atrial fibrillation, n (%)
19 (45.2)
7 (36.8)
0.539
Anticoagulation, n (%)
7 (16.7)
5 (26.3)
0.596
Smoking history, n (%)
15 (35.7)
6 (31.6)
0.753
Intravenous thrombolysis, n (%)
13 (31.0)
3 (15.8)
0.351
Large-artery atherosclerosis, n (%)
28 (66.7)
7 (36.8)
0.029
Cardioembolism, n (%)
11 (26.2)
7 (36.8)
0.398
Stroke of other determined etiology, n (%)
0 (0)
3 (15.8)
0.045
Stroke of undetermined etiology, n (%)
3 (7.1)
2 (10.5)
1
Internal carotid artery occlusion, n (%)
18 (42.9)
8 (42.1)
0.956
Middle cerebral artery occlusion, n (%)
17 (40.5)
8 (42.1)
0.905
Anterior cerebral artery occlusion, n (%)
2 (4.8)
2 (10.5)
0.777
Basilar artery occlusion, n (%)
5 (11.9)
1 (5.3)
0.732
Aspiration, n (%)
20 (47.6)
13 (68.4)
0.131
Stent Retriever, n (%)
12 (28.6)
2 (10.5)
0.221
Solumbra, n (%)
10 (23.8)
4 (21.1)
1
rHU (mean)
1.08 ± 1.45
1.17 ± 0.43
0.789
1HU (mean)
6.67 ± 26.34
5.97 ± 18.73
0.918
FIGURE 4
Selection of features based on the least absolute shrinkage and selection operator (LASSO) regression model. Extraction, selection, and construction of
the radiomics signatures Diferent models
Training cohort (n = 42)
Testing cohort (n = 19)
AUC (95%CI)
SEN
SPE
ACC
AUC (95%CI)
SEN
SPE
ACC
Clinical model
0.891(0.779–1.000)
0.800
0.926
0.833
0.637(0.339–0.935)
0.667
0.714
0.421
Radiomics model
0.911(0.792–1.000)
0.933
0.852
0.857
0.714(0.461–0.967)
0.833
0.571
0.579
Clinical radiomics nomogram
0.963(0.905–1.000)
0.933
0.926
0.810
0.762(0.507–1.000)
1.000
0.571
0.632
AUC, the area under the curve; SEN, sensitivity; SPE, specificity; ACC, accuracy; 95% CI, 95% confidence interval. FIGURE 6
Receiver operating characteristic curves of the radiomics model, clinical model, and the clinical radiomics nomogram for predicting the SE risk for
the training (A) and test (B) cohorts. FIGURE 5
Clinical radiomics nomogram. The radiomics (Rad-signature) and clinical signatures values can be converted into quantitative values according to
the corresponding points indicated on the axis. The total risk value was calculated by summing up the individual points. The final sum shown on the
total points axis is then used to calculate the overall SE risk. Clinical radiomics nomogram. The radiomics (Rad-signature) and clinical signatures values can be converted into quantitative values according to
the corresponding points indicated on the axis. The total risk value was calculated by summing up the individual points. The final sum shown on the
total points axis is then used to calculate the overall SE risk. LE 2 Performance of the clinical model, radiomics model, and clinical radiomics model in the training and testing cohorts. TABLE 2 Performance of the clinical model, radiomics model, and clinical radiomics model in the training and testing cohorts. e o
a ce o
e c
ca
ode , ad o
cs
ode , a d c
ca
ad o
cs
ode
e
a
g a d es
g co o s
Diferent models
Training cohort (n = 42)
Testing cohort (n = 19)
AUC (95%CI)
SEN
SPE
ACC
AUC (95%CI)
SEN
SPE
ACC
Clinical model
0.891(0.779–1.000)
0.800
0.926
0.833
0.637(0.339–0.935)
0.667
0.714
0.421
Radiomics model
0.911(0.792–1.000)
0.933
0.852
0.857
0.714(0.461–0.967)
0.833
0.571
0.579
Clinical radiomics nomogram
0.963(0.905–1.000)
0.933
0.926
0.810
0.762(0.507–1.000)
1.000
0.571
0.632
AUC, the area under the curve; SEN, sensitivity; SPE, specificity; ACC, accuracy; 95% CI, 95% confidence interval. FIGURE 6
Receiver operating characteristic curves of the radiomics model, clinical model, and the clinical radiomics nomogram for predicting the SE risk for
the training (A) and test (B) cohorts. Extraction, selection, and construction of
the radiomics signatures (A) shows a representative LASSO
coefcient distribution map. The vertical dashed line indicates the value chosen after 10 rounds of cross-validation following the coefcient
distribution map produced by the λ sequence. (B) LASSO model with the adjusted λ parameter following 10 rounds of cross-validation performed to
pass the minimum standard. The optimal λ value was 0.059636 and is indicated by the vertical dashed line. (C) The selected radiomics features and
their corresponding coefcients. FIGURE 4
Selection of features based on the least absolute shrinkage and selection operator (LASSO) regression model. (A) shows a representative LASSO
coefcient distribution map. The vertical dashed line indicates the value chosen after 10 rounds of cross-validation following the coefcient
distribution map produced by the λ sequence. (B) LASSO model with the adjusted λ parameter following 10 rounds of cross-validation performed to
pass the minimum standard. The optimal λ value was 0.059636 and is indicated by the vertical dashed line. (C) The selected radiomics features and
their corresponding coefcients. FIGURE 4 FIGURE 4
Selection of features based on the least absolute shrinkage and selection operator (LASSO) regression model. (A) shows a representative LASSO
coefcient distribution map. The vertical dashed line indicates the value chosen after 10 rounds of cross-validation following the coefcient
distribution map produced by the λ sequence. (B) LASSO model with the adjusted λ parameter following 10 rounds of cross-validation performed to
pass the minimum standard. The optimal λ value was 0.059636 and is indicated by the vertical dashed line. (C) The selected radiomics features and
their corresponding coefcients. 06 Frontiers in Neurology frontiersin.org Yusuying et al. 10.3389/fneur.2023.1152730 FIGURE 5
Clinical radiomics nomogram. The radiomics (Rad-signature) and clinical signatures values can be converted into quantitative values according to
the corresponding points indicated on the axis. The total risk value was calculated by summing up the individual points. The final sum shown on the
total points axis is then used to calculate the overall SE risk. FIGURE 5
Clinical radiomics nomogram. The radiomics (Rad-signature) and clinical signatures values can be converted into quantitative values according to
the corresponding points indicated on the axis. The total risk value was calculated by summing up the individual points. The final sum shown on the
total points axis is then used to calculate the overall SE risk. TABLE 2 Performance of the clinical model, radiomics model, and clinical radiomics model in the training and testing cohorts. Discussion Secondary embolization is a common complication of MT. Therefore, there is a need to identify patients at risk of developing
SE to optimize the surgical procedure for stroke patients. Previous
studies (14, 29) have attempted to develop risk prediction models
based on clinical and thrombus features. However, the accuracy of
these prediction models varied. To the best of our knowledge, this
is the first study using both clinical variables and pre-interventional
CT radiomics to identify patients at risk of developing SE. We acknowledge that our study has several limitations. The
incidence of SE in our study was 43.3% higher than the 35.2% of
patients reported by Gengfan et al. (29), possibly as a result of the
selection bias introduced during the retrospective data collection
process. The sample was small, and all the data were obtained
from one institute. Therefore, a larger multicenter study is required
to validate the prediction ability of the nomogram. The manual
segmentation of the thrombus is prone to inter-observer and intra-
observer variation due to which it can be easily affected by the
reader’s experience. Automatic or semiautomatic methods could
improve the accuracy of the thrombus delineation. Moreover, our
model was based on standard radiomics features. A deep learning
approach could improve the model’s prediction ability. Finally, the
underlying biologic meaning of the radiomics features is difficult to
interpret. Further studies are required to investigate the correlation
between the thrombus histological and radiomics features. Studies have shown that the risk of SE is affected by both clinical
and thrombus features (32). The clinical information provides
important information about the common risk factors, such as
the thrombus location and surgical procedure for developing SE. Various studies also evaluated the impact of specific thrombus
features on developing SE. In the study of Gengfan et al. (30),
thrombus density was identified as an independent predictor of
SE. However, this feature does not reflect the full heterogeneities
of the different thrombi. The advantage of radiomics is that it can
capture a wide range of thrombus features, thus better reflecting
the heterogeneity of the thrombus than the conventional thrombus
density feature. As a result, under the same SVM machine-learning
model, our radiomics model achieved a better performance than
the clinical model in both training and testing cohorts. However,
the best performance was achieved by the combined clinical
radiomics nomogram. Extraction, selection, and construction of
the radiomics signatures FIGURE 6
Receiver operating characteristic curves of the radiomics model, clinical model, and the clinical radiomics nomogram for predicting the SE risk for
the training (A) and test (B) cohorts. of
the
nomogram
and
the
clinical
model
(p-value
=
0.266) or the nomogram and the radiomics model (p-value
= 0.422). =
0.191),
the
nomogram,
and
the
radiomics
model
(p-
value = 0.132). In the testing set, there were either no
significant
difference
was
noted
between
the
AUC
values Frontiers in Neurology
0 07 Frontiers in Neurology frontiersin.org Yusuying et al. 10.3389/fneur.2023.1152730 FIGURE 7
Decision curve analysis for the three models for the training (A) and test cohorts (B). The y-axis represents the net benefit, and the x-axis represents
the threshold probability. Decision curve analysis for the three models for the training (A) and test cohorts (B). The y-axis represents the net benefit, and the x-axis represents
the threshold probability. Apart from clinical and thrombus features, other studies used
histological thrombus analysis to assess the risk of developing
SE. Sporn et al. (18) found that SE occurred more frequently in
thrombi with a small fraction of red blood cells. However, the
results of the histopathology analysis can only be obtained after
the MT procedure; therefore, this data cannot be used to optimize
the surgical procedure. Radiomics analysis has the advantage of
providing a fast, non-invasive method for neuro-interventional
surgeons to objectively evaluate the risk of developing SE (33)
before the surgical procedure rather than solely relying on their
clinical judgment. The high-risk patients could then have their
surgical technique optimized, which may eventually reduce the
incidence of SE. The DCA for the three models for the training and testing
cohorts are illustrated in Figures 7A, B. Compared with the clinical
and radiomics nomograms, the DCA revealed that the combined
nomogram added a net clinical prediction benefit for most of the
threshold probabilities. Discussion The net clinical prediction benefit of the
combined nomogram was further confirmed by DCA. Frontiers in Neurology frontiersin.org Conclusion This study proposed a new clinical radiomics model to
predict the risk of SE. The radiomics model showed higher 08 frontiersin.org Yusuying et al. 10.3389/fneur.2023.1152730 accuracy than the clinical model, and the clinical radiomics
nomogram outperformed both the radiomics and clinical models. Our proposed clinical radiomics model could be used by neuro-
interventional surgeons to predict the risk of SE, thus allowing
them to optimize the surgical procedure according to the
patient’s needs. Capital’s Funds for Health Improvement and Research (Grant No. 2020-4-4053). Ethics statement The
authors
declare
that
the
research
was
conducted
in the absence of any commercial or financial relationships
that
could
be
construed
as
a
potential
conflict
of interest. The studies involving human participants were reviewed and
approved by the Ethics Committee of Beijing Hospital (2019S-
174). Written informed consent to participate in this study was
provided by the patient/participants or patient/participants’ legal
guardian/next of kin. Data availability statement The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation. Author contributions SY, PQ, JL, and DW participated in the study design. JW, PQ,
and JL were the surgeons and were in charge of patient care. SY and
SZ participated in clinical data collection. YL and JC participated
in imaging evaluation for AIS patients, delineation of the region
of interest, and thrombus density measurement in images. SY,
SZ, and JW participated in data analysis, data interpretation,
and statistical analysis. SY drafted the manuscript. PQ, JL, and
DW revised the manuscript. DW was responsible for the overall
supervision. All the authors gave final approval for the version to
be published. Publisher’s note All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher. Acknowledgments We
thank
TopEdit
(http://www.topeditsci.com)
for
its
linguistic assistance during the preparation of this manuscript,
and OnekeyAI and their developer’s help in this scientific
research study. Supplementary material The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fneur.2023. 1152730/full#supplementary-material Funding Receiver operating characteristic (ROC) curve (AUC) on the testing set of
eight machine learning classification algorithms. ROC, receiver operating
characteristic; AUC, the area under the curve; LR, logistic regression; SVM,
support vector machine; KNN, K nearest neighbor; RF, random forest;
extra-trees, extremely randomized trees; XGBoost, eXtreme Gradient
Boosting; LightGBM, light gradient boosting machine; MLP,
multilayer perceptron. This study was supported by Beijing Hospital Clinical Research
121 Project BJ-2021-234, the CMAS Innovation Fund for Medical
Sciences (CIFMS) (Grant No. 2021-I2M-C&T-B-092), and the References 8. Lee DH, Sung JH, Kim SU Yi HJ, Hong JT, Lee SW. Effective use of balloon
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and
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of
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during
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with
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stroke. Neurosurgery. (2016)
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50. doi: 10.1227/NEU.0000000000001135 2. Berkhemer OA, Fransen PSS, Beumer D, van den Berg LA, Lingsma HF, Yoo AJ,
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thrombectomy
versus
stent
retriever
thrombectomy
as
first-
line
approach
for
large
vessel
occlusion. (COMPASS):
a
multicentre,
randomised, open label, blinded outcome, non-inferiority trial. Lancet. (2019)
393:998–1008. doi: 10.1016/S0140-6736(19)30297-1 10. Fargen
KM,
Mocco
J,
Gobin
YP. The
Lazarus
Funnel:
a
blinded
prospective randomized in vitro trial of a novel CE-marked thrombectomy
assist device. J Neurointerv Surg. (2016) 8:66–8. doi: 10.1136/neurintsurg-2014-
011432 10. Fargen
KM,
Mocco
J,
Gobin
YP. The
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blinded
prospective randomized in vitro trial of a novel CE-marked thrombectomy
assist device. J Neurointerv Surg. (2016) 8:66–8. doi: 10.1136/neurintsurg-2014-
011432 09 Frontiers in Neurology frontiersin.org Yusuying et al. occlusion stroke. Neuroimage Clin. (2022) 34:103034. doi: 10.1016/j.nicl.2022.
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Виды дискурса, повышающие эффективность кооперативного обучения
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Zenodo (CERN European Organization for Nuclear Research)
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Рафикова А.С. Рафикова Антонина Семеновна, кандидат психологических наук, старший научный сотруд-
ник, Государственный академический университет гуманитарных наук, Россия, 119049, г. Москва, Мароновский пер., 26. E-mail: antoninaraf@yandex.ru. Гуманитарный научный вестник. 2021. №10 Гуманитарный научный вестник. 2021. №10 81 Виды дискурса, повышающие эффективность
кооперативного обучения Аннотация. В статье представлен обзор работ, посвященных изучению форм дискурса, способству-
ющих повышению эффективности кооперативного обучения. Дано определение понятию коопера-
тивного обучения, раскрыты некоторые особенности этого образовательного подхода. Описаны
наиболее эффективные для кооперативного обучения формы использования языковых средств. Пред-
ставлена концепция Н. Мерсера, согласно которой существует три типа обсуждения, характерные для
совместного обучения: кумулятивное, диспутивное и исследовательское. Раскрыто понятие прогрес-
сивного дискурса, предложенного К. Берейтером. Перечислены основные свойства прогрессивного
дискурса: фокус на идеях как концептуальных артефактах; приоритет достижения общего понимания
перед единством мнений; стремление расширить фактологическую базу; выборочная критика; откры-
тость идеям. Отмечается, что прогрессивный дискурс и исследовательское обсуждение позволяют
максимизировать эффективность кооперативного обучения. Ключевые слова: кооперативное обучение, дискурс, исследовательское обсуждение, прогрессивный
дискурс. Rafikova A. S. Rafikova Antonina Semenovna, candidate of psychological sciences, senior researcher, State aca-
demic university for the humanities, Russia, 119049, Moscow, Maronovskiy per., 26. E-mail: anto-
ninaraf@yandex.ru. Discourse types that enhance the effectiveness of cooperative
learning Abstract. The article presents a review of researches devoted to the study of discourse types that enhance the
effectiveness of cooperative learning. The definition of the concept of cooperative learning is given, some
features of this educational approach are revealed. The most effective uses of language tools for cooperative
learning are described. The concept of N. Mercer is presented according to which there are three types of talk
that are characteristic of cooperative learning: cumulative, disputational and exploratory. The concept of
progressive discourse proposed by K. Bereiter is revealed. The main properties of progressive discourse are
listed: a focus on ideas as conceptual artifacts, common understanding given priority over agreement, expan-
sion of the factual base, selective criticism, non-sectarianism. The conclusions are based on the results of a
review of studies devoted to the study of discourse types that increase the effectiveness of cooperative learn-
ing. g
Key words: cooperative learning, discourse, exploratory talk, progressive discourse. ISSN 2541-7509 82 Педагогические науки могут оказывать помощь группам студен-
тов [7]. овышение эффективности обуче-
ния является одним из главных во-
просов, стоящих перед современ-
ной методикой преподавания. Одним из
трендов современного образования являет-
ся активное обучение, одной из форм ко-
торого, в свою очередь является коопера-
тивное обучение. Основными характери-
стиками кооперативной среды являются
содействие участию и взаимодействие сту-
дентов между собой. Создание подобной
среды требует применения специальных
форм общения между учащимися. П П Большое количество обзорных и ме-
тааналитических исследований, проведен-
ных на протяжении нескольких десятиле-
тий, подтверждают преимущество коопе-
ративного обучения в сравнении с конку-
рентным и индивидуальным в показателях
академической успеваемости, уровня мо-
тивации, отношения к работе с другими
студентами, управления конфликтными
ситуациями и когнитивных переменных
(например, концептуальное развитие, ре-
шение проблем, когнитивные навыки вы-
сокого уровня) [4; 11; 10; 6; 12]. Кооперативное обучение Эффективный процесс обучения тре-
бует прямого и активного участия студен-
тов и может быть реализован в совместной
работе для достижения общих целей. В об-
становке сотрудничества студенты стре-
мятся достичь результатов, полезных для
них самих и для всех членов группы. Ко-
оперативное обучение представляет собой
вид образовательной деятельности, в кото-
рой цели участников тесно связаны таким
образом, что каждый из них «может до-
стичь своих целей только тогда, когда дру-
гим членам группы удастся достичь сво-
их» [5, c. 786]. При кооперативном обуче-
нии преподаватели формируют группы по
2-5 студента и предлагают структуру зада-
ния (например, предоставляют инструкции
по выполнению задачи, назначают группо-
вые роли, требуют отчетности). Цель со-
стоит в том, чтобы учащиеся работали
вместе для достижения общей цели на вза-
имозависимой и взаимовыгодной основе. По мере необходимости преподаватели Постановка проблемы Несмотря на актуальность данного
вопроса, тема дискурса в кооперативном
обучении остается на данный момент не-
достаточно изучена в работах российских
авторов. Целью данной работы является
представить обзор основных зарубежных
исследований на тему роли дискурса в ко-
оперативном обучении. Одним из важных аспектов коопера-
тивного обучения являются типичные для
него формы использования языка, то есть
особые формы обсуждения, с помощью
которых люди могут совместно осваивать
новые знания и взаимодействовать в сов-
местных учебных ситуациях. В этом смыс-
ле многие авторы пытались найти ответ на
вопрос о том, какие формы использования
языка позволяют продуктивно взаимодей-
ствовать между членами группы в коопе-
ративных ситуациях. В данном обзоре бу-
дут представлены два подхода к изучению
данного аспекта кооперативного обучения:
концепция «исследовательского обсужде-
ния» [9] и идея о «прогрессивном дискур-
се» [3]. Методология Исследование было проведено на ос-
нове нарративно-описательного обзора. Исследовательское обсуждение Согласно Н.Мерсеру [8], существует
три типа обсуждения, характерных для
общения между учащимися в условиях
совместного обучения. Первый - это «ку-
мулятивное
обсуждение»,
в
котором
участники используют языковые средства,
чтобы суммировать вклад других членов
группы, не подвергая критики их идеи. Для этого типа обсуждения типичны по-
вторения и подтверждения того, что сказа-
ли другие, и группа, кажется, пытается
минимизировать различия, уделяя больше
внимания поддержанию внутренней спло-
ченности, чем совместному построению
идей. Второй - «диспутивное обсуждение»,
в которой участники используют языковые
средства, чтобы выразить свои разногласия http://naukavestnik.ru/ Гуманитарный научный вестник. 2021. №10 83 и отстаивать свою позицию, и обсуждение
часто включает повторное подтверждение
их собственной точки зрения и опровер-
жение идей других. Этот тип обсуждения
направлен не на построение объединяю-
щей идеи, а скорее на защиту собственного
мнения и принятие собственных решений,
что создает скорее соревновательную, а не
кооперативную обстановку. Третий тип -
это «исследовательское обсуждение», и в
отличие от предыдущих, он ориентирован
на совместное построение идей и прогресс
в процессе построения знаний. По этой
причине, по мнению Н. Мерсера [8], имен-
но этот тип обсуждения следует использо-
вать в образовательном процессе. В этом
типе обсуждения участники используют
язык, чтобы критически, но конструктивно
высказываться о своем собственном вкладе
и вкладе других людей, четко и ясно изла-
гая свои идеи для их совместного оцени-
вания и анализирования. Для этого обсуж-
дение часто включает предложения, кото-
рые предлагаются для совместного рас-
смотрения участниками, а также аргумен-
ты, доводы и обоснования. Исследователь-
ское обсуждение не развивается в группе
спонтанно, и студенты могут усваивать его
навыки во время специальных тренингов
[9]. Использование
исследовательского
обсуждения положительно влияет как на
групповое обучение, так и на последующее
индивидуальное обучение и академиче-
ские показатели. вень понимания, который, по мнению всех
участников,
превосходит
уровень
их
предыдущего понимания» [2, с. 9]. Для
прогрессивного дискурса характерны: фо-
кус на идеях как концептуальных артефак-
тах (т.е. объектах абстрактного знания, ко-
торые могут быть воспроизведены, проте-
стированы и улучшены [1]); улучшаемость
как важное и положительное свойство
концептуальных артефактов; достижение
общего понимания всеми членами группа-
ми имеет приоритет перед единством мне-
ний; стремление расширить фактологиче-
скую базу; выборочная критика, основан-
ная на целях развития знаний; открытость
идеям. Этот тип дискурса позволяет вос-
принимать знания и относиться к ним не
как к чему-то статическому и неизменно-
му, а как к результату деятельности, кото-
рый можно улучшать и воссоздавать, и ко-
торый можно использовать различными и
более эффективными способами благодаря
сотрудничеству и посредством сотрудни-
чества. Заключение Кооперативное обучение представля-
ет собой процесс, успешность которого
зависит от усвоения и реализации учащи-
мися определенных способов использова-
ния языковых средств. Такие формы дис-
курса, как прогрессивный дискурс и ис-
следовательское обсуждение позволяют
максимизировать эффективность коопера-
тивного обучения. Исследовательское об-
суждение способствует кооперативному
построению идей, обмену информацией,
критической и конструктивной манере вы-
сказывания своей точки зрения. Основой
прогрессивного дискурса являются систе-
матизация и анализ точек зрения учащих-
ся. Данный тип дискурса предполагает
восприятие знаний не как статичных и
неизменных конструкций, а как улучшае-
мых и воссоздаваемых динамичных струк-
тур. Овладение этими двумя формами ис-
пользования языковых средств может быть
реализовано с помощью специальных
учебных программ. CПИСОК ЛИТЕРАТУРЫ 1. Alter S. Nothing is more practical than a good conceptual artifact… which may be a theory, frame-
work, model, metaphor, paradigm or perhaps some other abstraction // Information Systems Journal. 2016. V. 27. N.5. P. 671–693. doi:10.1111/isj.12116 j
2. Bereiter C. Implications of postmodernism for science, or, science as progressive discourse // Educa-
tional Psychologist. 1994. V. 29. N. 1. P. 3-12. DOI: 10.1207/s15326985ep2901_1 3. Bereiter C., Scardamalia M. Learning to work creatively with knowledge // Powerful learning envi-
ronments: Unravelling basic components and dimensions / E. De Corte, L. Verschaffel, N. Entwistle,
J. van Merrienboer (Eds.). Oxford: Pergamon, 2003. P. 55-68. g
4. Effects of cooperative, competitive, and individualistic goal structures on achievement: A meta-
analysis / D.W. Johnson et al. // Psychological Bulletin. 1981. V. 89. N. 1. P. 47–62. https://doi.org/10.1037/0033-2909.89.1.47 5. Johnson D.W., Johnson R.T. Cooperation and the use of technology // Handbook of research for edu-
cational communications and technology / D.H. Johassen (Ed.). New York: Simon and Schuster
Macmillan,
1996. P. 785-812. https://www.researchgate.net/publication/243671476
_Cooperation_and_the_use_of_technology 6. A meta-analysis of the effects of face-to-face cooperative learning. Do recent studies falsify or verify
earlier findings? / Eva Kyndt et al. // Educational Research Review. 2013. V. 10. P. 133–149. https://doi.org/10.1016/j.edurev.2013.02.002 7. Major C. Collaborative Learning: A Tried and True Active Learning Method for the College Class-
room // New Directions for Teaching and Learning. 2020. V. 2020. N. 164. P. 19-28. https://doi.org/10.1002/tl.20420 p
g
8. Mercer N. The Guided Construction of Knowledge: Talk amongst Teachers and Learners. Bristol:
Multilingual matters, 1994. 135 p. 9. Mercer N., Dawes L. The value of exploratory talk // Exploring talk in school: Inspired by the work of
Douglas Barnes / N. Mercer, S. Hodgkinson (Eds.). SAGE Publications Ltd, 2008. P. 55-72. https://www.doi.org/10.4135/9781446279526.n4 10. Roseth C. J., Johnson D. W., Johnson R. T. Promoting early adolescents' achievement and peer rela-
tionships: The effects of cooperative, competitive, and individualistic goal structures // Psychological
Bulletin. 2008. V. 134. N. 2. P. 223–246. https://doi.org/10.1037/0033-2909.134.2.223 p
g
11. Slavin R. E. Research on cooperative learning and achievement: What we know, what we need to
know
//
Contemporary
Educational
Psychology. 1996. V. 21. N. 1. P. 43–69. https://doi.org/10.1006/ceps.1996.0004 p
g
p
12. Vass E., Littleton K. Peer collaboration and learning in the classroom // International Handbook of
Psychology in Education / K. Littleton, C. Wood, J. Kleine Staarman (Eds.). Leeds: Emerald, 2010. pp. 105–136. https://www.researchgate.net/publication/45714612_Peer_collaboration_and_learning
_in_the_classroom Прогрессивный дискурс Прогрессивный дискурс По мнению К. Берейтера [2], для то-
го, чтобы учащиеся могли участвовать в
процессе исследования и совместного кон-
струирования знаний, им недостаточно де-
литься мнениями, они должны системати-
чески анализировать и подвергать сомне-
нию их, стремясь достичь лучшего пони-
мания содержания обучения. Эта цель мо-
жет быть достигнута с помощью овладе-
ния определенной формой использования
языковых средств - прогрессивной дискур-
сом. Посредством прогрессивного дискур-
са члены группы развивают «новый уро- ISSN 2541-7509 84 Педагогические науки REFERENCES (TRANSLITERATED) 1. Alter S. Nothing is more practical than a good conceptual artifact… which may be a theory,
framework, model, metaphor, paradigm or perhaps some other abstraction // Information Systems
Journal. 2016. V. 27. N.5. P. 671–693. doi:10.1111/isj.12116 1. Alter S. Nothing is more practical than a good conceptual artifact… which may be a theory,
framework, model, metaphor, paradigm or perhaps some other abstraction // Information Systems
Journal. 2016. V. 27. N.5. P. 671–693. doi:10.1111/isj.12116 j
2. Bereiter C. Implications of postmodernism for science, or, science as progressive discourse //
Educational Psychologist. 1994. V. 29. N. 1. P. 3-12. DOI: 10.1207/s15326985ep2901_1 2. Bereiter C. Implications of postmodernism for science, or, science as progressive discourse //
Educational Psychologist. 1994. V. 29. N. 1. P. 3-12. DOI: 10.1207/s15326985ep2901_1 3. Bereiter C., Scardamalia M. Learning to work creatively with knowledge // Powerful learning
environments: Unravelling basic components and dimensions / E. De Corte, L. Verschaffel, N. Entwistle, J. van Merrienboer (Eds.). Oxford: Pergamon, 2003. P. 55-68. 3. Bereiter C., Scardamalia M. Learning to work creatively with knowledge // Powerful learning
environments: Unravelling basic components and dimensions / E. De Corte, L. Verschaffel, N. Entwistle, J. van Merrienboer (Eds.). Oxford: Pergamon, 2003. P. 55-68. g
4. Effects of cooperative, competitive, and individualistic goal structures on achievement: A meta-
analysis / D.W. Johnson et al. // Psychological Bulletin. 1981. V. 89. N. 1. P. 47–62. https://doi.org/10.1037/0033-2909.89.1.47 4. Effects of cooperative, competitive, and individualistic goal structures on achievement: A meta-
analysis / D.W. Johnson et al. // Psychological Bulletin. 1981. V. 89. N. 1. P. 47–62. https://doi.org/10.1037/0033-2909.89.1.47 p
g
5. Johnson D.W., Johnson R.T. Cooperation and the use of technology // Handbook of research for
educational communications and technology / D.H. Johassen (Ed.). New York: Simon and Schuster p
g
5. Johnson D.W., Johnson R.T. Cooperation and the use of technology // Handbook of research for
educational communications and technology / D.H. Johassen (Ed.). New York: Simon and Schuster http://naukavestnik.ru/ Гуманитарный научный вестник. 2021. №10 85 Macmillan,
1996. P. 785-812. https://www.researchgate.net/publication/243671476
_Cooperation_and_the_use_of_technology Macmillan,
1996. P. 785-812. https://www.researchgate.net/publication/243671476
_Cooperation_and_the_use_of_technology 6. A meta-analysis of the effects of face-to-face cooperative learning. Do recent studies falsify or verify
earlier findings? / Eva Kyndt et al. // Educational Research Review. 2013. V. 10. P. 133–149. https://doi.org/10.1016/j.edurev.2013.02.002 7. Major C. Collaborative Learning: A Tried and True Active Learning Method for the College
Classroom // New Directions for Teaching and Learning. 2020. V. 2020. Для цитирования:
Рафикова А.С. Виды дискурса, повышающие эффективность кооперативного обучения //
Гуманитарный научный вестник. 2021. №10. С. 81-85. URL: http://naukavestnik.ru
/doc/2021/10/Rafikova.pdf REFERENCES (TRANSLITERATED) N. 164. P. 19-28. https://doi.org/10.1002/tl.20420 8. Mercer N. The Guided Construction of Knowledge: Talk amongst Teachers and Lea
Multilingual matters, 1994. 135 p. 9. Mercer N., Dawes L. The value of exploratory talk // Exploring talk in school: Inspired by the work of
Douglas Barnes / N. Mercer, S. Hodgkinson (Eds.). SAGE Publications Ltd, 2008. P. 55-72. https://www.doi.org/10.4135/9781446279526.n4 p
g
10. Roseth C. J., Johnson D. W., Johnson R. T. Promoting early adolescents' achievement and peer
relationships: The effects of cooperative, competitive, and individualistic goal structures //
Psychological Bulletin. 2008. V. 134. N. 2. P. 223–246. https://doi.org/10.1037/0033-2909.134.2.223
11. Slavin R. E. Research on cooperative learning and achievement: What we know, what we need to 10. Roseth C. J., Johnson D. W., Johnson R. T. Promoting early adolescents' achievement and peer
relationships: The effects of cooperative, competitive, and individualistic goal structures //
Psychological Bulletin. 2008. V. 134. N. 2. P. 223–246. https://doi.org/10.1037/0033-2909.134.2.223 11. Slavin R. E. Research on cooperative learning and achievement: What we know, what we need to
know
//
Contemporary
Educational
Psychology. 1996. V. 21. N. 1. P. 43–69. https://doi.org/10.1006/ceps.1996.0004 12. Vass E., Littleton K. Peer collaboration and learning in the classroom // International Handbook of
Psychology in Education / K. Littleton, C. Wood, J. Kleine Staarman (Eds.). Leeds: Emerald, 2010. pp. 105–136. https://www.researchgate.net/publication/45714612_Peer_collaboration_and_learning
_in_the_classroom 12. Vass E., Littleton K. Peer collaboration and learning in the classroom // International Handbook of
Psychology in Education / K. Littleton, C. Wood, J. Kleine Staarman (Eds.). Leeds: Emerald, 2010. pp. 105–136. https://www.researchgate.net/publication/45714612_Peer_collaboration_and_learning
_in_the_classroom Поступила в редакцию 18.10.2021. Принята к публикации 21.10.2021. ISSN 2541-7509
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Rastreabilidade de pêssegos produzidos no sistema de produção integrada no Sul do Brasil
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10 10 10 Casiane Salete Tibola1*; José Carlos Fachinello2; Cesar Valmor Rombaldi2; Leonardo Nora2;
Andrea de Rossi Rufato2; Leo Rufato3 1Embrapa Trigo, Rod. BR 285 km 294, C.P. 451 - 99001-970 - Passo Fundo, RS - Brasil. 2UFPEL/FAEM - C.P. 354 - 96010-900 - Pelotas, RS - Brasil. 3UDESC/CAV - 88.520-000 - Lages, SC - Brasil. *Corresponding author <casiane@cnpt.embrapa.br > ABSTRACT: Traceability is becoming the most effective method to provide a safer food chain and
connection producers to consumers. This paper report the application and validation of a traceability
system on the production chain of peaches, according the rules for Integrated Production of Peach (IP)
and a Hazard Analysis and Critical Control Point (HACCP) systems. The harvesting plots were
discriminated using a Global Positioning System (GPS) device. The horticultural practices were registered
in a field book according to the Brazilian IP rules. Boxes to transport the fruit, from the orchard on, were
barcode labelled to identify the fruits in terms of origin (orchard and harvesting plot), cultivar, quality,
picking date and time. Arriving in the factory, by an optical barcode reading device, the fruits in the boxes
were assigned to homogeneous batches. Peach cans were labelled according to their corresponding
batch number and monitored based on physical and chemical analysis as preconized by the IP rules and
HACCP system. An electronic data base was set up and placed over the Internet. Using the batch
number, the history of each peaches can could be traceable back to their harvesting plot. Therefore,
manufacturers can monitor the product at any time and take any necessary action, such as product recall
and/or product reprocessing. Key words: traceability, quality, food safety RASTREABILIDADE DE PÊSSEGOS PRODUZIDOS NO SISTEMA DE
PRODUÇÃO INTEGRADA NO SUL DO BRASIL RESUMO: A rastreabilidade está se tornando o método mais efetivo para assegurar uma cadeia alimentar
mais segura e conectar produtores e consumidores. Nesse trabalho relatamos a aplicação e validação de
um sistema de rastreabilidade na cadeia produtiva do pêssego, de acordo com as Normas Brasileiras para
Produção Integrada de Pêssego (PIP) e Análises de Perigos e Pontos Críticos de Controle (APPCC). As
parcelas de colheita foram discriminadas utilizando-se um aparelho de GPS (Global Positioning System). As práticas horticulturais foram registradas no caderno de campo, de acordo com as normas da PIP. As
caixas para transportar as frutas, desde a lavoura, foram etiquetadas com código de barra para identificar
as frutas em termos de origem (pomar e parcela de colheita), cultivar, qualidade, data e hora de colheita. Na indústria, utilizando-se um leitor ótico de código de barras, as frutas de cada caixa foram alocadas
para um determinado lote homogêneo para fins de processamento. As latas de pêssego foram etiquetadas
com o número do lote homogêneo correspondente, foi monitorado com base em análise físicas e químicas,
de acordo com as regras da PIP e da APPCC. Uma base d e dados eletrônica foi construída e disponibilizada
através da Internet. Utilizando-se o número do lote, foi possível obter todas as informações desde a(s)
parcela(s) de colheita correspondente(s) a uma determinada lata de pêssego. Palavras-chave: rastreabilidade, qualidade, alimento seguro INTRODUCTION cepts of Integrated Production (IP), traceability sys-
tems and Hazard Analysis and Critical Control Point
(HACCP), competitiveness is decisive to the
primary sector (Fachinello et al., 2004; Regattieri et
al., 2007). Recent food safety records show that about
seven million people per year are affected
by foodborne illnesses. As a result, consumers re-
quire suitable information about food origin, compo-
sition and safeness, which can only be guaranteed In the food business assurance of quality is
becoming a central part of all activities focusing on
safety. The consumer’s interest in a better life qual-
ity is inducing the development of technologies that
protect the environment and their health. This
behaviour contributes for the production of safer
foods of recognized quality. According to the con- Sci. Agric. (Piracicaba, Braz.), v.65, n.1, p.10-15, January/February 2008 Traceability of peaches from integrated production 11 by an efficient tracking-tracing system (Sarig et al.,
2006; Regattieri et al., 2007). visits to the factory while the peach industrial process-
ing period. The consumers concern about food safety and
quality has led to the use of the new buzzword: ‘trace-
ability’, triggered primarily by consumers concerns
about Bovine Spongiform Encephalopathy (BSE), di-
oxin in chicken feed, Salmonella and Listeria in fresh
produce food and bioengineered food products, among
other problems (Kuiper et al., 2004; Nilsson et al.,
2004). Since then, traceability has become more than
a food production buzzword, and is now a necessary
component of the food production process (Sarig et
al., 2006). Orchard traceability Each farm was individually identified and the
orchards were divided and characterized in plots ac-
cording to their cultivar and implantation year. A GPS
(Global Positioning System) was used to locate the
plots. Plots were described on the field book by trained
fruit growers and/or agronomy technicians. The de-
scription of each plot comprised: (i) plot identification;
(ii) weekly monitoring of oriental fruit moth
(Grapholita molesta Busk), (iii) fruit fly (Anastrepha
fraterculus Wied) and other pests; (iv) agrochemical
application of legally authorized agrochemicals for pest
and diseases control; (v) fertilizers application; (vi)
pruning and thinning; (vii) soil management and mow-
ing or weeding of herbaceous plants under the tree
canopy; (viii) agrochemicals storing and (ix) disposal
of empty agrochemical containers. Risk assessment in
the peach production (Table 2) was performed and pre-
venting and monitoring procedures were established
according to IP rules for peaches (Fachinello et al.,
2003). During the harvest, fruit boxes were barcode
labelled to identify the corresponding fruit producer,
plot, cultivar, date and the time of picking. The total Brazilian peach production is around
150 thousand tons per year. In 1999 a project was ini-
tiated in order to establish the Integrated Peach Pro-
duction (IP), which was adopted by 105 peach pro-
ducers, corresponding to a production of 6.240 tons
from 520 ha in 2005/2006 (Andrigueto & Kososki,
2004). The aim of this study was to establish, evalu-
ate and validate a traceability system in a canned peach
production chain, supported by the IP rules for
peaches and by the HACCP rules, therefore in a pro-
cess where good manufacture practices are expected
to be in place. Factory traceability The project was started in 2002 with five
peach growers and one canning factory. Nowadays,
two canning industries and 25 peach producers from
a fruit growers association participate on the project. The project was started in 2002 with five
peach growers and one canning factory. Nowadays,
two canning industries and 25 peach producers from
a fruit growers association participate on the project. The peaches for the present study were produced and
processed according to the IP rules. The main char-
acteristics of the orchards comprised in the present
study are in Table 1. The assistance to establish the
Integrated Peach Production and the Traceability Sys-
tem comprised monthly visits to the orchards and daily In the factory batches including at least 100
fruit boxes were prepared. The size of the batches was
adequate according to the production line. The batches
preferably included similar quality peaches from the
same cultivar and orchard. Only eventually it was nec-
essary to include fruit from different quality and/or
from more than one orchard. The labels were read us-
ing a barcode optical reading device in order to assign
the fruits to a specific processing batch. The infor-
mation stored in the barcode reading device was trans- Table 1 - Main aspects of the peach production in the farms that supplied the fruit, according to the integrated production
rules, for a study aiming the implementation of a traceability system in the production of canned peaches in
Pelotas, RS, Brazil, in 2002 to 2005 years. Table 1 - Main aspects of the peach production in the farms that supplied the fruit, according to the integrated production
rules, for a study aiming the implementation of a traceability system in the production of canned peaches in
Pelotas, RS, Brazil, in 2002 to 2005 years. Sci. Agric. Factory traceability (Piracicaba, Braz.), v.65, n.1, p.10-15, January/February 2008
n
oitc
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e
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h
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r
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6
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e
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c
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o
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e
r
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5
7
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p
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s
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tn
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p
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1
9 Sci. Agric. (Piracicaba, Braz.), v.65, n.1, p.10-15, January/February 2008 Tibola et al. 12 ferred to an electronic database for storage and analysis
by means of a specialized computer program (Fruit
tracing
®)
developed for the present study. Each pro-
cessed batch fruit passed the processing line with a
five min interval between them. Electronic data handling p
m
u
d
la
ci
m
e
h
c
a
s
a
a
e
r
a
e
h
t
f
o
e
s
u
s
u
oiv
e
r
p
s
n
oita
cil
p
p
a
la
ci
m
e
h
c
o
r
g
a
la
ci
m
e
h
c
o
n
r
o
/
d
n
a
s
e
s
o
d
tc
e
rr
o
c
ni
r
o
/n
a
tn
ei
d
e
r
g
ni
e
vitc
a
d
e
d
n
e
m
m
o
c
e
r
t
o
n
a
f
o
e
s
u
.s
s
e
n
e
vitc
eff
e
la
u
dis
e
r
e
h
t
f
o
e
c
n
a
v
r
e
s
b
o
s
n
oita
cil
p
p
a
la
ci
m
e
h
c
o
r
g
a
la
cig
ol
oi
b
.)s
air
etc
a
b
d
n
a
s
etis
a
r
a
p
s
cin
e
g
o
h
ta
p
o
r
etn
e
(
r
eta
w
d
eta
ni
m
atn
o
c
f
o
e
s
u
s
e
r
u
n
a
m
la
ci
m
e
h
c
la
ci
m
e
h
c
.n
oita
zilitr
ef
n
e
g
o
rtin
f
o
s
s
e
c
x
e
s
e
r
u
n
a
m
cin
a
g
r
o
la
ci
m
e
h
c
.slate
m
y
v
a
e
h
r
o
/
d
n
a
s
eta
rtin
f
o
s
s
e
c
x
e
tn
e
m
piu
q
e
p
o
r
c
la
cig
ol
oi
b
.s
air
etc
a
b
r
o
/
d
n
a
s
etis
a
r
a
p
s
cin
e
g
o
h
ta
p
o
r
etn
e
y
b
n
oita
ni
m
atn
o
c
Table 2 - Steps and risks monitored and registered in the field book of Integrated Peach Production. Pelotas, 2005. Sci. Agric. Electronic data handling (Piracicaba, Braz.), v.65, n.1, p.10-15, January/February 2008
sr
otc
af
k
si
R
k
sir
f
o
e
p
y
T
e
s
u
a
C
n
oita
cifis
s
alc
d
n
a
n
oit
p
e
c
e
r
la
cig
ol
oi
b
.stiu
rf
n
ett
o
r
g
nitti
p
la
cis
y
h
p
. plu
p
e
ht
ni
g
ninia
m
e
r
s
e
c
ei
p
e
n
ots
g
nile
e
p
la
ci
m
e
h
c
.sla
u
dis
e
r
e
dix
o
r
d
y
h
m
ui
d
o
s
g
nillif
stn
ei
pic
e
r
la
cis
y
h
p
lair
eta
m
n
gie
r
of
r
o/
d
n
a
ytir
u
p
m
i
f
o
e
c
n
e
s
e
r
p
)s
dil
r
o
s
n
a
c
(
stn
ei
pic
e
r
la
ci
m
e
h
c
. Electronic data handling d
ets
u
r
r
o
d
n
a
slate
m
y
v
a
e
h
)s
dil
r
o
s
n
a
c
(
stn
ei
pic
e
r
la
cis
y
h
p
lair
eta
m
n
gie
r
of
f
o
e
c
n
e
s
e
r
p
r
a
g
u
s
la
cis
y
h
p
lair
eta
m
n
gie
r
of
r
o/
d
n
a
ytir
u
p
m
i
s
n
oiti
d
n
o
c
n
oita
zilir
ets
la
cig
ol
oi
b
.)la
viv
r
u
s
's
m
sin
a
g
r
o
o
r
ci
m
(
e
r
uliaf
n
oita
zilir
ets
r
eta
w
la
ci
m
e
h
c
.n
oita
ni
m
atn
o
c
s
e
c
n
ats
b
u
s
la
ci
m
e
h
c
r
eta
w
la
cig
ol
oi
b
.s
m
sin
a
g
r
o
o
r
ci
m
r
o/
d
n
a
s
etis
a
r
a
p
cin
e
g
o
hta
p
o
r
etn
e
f
o
n
oita
ni
m
atn
o
c
hta
b
r
eta
w
g
nil
o
o
c
la
cig
ol
oi
b
.n
oita
ni
m
atn
o
c
e
r
te
k
r
a
m
liate
r
d
n
a
tr
o
p
s
n
a
rt
,e
g
a
r
ots
la
ci
m
e
h
c
.g
nits
u
r
n
a
c
te
k
r
a
m
liate
r
d
n
a
tr
o
p
s
n
a
rt
,e
g
a
r
ots
la
cis
y
h
p
n
oita
m
r
of
e
d
n
a
c
Table 3 - Risk assessment in the production of canned peaches according to the Integrated Peach Production rules (Fachinello
et al., 2003). Electronic data handling la
c
ol/
p
et
S
k
si
R
s
e
s
u
a
C
a
e
r
a
d
r
a
h
c
r
o
la
cig
ol
oi
b
,stn
e
m
ta
e
rt
,
d
e
s
u
sr
e
zilitr
ef
,a
e
r
a
e
h
t
f
o
la
cir
o
tsih
,n
oita
zila
c
ol
d
r
a
h
c
r
o
s
m
sin
a
g
r
o
s
cin
e
g
o
h
ta
p
o
r
etn
e
f
o
e
c
n
e
rr
u
c
c
o
a
e
r
a
d
r
a
h
c
r
o
la
ci
m
e
h
c
. Electronic data handling p
m
u
d
la
ci
m
e
h
c
a
s
a
a
e
r
a
e
h
t
f
o
e
s
u
s
u
oiv
e
r
p
s
n
oita
cil
p
p
a
la
ci
m
e
h
c
o
r
g
a
la
ci
m
e
h
c
o
n
r
o
/
d
n
a
s
e
s
o
d
tc
e
rr
o
c
ni
r
o
/n
a
tn
ei
d
e
r
g
ni
e
vitc
a
d
e
d
n
e
m
m
o
c
e
r
t
o
n
a
f
o
e
s
u
.s
s
e
n
e
vitc
eff
e
la
u
dis
e
r
e
h
t
f
o
e
c
n
a
v
r
e
s
b
o
s
n
oita
cil
p
p
a
la
ci
m
e
h
c
o
r
g
a
la
cig
ol
oi
b
.)s
air
etc
a
b
d
n
a
s
etis
a
r
a
p
s
cin
e
g
o
h
ta
p
o
r
etn
e
(
r
eta
w
d
eta
ni
m
atn
o
c
f
o
e
s
u
s
e
r
u
n
a
m
la
ci
m
e
h
c
la
ci
m
e
h
c
.n
oita
zilitr
ef
n
e
g
o
rtin
f
o
s
s
e
c
x
e
s
e
r
u
n
a
m
cin
a
g
r
o
la
ci
m
e
h
c
.slate
m
y
v
a
e
h
r
o
/
d
n
a
s
eta
rtin
f
o
s
s
e
c
x
e
tn
e
m
piu
q
e
p
o
r
c
la
cig
ol
oi
b
.s
air
etc
a
b
r
o
/
d
n
a
s
etis
a
r
a
p
s
cin
e
g
o
h
ta
p
o
r
etn
e
y
b
n
oita
ni
m
atn
o
c
Table 2 - Steps and risks monitored and registered in the field book of Integrated Peach Production. Pelotas, 2005. ored and registered in the field book of Integrated Peach Production. Pelotas, 2005. Sci. Agric. Electronic data handling (Piracicaba, Braz.), v.65, n.1, p.10-15, January/February 2008
sr
otc
af
k
si
R
k
sir
f
o
e
p
y
T
e
s
u
a
C
n
oita
cifis
s
alc
d
n
a
n
oit
p
e
c
e
r
la
cig
ol
oi
b
.stiu
rf
n
ett
o
r
g
nitti
p
la
cis
y
h
p
. plu
p
e
ht
ni
g
ninia
m
e
r
s
e
c
ei
p
e
n
ots
g
nile
e
p
la
ci
m
e
h
c
.sla
u
dis
e
r
e
dix
o
r
d
y
h
m
ui
d
o
s
g
nillif
stn
ei
pic
e
r
la
cis
y
h
p
lair
eta
m
n
gie
r
of
r
o/
d
n
a
ytir
u
p
m
i
f
o
e
c
n
e
s
e
r
p
)s
dil
r
o
s
n
a
c
(
stn
ei
pic
e
r
la
ci
m
e
h
c
. Factory traceability The quality control at
the factory was performed according to the IP rules
for peaches including several measurements: firmness
and total soluble sugar content in the fruit (°Brix), so-
dium hydroxide concentration and temperature of the
peeling solution, sugar content in the syrup, steriliza-
tion temperature and time, can lid riveting quality, gross
weight, net weight; drained net weight, can internal
vacuum and other information to access the quality and
safety of the canned peaches. The risk assessment per-
formed in the postharvest operations is described in
Table 3. program was designed to allow access to the stored
information over the Internet by whoever in it is in-
terested. The database generated by the Fruit tracing
®
allows the identification of products labelled with a per-
tinent barcode type EAN/UCC-128 (GS1-Belgium &
Luxembourg, 2003). This barcode has prefixes called
Application Identifiers (AI’s) that determine the signifi-
cance and the data format and/or type and facilitates
the buyers to get information about the correspond-
ing batch for a specific food product. The barcode
contains the following information: product, company,
category (special or extra), drained net weight, date
and time of processing, expiring date, and batch num-
ber. Each company has a database including quality
control, production reports and documents about the
origin of raw matter. This makes it possible to obtain
detailed information about processed peaches accord-
ing to each cultivar, plot and/or farmer. In electronic
tracking and tracing systems, the EAN.UCC System
(GS1-Belgium&Luxembourg, 2003) is universally ac-
cepted as an identification and communication system
that facilitates efficient global commerce and improves Electronic data handling A computer program denominated Fruit trac-
ing
® was developed for electronic data handling ac-
cording to the main fruit classification systems and
fruit canning processes used in Brazil. This computer Sci. Agric. (Piracicaba, Braz.), v.65, n.1, p.10-15, January/February 2008
sr
otc
af
k
si
R
k
sir
f
o
e
p
y
T
e
s
u
a
C
n
oita
cifis
s
alc
d
n
a
n
oit
p
e
c
e
r
la
cig
ol
oi
b
.stiu
rf
n
ett
o
r
g
nitti
p
la
cis
y
h
p
. plu
p
e
ht
ni
g
ninia
m
e
r
s
e
c
ei
p
e
n
ots
g
nile
e
p
la
ci
m
e
h
c
.sla
u
dis
e
r
e
dix
o
r
d
y
h
m
ui
d
o
s
g
nillif
stn
ei
pic
e
r
la
cis
y
h
p
lair
eta
m
n
gie
r
of
r
o/
d
n
a
ytir
u
p
m
i
f
o
e
c
n
e
s
e
r
p
)s
dil
r
o
s
n
a
c
(
stn
ei
pic
e
r
la
ci
m
e
h
c
. Electronic data handling d
ets
u
r
r
o
d
n
a
slate
m
y
v
a
e
h
)s
dil
r
o
s
n
a
c
(
stn
ei
pic
e
r
la
cis
y
h
p
lair
eta
m
n
gie
r
of
f
o
e
c
n
e
s
e
r
p
r
a
g
u
s
la
cis
y
h
p
lair
eta
m
n
gie
r
of
r
o/
d
n
a
ytir
u
p
m
i
s
n
oiti
d
n
o
c
n
oita
zilir
ets
la
cig
ol
oi
b
.)la
viv
r
u
s
's
m
sin
a
g
r
o
o
r
ci
m
(
e
r
uliaf
n
oita
zilir
ets
r
eta
w
la
ci
m
e
h
c
.n
oita
ni
m
atn
o
c
s
e
c
n
ats
b
u
s
la
ci
m
e
h
c
r
eta
w
la
cig
ol
oi
b
.s
m
sin
a
g
r
o
o
r
ci
m
r
o/
d
n
a
s
etis
a
r
a
p
cin
e
g
o
hta
p
o
r
etn
e
f
o
n
oita
ni
m
atn
o
c
hta
b
r
eta
w
g
nil
o
o
c
la
cig
ol
oi
b
.n
oita
ni
m
atn
o
c
e
r
te
k
r
a
m
liate
r
d
n
a
tr
o
p
s
n
a
rt
,e
g
a
r
ots
la
ci
m
e
h
c
.g
nits
u
r
n
a
c
te
k
r
a
m
liate
r
d
n
a
tr
o
p
s
n
a
rt
,e
g
a
r
ots
la
cis
y
h
p
n
oita
m
r
of
e
d
n
a
c
Table 3 - Risk assessment in the production of canned peaches according to the Integrated Peach Production rules (Fachinello
et al., 2003). Electronic data handling la
c
ol/
p
et
S
k
si
R
s
e
s
u
a
C
a
e
r
a
d
r
a
h
c
r
o
la
cig
ol
oi
b
,stn
e
m
ta
e
rt
,
d
e
s
u
sr
e
zilitr
ef
,a
e
r
a
e
h
t
f
o
la
cir
o
tsih
,n
oita
zila
c
ol
d
r
a
h
c
r
o
s
m
sin
a
g
r
o
s
cin
e
g
o
h
ta
p
o
r
etn
e
f
o
e
c
n
e
rr
u
c
c
o
a
e
r
a
d
r
a
h
c
r
o
la
ci
m
e
h
c
. Peach production - agreement with the IP rules
for peaches The IP directives on soil management were
well accepted by the fruit growers that participated
in the present study. Soil conditioning and fertiliza-
tion were based on soil analysis and eventually also
on leaf analysis. The southern region of Rio Grande
do Sul state (Brazil) is affected by strong winds. Therefore, according to the IP rules, wind barriers
are recommended to decrease mechanical damage. This practice is particularly important to reduce in-
fection by pathogens through wounded tissues and
has been successfully employed. Summer pruning
was not widely used by the fruit growers because it
demands extra hand-work. Field days on summer
pruning in demonstrative orchards was organized to
encourage the fruit growers to adopt the summer
pruning. Growers were oriented about the time and
intensity of fruit thinning. Such practice was dis-
cussed with the fruit growers and differently used
due to the absence of a pre-definition by the market
of size and quality of the fruits. In terms of pests monitoring, the obstacles are
pest identification and an increase in labour costs, since
monitoring must be done weekly. Although monitor-
ing allows a rational use of agrochemicals it is neces-
sary to increase the confidence of the growers in the
pest monitoring system associated with pest control
system. Agrochemical molecules that can be used le-
gally are very limited, especially for pest control. Al-
ternatives are not available to control neither maize
weevil (Sitophilus zeamais Mots.), a common pest of
stored grains that attaches peaches; nor bacterial spot,
that in the high humidity and high incidence of winds
years causes up to 50% of production losses. For the new orchards it has been used a high
density planting and the trees are trimmed as a “Y” or
“V” systems, which allows a better light distribution
inside the canopy, earlier production and facilitate crop
treatments. However, in some orchards, due to the lack
of qualified labour on summer pruning, the plants were
trimmed as a “vase” system, which promotes exces-
sive shading affecting the productivity and fruit qual-
ity. There are many claims aiming to increase the
number of allowed agrochemicals and registered ac-
tive ingredients in the IP for a more efficient control
of pests and diseases. Electronic data handling d
ets
u
r
r
o
d
n
a
slate
m
y
v
a
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h
)s
dil
r
o
s
n
a
c
(
stn
ei
pic
e
r
la
cis
y
h
p
lair
eta
m
n
gie
r
of
f
o
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c
n
e
s
e
r
p
r
a
g
u
s
la
cis
y
h
p
lair
eta
m
n
gie
r
of
r
o/
d
n
a
ytir
u
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m
i
s
n
oiti
d
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n
oita
zilir
ets
la
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ol
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's
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uliaf
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ets
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.n
oita
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atn
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o/
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etn
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te
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Table 3 - Risk assessment in the production of canned peaches according to the Integrated Peach Production rules (Fachinello
et al., 2003). Table 3 - Risk assessment in the production of canned peaches according to the Integrated Peach Pro
et al., 2003). nt in the production of canned peaches according to the Integrated Peach Production rules (Fachinello isk assessment in the production of canned peaches according to the Integrated Peach Production rules (
al., 2003). Sci. Agric. Electronic data handling (Piracicaba, Braz.), v.65, n.1, p.10-15, January/February 2008 Traceability of peaches from integrated production 13 After triple washing, the agrochemical contain-
ers were delivered to an officially designated collect-
ing centre. Agrochemicals were used properly in most
orchards. However, the use of agrochemicals not li-
censed for peach orchards, which were highly toxic
or of low selectivity, was eventually observed. Ex-
amples of agrochemicals misuse on the peach orchards
were deltametrin and fenthion (not a selective pesti-
cide), dimethoate (pesticide), oxitetraclin (bactericide)
and benomyl (ineffective fungicide due to resistance
problems). the effectiveness of recording and exchanging of in-
formation between supply chain participants. This sys-
tem uniquely identifies products, locations, services and
assets and also includes a series of standard data struc-
tures known as Application Identifiers (AI’s), which
allow secondary information about a product such as
batch, expiring date and batch number to be encoded
(Schwägele, 2005). Besides, the information encoded
in barcode label, was printed on each can including
fabrication and expire dates, batch number and the
word traceability. Main limitations for the production of peaches ac-
cording to the IP rules In the peach production, the guidelines to con-
trol pests and diseases are not sufficient to support a
more rational use of agrochemicals. Spilling of agro-
chemicals in water sources is another important limi-
tation in the peach production in order to comply with
the IP rules. About 80% of the fruit growers do not
have a safe system to refill their sprayer tanks that can
prevent agrochemicals spillage in the water sources
such as rivers, streams and dams. Lack of proper cali-
bration of equipments to apply agrochemical is another
common problem. As a consequence of this reduc-
tions in agrochemicals effectiveness, production losses,
and an increased environmental pollution and higher
costs may be observed when reapplications are per-
formed. Peach production - agreement with the IP rules
for peaches Peach production - agreement with the IP rules
for peaches Brazilian fruit growers have
found difficulties to get in the foreign market because
there is not a common rule between countries regard-
ing to prohibition of certain of their active ingredients
(Iba, 2003). The fruit growers were advised to apply only
the legally and agronomically recommended agrochemi-
cals, at the correct dosage, using clean water (with
appropriate pH), the correct and properly adjusted
equipment at the right period of the day under adequate
weather conditions and wearing the individual protec-
tive equipment. The agrochemical containers were
properly disposed according to a Brazilian law
(n°.7.802/89). Traceability information management An efficient and effective traceability system
should be capable to trace the complete history of the Sci. Agric. (Piracicaba, Braz.), v.65, n.1, p.10-15, January/February 2008 Tibola et al. 14 product, including production conditions an added in-
gredients (Bertolini et al., 2006; Sarig et al., 2006;
Regattieri et al., 2007). In the present study, we have
noticed that the fruit grower attitude on recording in-
formation in the field book was a barrier to implement
the IP rules, the HACCP and the traceability system. Frequently, important information, such as the ones
related to agrochemical applications, was not recorded
properly, mainly during the busiest fruit production
periods. Fortunately, this behaviour is changing since
in the 2005 year, the majority of fruit growers recorded
the information correctly. any specific processing batch, the information recorded
in the traceability system database during the fruit pro-
duction and fruit processing was made accessible from
a public Internet server. A product traceability system requires the iden-
tification of all the locations from which the product
originates, that is to say, where it is processed, pack-
aged, and stocked, and so this includes every agent
in the supply chain (Regattieri et al., 2007). In the
factory, accessing the traceability system from a lo-
cal database was possible to retrieve detailed infor-
mation for each cultivar, plot and/or fruit grower, ac-
cording to the processing batch identification. The
availability and easy access to all these information
improved the factory capability to monitor the pro-
duction line and to deliver canned peaches of higher
quality. Peaches that arrived in the factory usually
were diverse in terms of producer, production sys-
tem, plot, cultivar, quality and quantity. As a basic
requirement in the processing line to produce canned
peaches, homogeneous batches were prepared. This
was necessary because the unitary operations and the
corresponding equipments need to be set according
to the fruit cultivar, size and maturation stage. In ad-
dition, in order to implement the traceability system,
the batches were prepared taking in account the
peaches origin. After transporting the barcoded la-
belled boxes from one control point to another, they
had to be set in a position were the labels could be
accessible for reading. In addition, every time the
peaches were transferred from one box to another,
the barcode label had to be removed and replaced. Traceability information management Because this operation requires human intervention,
there is room for error and inefficiency (Regattieri,
2007). Apart from that, the barcode system was con-
sidered attractive to the sector, mainly because the
efficiency in collecting and transferring information
to the system. CONCLUSIONS The implementation of a traceability system
supported by the IP and HACCP was successfully used
in the production of canned peaches. The increasing
demands from the international and internal market for
high quality and safe foods, produced under environ-
mental friendly conditions, respecting local and inter-
national social rules and able to be traced throughout
the production chain, gives no other alternative to the
peach production sector in Brazil but comply with these
tendencies in order to maintain and increase the com-
petitiveness. In order to comply with these tendencies
a permanent effort from both the private and govern-
mental sectors is necessary. In the present study, the
most significant results from implementing the trace-
ability system in the production of canned peaches
supported by the IP and HACCP are: (i) IP and HACCP
rules were more consistently applied; (ii) the amount
of useful and accessible information for the decision
making process significantly increased; (iii) the qual-
ity control system was improved; (iv) attainment of
competitive advantages in the market due to the in-
creases in the product quality and credibility; (v) in-
formation was made available in silico for manufac-
turers to check the progress in the production and to
perform recall and/or reprocessing of products if nec-
essary; (vi) information was made available in silico
for consumers, guaranteeing food quality, safety and
transparency. In the preparation of homogeneous batches of
peaches, suitable for both the processing line and the
traceability system implementation, two procedures
had to be used: (i) For fruit loads of the same culti-
var, class and plot, it was possible to form process-
ing batches where the fruit could be traced back to
the plot. (ii) For fruit loads from diverse growers, plots
and classes, it was more difficult to get a homoge-
neous processing batch. In this case fruit from the
same cultivar and with equivalent history were pooled
to form the processing batch. It was possible to trace
the fruit back to the processing batch, but not to the
plot or to a specific fruit grower. Physical, chemical, sanitary and sensorial pa-
rameters were determined for all processing batches
of peaches and the results were transferred immedi-
ately to the traceability system database. Detailed in-
formation about the can, lid, sugar and sodium hydrox-
ide used to prepare each batch of canned peaches were
also included in the traceability system database. For Sci. Agric. (Piracicaba, Braz.), v.65, n.1, p.10-15, January/February 2008 ACKNOWLEDGMENTS This work was supported by Brazilian Govern-
mental Agencies: CNPq-MCT, MAPA and CAPES-ME-
Brazil. Sci. Agric. (Piracicaba, Braz.), v.65, n.1, p.10-15, January/February 2008 Traceability of peaches from integrated production 15 Sci. Agric. (Piracicaba, Braz.), v.65, n.1, p.10-15, January/February 2008 Received October 17, 2006
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Poor correlation between large-scale environmental flow violations and freshwater biodiversity: implications for water resource management and the freshwater planetary boundary
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Hydrol. Earth Syst. Sci., 26, 6247–6262, 2022
https://doi.org/10.5194/hess-26-6247-2022
© Author(s) 2022. This work is distributed under
the Creative Commons Attribution 4.0 License. Poor correlation between large-scale environmental flow violations
and freshwater biodiversity: implications for water resource
management and the freshwater planetary boundary Poor correlation between large-scale environmental flow violations
and freshwater biodiversity: implications for water resource
management and the freshwater planetary boundary
Chinchu Mohan1,2,3, Tom Gleeson2,4, James S. Famiglietti1,5, Vili Virkki6, Matti Kummu6, Miina Porkka6,7,
Lan Wang-Erlandsson8,9,10, Xander Huggins1,2, Dieter Gerten10,11, and Sonja C. Jähnig11,12
1Global Institute for Water Security, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
2Department of Civil Engineering, University of Victoria, Victoria, British Columbia, Canada
3Waterplan (YC S21), San Francisco, California, USA
4School of Earth and Ocean Sciences, University of Victoria, Victoria, British Columbia, Canada
5School of Environment and Sustainability, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
6Water and Development Research Group, Aalto University, Espoo, Finland
7Global Economic Dynamics and the Biosphere, Royal Swedish Academy of Sciences, Stockholm, Sweden
8Stockholm Resilience Centre, Stockholm University, Stockholm, Sweden
9Bolin Centre for Climate Research, Stockholm University, Stockholm, Sweden
10Potsdam Institute for Climate Impact Research (PIK), Member of the Leibniz Association, Potsdam, Germany
11Humboldt-Universität zu Berlin, Geography Department and Integrative Research Institute on Transformations
of Human–Environment Systems, Berlin, Germany
12L ib i I
tit t
f F
h
t
E
l
d I l
d Fi h i
Mü
l
d
310 B li
G 10Potsdam Institute for Climate Impact Research (PIK), Member of the Leibniz Association, Potsdam, Germany
11Humboldt-Universität zu Berlin, Geography Department and Integrative Research Institute on Transformations
of Human–Environment Systems, Berlin, Germany 10Potsdam Institute for Climate Impact Research (PIK), Member of the Leibniz Association, Potsdam, Germany
11Humboldt-Universität zu Berlin, Geography Department and Integrative Research Institute on Transformations
of Human–Environment Systems, Berlin, Germany Leibniz Institute of Freshwater Ecology and Inland Fisheries, Müggelseedamm 310, Berlin, Germa bniz Institute of Freshwater Ecology and Inland Fisheries, Müggelseedamm 310, Berlin, Germany Correspondence: Chinchu Mohan (chinchu.mohan@usask.ca) and Tom Gleeson (tgleeson@uvic.ca) Correspondence: Chinchu Mohan (chinchu.mohan@usask.ca) and Tom Gleeson (tgleeson@uvic.ca) Received: 2 March 2022 – Discussion started: 5 April 2022
Revised: 6 September 2022 – Accepted: 7 November 2022 – Published: 13 December 2022 Received: 2 March 2022 – Discussion started: 5 April 2022
Revised: 6 September 2022 – Accepted: 7 November 2022 – Published: 13 December 2022 Received: 2 March 2022 – Discussion started: 5 April 2022
Revised: 6 September 2022 – Accepted: 7 November 2022 – Published: 13 December 2022 been used in assessing a planetary boundary3 for freshwater. Therefore, this study intends to conduct an exploratory evalu-
ation of the relationship between EF violation and freshwater
biodiversity at globally aggregated scales and for freshwater
ecoregions. 2EF violations are deviations in streamflow beyond the upper
and lower boundaries of environmental flow envelopes (EFEs). The
EFEs establish an envelope for acceptable EF deviations based on
pre-industrial (1801–1860) stream discharge (see Sect. 2.2 for more
details) 3Planetary boundary: planetary boundary defines biogeophysi-
cal planetary-scale boundaries for Earth system processes that, if
violated, can irretrievably impair the Holocene-like stability of the
Earth system. Poor correlation between large-scale environmental flow violations
and freshwater biodiversity: implications for water resource
management and the freshwater planetary boundary Four EF violation indices (severity, frequency,
probability of shifting to a violated state, and probability of
staying violated) and seven independent freshwater biodiver-
sity indicators (calculated from observed biota data) were
used for correlation analysis. No statistically significant neg-
ative relationship between EF violation and freshwater bio-
diversity was found at global or ecoregion scales. These find-
ings imply the need for a holistic bio-geo-hydro-physical ap-
proach in determining the environmental flows. While our re-
sults thus suggest that streamflow and EF may not be the only Abstract. The freshwater ecosystems around the world are
degrading, such that maintaining environmental flow1 (EF)
in river networks is critical to their preservation. The rela-
tionship between streamflow alterations (subsequent EF vi-
olations2) and the freshwater biodiversity response is well
established at the scale of stream reaches or small basins
( ∼< 100 km2). However, it is unclear if this relationship is
robust at larger scales, even though there are large-scale ini-
tiatives to legalize the EF requirement. Moreover, EFs have 1Environmental flow (EF): “The quantity, timing, and quality
of water flows required to sustain freshwater and estuarine ecosys-
tems and the human livelihoods and well-being that depend on these
ecosystems.” – Arthington et al. (2018). 1Environmental flow (EF): “The quantity, timing, and quality
of water flows required to sustain freshwater and estuarine ecosys-
tems and the human livelihoods and well-being that depend on these
ecosystems.” – Arthington et al. (2018).
2EF violations are deviations in streamflow beyond the upper
and lower boundaries of environmental flow envelopes (EFEs). The
EFEs establish an envelope for acceptable EF deviations based on
pre-industrial (1801–1860) stream discharge (see Sect. 2.2 for more
details) 1
Introduction Water resources are inarguably one of the most important
natural resources in the Earth system for sustaining life. Nev-
ertheless, these resources and their associated ecosystems are
threatened by human actions (Bélanger and Pilling, 2019;
Clausen and York, 2008; Vörösmarty et al., 2010; Wilting
et al., 2017). Global freshwater covers up to 0.8 % of the to-
tal Earth’s surface (Gleick, 1996) and inhabits 6 % of all the
known species in the world, including 40 % of the total fish
diversity and nearly one-third of all vertebrates (Lundberg
et al., 2000). Since freshwater ecosystems have high species
richness in a relatively small area and are exposed to a high
level of pressure, they are more vulnerable to environmental
change and human actions than any other ecosystems (Dud-
geon et al., 2006). The rapid increase in the demand for natu-
ral resources is the fundamental cause of freshwater ecosys-
tem degradation (Darwall et al., 2018). Anthropogenic cli-
mate change (Allan and Flecker, 1993; Darwall and Freyhof,
2016; Knouft and Ficklin, 2017; Meyer et al., 1999), over-
exploitation (Allan et al., 2005), water pollution (Albert et
al., 2021; Dudgeon et al., 2006; Reid et al., 2019; Smith,
2003), flow alteration (Nilsson et al., 2005; Vörösmarty et
al., 2000), habitat destruction (Dudgeon, 2002), and the in-
troduction of alien species (Gozlan et al., 2010; Vitule et
al., 2009) are some of the manifestations of this increased
demand which directly threatens freshwater ecosystems. In
addition, increased water impoundment in large dams and
reservoirs has also led to an array of adversities for freshwa-
ter ecosystems, ranging from habitat destruction to irregular
flow alterations (Bergkamp et al., 2000). This situation is ag-
gravated by increasing pressure on related Earth system func-
tions, such as climate change and nutrient cycles, which are
articulated by their respective transgressions in the planetary
boundaries framework (Dudgeon, 2010). Freshwater ecosys-
tem processes that were previously governed by natural Earth
system facets such as temperature, rainfall, and relief are now
increasingly driven by demographic, social, and economic
drivers (Clausen and York, 2008; Kabat et al., 2004; Tyson
et al., 2002; Vitousek et al., 1997; Vörösmarty et al., 1997). Freshwater ecosystem health comprises both biotic factors
such as biodiversity and abiotic factors such as habitat in-
tegrity. 6248
C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity There has been an increased recognition in recent decades
of the need to maintain a natural flow regime in streams to
sustain healthy ecosystems (Horne et al., 2017; Poff et al.,
1997, 2017; Tickner et al., 2020; Tonkin et al., 2021). Despite
the indispensable role of aquatic biodiversity in maintaining
the quality of the system (Darwall et al., 2018), the inclu-
sion of such environmental flow (EF) in water management is
often controversial, particularly in regions where freshwater
availability is limited and is already a matter of severe com-
petition. These competitions have led to an increasing trend
for EF violation (insufficient streamflow compared to the rec-
ommended EF requirement; see Sect. 2.1 for more details)
in the past decade in terms of both severity and frequency
(Virkki et al., 2022). This wake-up call has led to several in-
ternational and national efforts to legalize EF requirements
through large-scale EF management schemes (Arthington
and Pusey, 2003; Richter et al., 1997, 2003). The Water and
Nature Initiative (Smith and Cartin, 2011), the Brisbane Dec-
laration (Brisbane Declaration, 2007), and the Global Ac-
tion Agenda (Arthington et al., 2018) are some of these ef-
forts. Nevertheless, there is a large gap in our understanding
of the relationship between EF requirements and biodiver-
sity responses at various spatial and temporal scales. Except
for a few (Domisch et al., 2017; Xenopoulos et al., 2005;
Yoshikawa et al., 2014), the majority of the studies exploring
this relation were conducted at smaller scales (Anderson et
al., 2006; Arthington and Pusey, 2003; Powell et al., 2008). Thus, there is a significant discrepancy in the scale at which
these processes are understood versus the scale at which the
policies are set (Thompson and Lake, 2010). Current knowl-
edge of how the small-scale processes scale up (e.g., valida-
tion of large-scale EF hydrologic methods using local data)
to a regional or global scale is thus limited, potentially under-
mining the scientific integrity of existing large-scale EF man-
agement schemes. determinant of freshwater biodiversity at large scales, they do
not preclude the existence of relationships at smaller scales
or with more holistic EF methods (e.g., including water tem-
perature, water quality, intermittency, connectivity, etc.) or
with other biodiversity data or metrics. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. https://doi.org/10.5194/hess-26-6247-2022 C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity
6 6249 phase 2b outputs of global daily discharge (available at
https://esg.pik-potsdam.de, last access: 27 January 2021)
(Warszawski et al., 2014). Monthly streamflow data (aver-
aged from the daily simulations) for two time periods were
used in this study: (1) data for the pre-industrial era (1800–
1860), which was considered the unaltered reference pe-
riod (Poff et al., 1997), and (2) data for the recent time pe-
riod (1976–2005). These monthly streamflow datasets were
used to calculate EF violations. To calculate the EF viola-
tion indices, the estimated EFEs for each basin were obtained
from Virkki et al. (2022). A total of four global hydrolog-
ical models (GHMs) (H08 – Hanasaki et al., 2018; LPJmL
– Schaphoff et al., 2018; PCR-GLOBWB – Sutanudjaja et
al., 2018; WaterGAP2 – Müller Schmied et al., 2016) were
used to obtain the monthly streamflow data. Each GHM was
forced with the outputs from four different global circula-
tion models (GCMs) (GFDL-ESM2M – Dunne et al., 2012;
HadGEM2-ES – Collins et al., 2011; The HadGEM2 De-
velopment Team, 2011; IPSL-CM5A-LR – Dufresne et al.,
2013; MICROC5 – Watanabe et al., 2010). All the GHM out-
puts used in this study were extensively validated and evalu-
ated in several previous studies (e.g., Zaherpour et al., 2018;
Gädeke et al., 2020). Moreover, as part of the ISIMIP impact
model intercomparison activity, all the GCM climate input
data were bias corrected using compiled reference datasets
covering the entire globe at 0.5◦resolution (Frieler et al.,
2017). Additionally, the GHM outputs were also validated
using historical data to better fit reality (Frieler et al., 2017). Therefore, no additional volition of the data was done in this
study. the EF–biodiversity relationship) in the planetary boundary
framework are also discussed. Introduction to the blue wa-
ter planetary boundary framework. The planetary boundaries
framework proposed by Rockström et al. (2009) and further
developed by Steffen et al. (2015) defines planetary-scale
biogeophysical boundaries for Earth system processes that,
if violated, can irretrievably impair the Holocene-like stabil-
ity of the Earth system. The framework establishes scientif-
ically determined safe operating limits for human perturba-
tions through control and response variable relationships, un-
der which humans and other life forms will coexist in equilib-
rium without jeopardizing the Earth’s resilience. Nine plane-
tary boundaries were defined to cover all independent signif-
icant Earth system processes. 2.1
Data In addition to the streamflow data, data on fish diversity were
also used in this study (Table 1). Freshwater biodiversity was
evaluated using seven indices estimated from the observed
biota data. The biodiversity indicators were obtained from
international agencies and the literature. The biodiversity in- C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity
6 Out of those nine, the freshwa-
ter planetary boundary quantifies the safe limits of the terres-
trial hydrosphere (Gleeson et al., 2020a, b). The freshwater
planetary boundary was originally defined using human wa-
ter consumption as the control variable, set at 4000 km3 yr−1
(with an uncertainty of 4000 to 6000 km3 yr−1) (Rockström
et al., 2009). Gerten et al. (2013) proposed a bottom-up, spa-
tially explicit quantification of EF violations as part of the
water boundary, while Gleeson et al. (2020b) subdivided the
water planetary boundary into six sub-boundaries and pro-
posed possible control and response variables for each, with
aquatic biosphere integrity (i.e., EF) as the potential control
variable for a surface water sub-boundary. Quantitative eval-
uation of the strength and scalability of the identified control
and response variables is still required. 2
Methodology and data The streamflow data were aggregated to the sub-basin
scale according to level 5 HydroBASIN version 1.0 (https://
www.hydrosheds.org/page/hydrobasins, last access: 27 Jan-
uary 2021) (Lehner and Grill, 2013). The data from ISIMIP
2b are representative of historical land use and other hu-
man influences, including dams and reservoirs (Frieler et al.,
2017). The maximum discharge cell value within the bound-
aries of each level 5 HydroBASIN was chosen to represent
the outlet discharge value. Any violations within the outlet
cell were regarded as indicative of the entire basin, even if
conditions could differ in various areas within the level 5 Hy-
droBASIN. As the spatial resolution of the study was level 5
HydroBASIN to allow a global analysis, we accept a cer-
tain homogenization of the local-scale characteristics. See
Sect. S2 of the Supplement for more details on the datasets
used in this study. The study was conducted at two spatially aggregated scales,
(1) global and (2) ecoregion, for a historic time pe-
riod of 30 years (1976–2005). All the underlying calcu-
lations were done at level 5 HydroBASIN (median basin
area = 19 600 km2) (Lehner and Grill, 2013) and were ag-
gregated to the corresponding spatial scale for further anal-
ysis. Level 5 HydroBASIN (also referred to as “basin” in
this paper) was selected as the smallest spatial unit as it is
the highest level of specificity that can be rasterized into a
0.5◦resolution grid without significantly reducing the num-
ber of sub-basins smaller than a grid cell (Virkki et al., 2022). The EF violation indices were calculated using the novel
environmental flow envelope (EFE) framework of Virkki et
al. (2022), and biodiversity was represented by a combina-
tion of relative and absolute value indices. The overall work-
flow for this manuscript is depicted in Fig. 1. 1
Introduction As any disruption in the abiotic factors is most likely
to be reflected in the biotic status of the freshwater ecosys-
tem, the scope of this paper is confined to the biotic dimen-
sion of the freshwater ecosystem (i.e., biodiversity) and not
the health of the entire ecosystem. In order to scientifically underpin large-scale EF policies,
the existing assumption of the inverse relationship between
freshwater biodiversity response and EF violation must be
tested at regional and global scales (see Sect. S1 in the Sup-
plement for more details). Therefore, in this study, we eval-
uate the relationship between EF violation and freshwater
biodiversity at two different spatial scales (freshwater ecore-
gion and global) using four EF violation indices (frequency,
severity, probability of moving to a violated state, and prob-
ability of staying violated) and seven freshwater biodiver-
sity indicators describing taxonomic, functional, and phy-
logenetic dimensions of the biodiversity. The paper is not
intended to be a definitive test of the relationship between
EF violation and aquatic biodiversity. It is rather intended to
be an exploratory analysis of the idea of conducting more
detailed evaluations of the EF–biodiversity relationship be-
fore formulating large-scale EF management policies. The
implications of the findings for large-scale water manage-
ment and the use of the relationship between environmen-
tal flows and freshwater biodiversity (hereafter referred to as https://doi.org/10.5194/hess-26-6247-2022 Hydrol. Earth Syst. Sci., 26, 6247–6262, 2022 C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity 2.1.1
Streamflow data Streamflow data used in the EFE (see Sect. 2.2 for more de-
tails) definition were obtained from the Inter-Sectoral Im-
pact Model Intercomparison Project (ISIMIP) simulation (a) Absolute biodiversity indicator The absolute biodiversity indicator consisted of freshwa-
ter fish richness (FiR). The fish richness data were com-
piled and processed from 1436 published papers, books, gray
literature, and web-based sources published between 1960
and 2014 (Tedesco et al., 2017). They cover 3119 basins all
over the world and account for 14 953 fish species perma-
nently or occasionally inhabiting freshwater systems. In ad-
dition to FiR, we used the RivFishTIME dataset by Comte
et al. (2021) – compiled from long-term riverine fish surveys
from 46 regional and national monitoring programmes and
from individual academic research efforts. Though the Riv-
FishTIME dataset is highly spatially skewed towards the al-
ready data-rich regions of Europe, North America (partic-
ularly the United States of America) and Australia and is
temporally discontinuous, it is the only species-specific fish
abundance time series data available and it is useful to have
an independent verification of the findings using FiR and rel-
ative biodiversity indicators. https://doi.org/10.5194/hess-26-6247-2022 https://doi.org/10.5194/hess-26-6247-2022 Hydrol. Earth Syst. Sci., 26, 6247–6262, 2022 6250
C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity 6250
C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity 6250
C. Mohan et al.: Poor correlation between large-scale environmental flow violation Figure 1. Outline of the methodologies used for (a, b) EF violation indicator calculation and (c) EF–biodiversity relationship evaluation. Figure 1. Outline of the methodologies used for (a, b) EF violation indicator calculation and (c) EF–biodiversity relationship evaluation. thodologies used for (a, b) EF violation indicator calculation and (c) EF–biodiversity relationship evaluation. Figure 1. Outline of the methodologies used for (a, b) EF violation indicator calculation and (c) EF–biodiver dicators consisted of six indices of relative change in biodi-
versity and one index of the absolute value of biodiversity. analysis to construct a holistic picture of the state of aquatic
biodiversity (see Fig. 1 in Su et al., 2021 for more details
on fish facet calculations). Each facet indicates the change
in the corresponding biodiversity component compared to
the 18th century (roughly the pre-industrial era). The taxo-
nomic facets measure the occurrence of fish in a riverine sys-
tem. Functional facets are calculated using the morphological
characteristics of each species that are linked to feeding and
locomotive functions, which in turn relate to larger ecosys-
tem functions such as food web control and nutrition trans-
port. Phylogenetic facets measure the total length of branches
linking all species from the assemblage on the phylogenetic
tree. The richness component of the three categories calcu-
lates the diversity among the assemblage, whereas the dis-
similarity accounts for the difference between each pair of
fish assemblages in one realm. All six fish facets were calcu-
lated at basin scale (2465 river basins), covering 10 682 fish
species all over the world. The scale at which the fish facets
are estimated does not necessarily align with the scale at
which the EF violations are estimated in all cases. The basin-
scale facet estimates were then matched with correspond-
ing EF violation indices using different aggregation/data-
matching methods (see Sect. 2.4 for more details). All six
facets are available as a single delta change in time and do
not cover multiple time steps. C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity
6251 Moreover, all the
basins with mean annual flow (MAF) < 10 m3 s−1 were ex-
cluded due to high uncertainty in EFE and streamflow esti-
mates (Gleeson et al., 2020a; Steffen et al., 2015; Virkki et
al., 2022). After this exclusion, a total of 3906 basins were discharge limits beyond which flow in the streams may not
meet freshwater biodiversity needs (Virkki et al., 2022). EFE
uses the pre-industrial (1801–1860) stream discharge to es-
tablish an upper and a lower boundary for EF deviations at
monthly time steps. This EFE is used to define the EF vio-
lation at the level 5 HydroBASIN scale. The EF violations
were calculated as the median ensemble of four global hy-
drological models (GHMs) (H08, LPJmL, PCR-GLOBWB,
WaterGAP2) and the mean ensemble of four global circula-
tion models (GCM) (GFDL-ESM2M, HadGEM2-ES, IPSL-
CM5A-LR, MICROC5). Moreover, five different EF calcula-
tion methods – the Smakhtin method (Smakhtin et al., 2004),
the Tennant method (Tennant, 1976), Q90–Q50 (Pastor et al.,
2014), the Tessmann method (Tessmann, 1979), and the vari-
able monthly flow method (Pastor et al., 2014)) were also
used in the EFE derivation (see Table S3 for more informa-
tion on EF methods) (Virkki et al., 2022). This approach ad-
dresses the uncertainty related to the outputs of models and
may eliminate the largest model-related extremes that might
cause results to be distorted (Virkki et al., 2022). In spite of
the uncertainty in hydrological estimates generated by using
different models, a simple ensemble matrix often produces
acceptable discharge at larger scales as the individual model
bias is removed (Zaherpour et al., 2018). Moreover, all the
basins with mean annual flow (MAF) < 10 m3 s−1 were ex-
cluded due to high uncertainty in EFE and streamflow esti-
mates (Gleeson et al., 2020a; Steffen et al., 2015; Virkki et
al., 2022). After this exclusion, a total of 3906 basins were Here we evaluate the EF violation by defining four dif-
ferent EF violation indices: violation severity (S), viola-
tion frequency (F), probability of shifting to a violated
state (P.shift), and probability of staying violated (P.stay). Out of the four EF violation indicators, two (S and F) were
modified from Virkki et al. (2022), and the other two (P.shift
and P.stay) were calculated based on the current EFE devia-
tions from Virkki et al. (2022). C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity
6251 6251 C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity
6251 a Results are only shown in the Supplement (see Sect. S8). b Variable for each species and sampling site. Each time series has a
minimum survey length of 2 years (mean = 8 years). a Results are only shown in the Supplement (see Sect. S8). b Variable for each species and sampling site. Each time series has a
minimum survey length of 2 years (mean = 8 years). considered for further analysis. However, many low flows
are seasonally observed, such that the MAF may be quite
large due to elevated wet season flows, with extremely low
flows occurring during a dry season (e.g., Eel River Basin,
California), making it difficult to model. In such cases with
higher intra-annual flow variability, it is appropriate to con-
sider more detailed discharge data (seasonal/sub-annual) to
gain more insight into the flow modeling uncertainties. discharge limits beyond which flow in the streams may not
meet freshwater biodiversity needs (Virkki et al., 2022). EFE
uses the pre-industrial (1801–1860) stream discharge to es-
tablish an upper and a lower boundary for EF deviations at
monthly time steps. This EFE is used to define the EF vio-
lation at the level 5 HydroBASIN scale. The EF violations
were calculated as the median ensemble of four global hy-
drological models (GHMs) (H08, LPJmL, PCR-GLOBWB,
WaterGAP2) and the mean ensemble of four global circula-
tion models (GCM) (GFDL-ESM2M, HadGEM2-ES, IPSL-
CM5A-LR, MICROC5). Moreover, five different EF calcula-
tion methods – the Smakhtin method (Smakhtin et al., 2004),
the Tennant method (Tennant, 1976), Q90–Q50 (Pastor et al.,
2014), the Tessmann method (Tessmann, 1979), and the vari-
able monthly flow method (Pastor et al., 2014)) were also
used in the EFE derivation (see Table S3 for more informa-
tion on EF methods) (Virkki et al., 2022). This approach ad-
dresses the uncertainty related to the outputs of models and
may eliminate the largest model-related extremes that might
cause results to be distorted (Virkki et al., 2022). In spite of
the uncertainty in hydrological estimates generated by using
different models, a simple ensemble matrix often produces
acceptable discharge at larger scales as the individual model
bias is removed (Zaherpour et al., 2018). 1. Violation severity (S): the annual violation severity was
calculated as the absolute mean of the magnitude of the
deviation of EF from the EFE lower or upper bound
in all the violated months. The magnitude of violation
was based on the violation ratio proposed by Virkki et 2.2
Environmental flow violation estimation The relative biodiversity indicators consisted of six freshwa-
ter fish facets. Six key facets of freshwater fish – taxonomic,
functional, and phylogenetic diversity (TR, FR, and PR, re-
spectively) as well as the dissimilarity of each of the three
groups (TD, FD, and PD, respectively) – were used in this The EFE framework proposed by Virkki et al. (2022) was
used to evaluate EF violations in this study. The EFE frame-
work establishes an envelope of variability constrained by https://doi.org/10.5194/hess-26-6247-2022 Hydrol. Earth Syst. Sci., 26, 6247–6262, 2022 C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity
6251
Table 1. Details of the different data used in this study. Data
Spatial resolution
Temporal resolution
Source/reference
(extent)
(extent)
Aquatic fish
30 arcsec (3119
Temporal aggregate
Observed/measured
richness data
drainage basins;
from data compiled
data
∼80 % of Earth’s land)
from reports between
Tedesco et al. (2017)
1960 and 2014
Freshwater fish
Basin scale (2465
Representative of 2015
Derived from observed
facets
drainage basins)
(change compared to
data
preindustrial era)
Su et al. (2021)
RivFishTIME
Stream reach (11 386
1951–2019b
Comte et al. (2021)
dataseta
sampling location)
EFE
Aggregated to level
Monthly (pre-
Model calculated
5 HydroBASIN
industrial: 1801–1860)
Virkki et al. (2022)
(global)
Streamflow
Aggregated to level
Monthly (pre-
Model calculated
5 HydroBASIN
industrial: 1801–1860,
Warszawski et al. (2014)
(global)
current: 1976–2005)
Basin
Level 5 HydroBASIN
Not applicable
Lehner and Grill (2013)
boundaries
(global)
a Results are only shown in the Supplement (see Sect. S8). b Variable for each species and sampling site. Each time series has a
minimum survey length of 2 years (mean = 8 years). 6252
C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity al. (2022) (see Table S4). The normalized value of S
was used in this study. al. (2022) (see Table S4). The normalized value of S
was used in this study. that includes only the biodiversity hotspots. Seven freshwa-
ter ecoregions in ecologically important regions were stud-
ied, and the EF–biodiversity relationship was evaluated sepa-
rately for each ecoregion type. Aggregating into major ecore-
gion types accounts for some of the data’s natural/spatial
variability, in addition to using an analysis of global data. 2. Violation frequency (F): the frequency of violation is
a measure of the proportion of months in which a
basin violated the EFE lower or upper bound in a year. Frequency is calculated as the percentage of violated
months per year. The normalized value of F is used in
this study. y,
g
y
g
One of the major limitations in conducting an aggregated
evaluation was the loss of heterogeneity. Aggregation at any
scale will lead to some level of homogenization of the data. A reach-by-reach evaluation is an ideal solution to capture
all the heterogeneity. However, this is not very practical for
a global study due to data and computational limitations. Therefore, to partially address this challenge, two different
aggregation/data-matching methods were employed: case 1,
where level 5 HydroBASIN data (EF violation indices) were
matched to biodiversity data, and case 2, where biodiver-
sity data were matched to level 5 HydroBASIN data (see
Sect. S5). In the first case, every level 5 HydroBASIN (i.e.,
EF violation indices) was matched with the nearest centroid
of the biodiversity data point, whereas in the second case,
there were three possible scenarios (see Fig. S4): (1) the bio-
diversity basin was smaller than level 5 HydroBASIN, in
which case all the biodiversity basins within one level 5 Hy-
droBASIN were matched with the same EF violation value;
(2) the biodiversity basin was equal in size to a level 5 Hy-
droBASIN, in which case the biodiversity basins and level 5
HydroBASIN had a one-to-one match; and (3) the biodiver-
sity basin was larger than a level 5 HydroBASIN. 6252
C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity 2), whereas basins with lower P.stay values
(closer to 0) tend to remain in the violated state only
for a brief period of time. In other words, the number
of consecutive violated years is much lower for basins
with lower P.stay values. 4. Probability of staying violated (P.stay): once shifted to
a violated state, the tendency of a basin to remain in that
state or switch to a non-violated state is determined by
this indicator. If a basin has a higher P.stay (closer to 1),
then the basin continues to remain in the violated state
for a longer time before switching to a non-violated
state (Eq. 2), whereas basins with lower P.stay values
(closer to 0) tend to remain in the violated state only
for a brief period of time. In other words, the number
of consecutive violated years is much lower for basins
with lower P.stay values. Pstay =
number of violated years with at least one consecutive year violated
total number of violated years
(2 (2) 3
Results and interpretations The relationship between freshwater biodiversity and EF vi-
olation was evaluated using regression analysis. None of the
relationships explored in this study exhibited any nonlinear-
ity, and hence first-order single-variate and multivariate lin-
ear regression analysis was opted for in this study for rea-
sons of parsimony and to achieve reasonable correlation ac-
curacy. Further analysis was carried out by aggregating the
level 5 HydroBASIN scale values to global level, the World
Wide Fund for Nature’s (WWF’s) freshwater ecoregions ma-
jor habitat type scale (see the results given in the Supple-
ment) (Abell et al., 2008), and the G200 freshwater ecore-
gion level (Olson and Dinerstein, 2002). The G200 freshwa-
ter ecoregion is a subset of the WWF’s freshwater ecoregion C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity
6251 P.shift and P.stay measure the
likelihood of shifting to or staying in a violated state during
a given year. The state of a basin (violated or non-violated)
was identified at annual time steps and the mean probability
of shifting or remaining in that state was calculated. The detailed definitions of the EF violation indicators are
as follows: 1. Violation severity (S): the annual violation severity was
calculated as the absolute mean of the magnitude of the
deviation of EF from the EFE lower or upper bound
in all the violated months. The magnitude of violation
was based on the violation ratio proposed by Virkki et https://doi.org/10.5194/hess-26-6247-2022 Hydrol. Earth Syst. Sci., 26, 6247–6262, 2022 6252
C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity In the last
case, two methods were used for data mapping: (1) outlet
matching, where each biodiversity basin was mapped with
the EF violation value from the level 5 HydroBASIN closest
to the outlet, and (2) mean matching, where each biodiversity
basin was mapped with the mean EF violation values of all
level 5 HydroBASINs within it. Data matching methods were
employed to partially understand the uncertainty due to scale
discrepancy between datasets. As the results were insensitive
to the aggregation method, only the results obtained using
case 1 (matching level 5 HydroBASIN data to biodiversity
data) are discussed in this paper. 3. Probability of shifting to a violated state (P.shift): the
P.shift is defined in this paper as the probability of a
basin shifting to a violated state from a non-violated
state (Eq. 1). This indicator, along with P.stay, gives a
measure of the stability of violation in each level 5 Hy-
droBASIN. The violated/non-violated state of a basin is
calculated annually based on the violations in the low-
flow months. If a basin violates the EFE lower or upper
bound for at least 3 consecutive months during the low
flow period (Q < 0.4 MAF) in a year, then the basin is
considered to be in a violated state: (1) 4. Probability of staying violated (P.stay): once shifted to
a violated state, the tendency of a basin to remain in that
state or switch to a non-violated state is determined by
this indicator. If a basin has a higher P.stay (closer to 1),
then the basin continues to remain in the violated state
for a longer time before switching to a non-violated
state (Eq. 2), whereas basins with lower P.stay values
(closer to 0) tend to remain in the violated state only
for a brief period of time. In other words, the number
of consecutive violated years is much lower for basins
with lower P.stay values. 4. Probability of staying violated (P.stay): once shifted to
a violated state, the tendency of a basin to remain in that
state or switch to a non-violated state is determined by
this indicator. If a basin has a higher P.stay (closer to 1),
then the basin continues to remain in the violated state
for a longer time before switching to a non-violated
state (Eq. C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity 6253 an independent dataset, RivFishTIME (see Figs. S8 and S9)
(Comte et al., 2021). The increase in the fish richness facets
primarily stems from the introduction of alien species into
streams for commercial purposes (Su et al., 2021). The in-
vasion of alien species can tamper with the existing natu-
ral ecosystem equilibrium, resulting in further degradation
of the overall ecosystem health. The results obtained using
RivFishTIME datasets were also consistent with the findings
obtained using FiR and six relative biodiversity indicators,
and there was no significant correlation between EF violation
indicators and fish abundance data over time (see the results
for five selected fish species based on data completeness and
geographical distribution shown in Sect. S8; Fig. S8). to a violated state (Fig. 2c and d). EF violations are very
frequent and severe in mostly arid/semi-arid regions such as
the Middle East, Pakistan, India, Australia, the Sahara, Sub-
Saharan Africa, Southern Africa, and the southernmost part
of North America. On the other hand, regions with a higher
probability of shifting to a violated state (P.shift) were not
limited to the low-precipitation and low-streamflow regions. p
p
g
Although the majority of regions with high P.shift values
were arid or semi-arid, some exceptions included southeast-
ern Asia and Central South America. The non-arid regions
with higher P.shift values also have extremely high water
withdrawal in all sectors (agriculture, domestic, and indus-
try). This spatial concurrence suggests that human activities,
as well as hydroclimatic influences, play a significant role
in deciding a region’s P.shift. However, once in the violated
state, the flow variability regimes in the catchment determine
the probability of remaining (P.stay) in the violated state. Catchments with highly variable flow regimes (i.e., that re-
ceive most of the annual flow as floods; see Fig. S2 in the
Supplement for a classification map) have higher probabil-
ities of staying violated once shifted, whereas catchments
with stable flow regimes (year-round steady high baseflow)
have a higher tendency to revert to a non-violated state. An
example of this behavior can be seen in the Australian basins. Though almost all the Australian basins have a very high
P.shift, only the highly variable flow regime northern catch-
ments have a high probability of staying violated. Despite
having an exceedingly high P.shift, the southern stable catch-
ments swiftly shift back to a non-violated state. C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity g
g p
;
g
)
Correlations between EF and biodiversity are generally
weak at the scale of G200 freshwater ecoregions as well (see
Sect. 2.2, Olson and Dinerstein, 2002). In G200 freshwa-
ter ecoregions (see Table S6 for the full freshwater ecore-
gion results), the nature of the EF–biodiversity relationship
greatly varied between different ecoregions (Fig. 4). In large
lakes, large rivers, and small lakes the richness indicators ob-
tained from Su et al. (2021) (TR, FR, PR) showed a strong
and significant positive correlation with most of the EF vi-
olation indices. The increase in biodiversity despite an in-
crease in EF violation could be a signal of the introduction
of nonnative species for commercial purposes, whereas, in
large rivers, large river deltas, and xeric basins, the dissimi-
larity indices and FiR show a negative correlation. However,
in most ecoregions, the EF–biodiversity relationship is in-
significant (p value > 0.05). Similar analysis using different
aggregation/scale matching methods also yielded compara-
ble results at the G200 ecoregion scale (see Figs. S5 and S6). In addition to this, the multivariate regression analysis results
(Fig. 5) also show a very low correlation between EF viola-
tion indicators and biodiversity indices in most G200 ecore-
gions except small lakes, where the coefficient of determi-
nation is between 0.25–0.4 for the richness indicators (TR,
FR, PR). The mean coefficient of determination (r2) is ap-
proximately 0.1. These results corroborate the above findings
that EF violations are not significantly inversely correlated
with biodiversity, regardless of the ecoregion, for the current
dataset. 3.2
Relationship between EF violation and freshwater
biodiversity The aggregated analysis was carried out at global and ecore-
gion scales. Multiple aggregation methods (Sect. 2.3) yielded
comparable results, so only case 1 (level 5 HydroBASIN
matched with biodiversity data) results are discussed further
(see Figs. S5 and S6 for results obtained using other aggre-
gation methods). At the global scale, none of the biodiversity
indicators correlated (significance of p value was < 0.05)
with any EF violation indices (Fig. 3). The biodiversity in-
dicators do not exhibit any strong trend in either the pos-
itive or the negative direction. The correlation coefficient
value (R value) for the remaining biodiversity indicators only
ranges from −0.2 to 0.17 (Fig. 3b). The three fish dissimilar-
ity facets (TD, FD, and PD) show a slight negative correla-
tion whereas the richness facets (TR, FR, and PR) display
a slight positive correlation with EF violation. The positive
correlation of the richness indicators is attributed to an over-
all increase in the assemblage in most of the basins despite
the increase in EF violation. Moreover, (relative) TR and (ab-
solute) FiR show opposite trends. The positive trend in TR
could be attributed to changes involving nonnative species,
whereas the FiR describes the current deteriorated state. The
increase in the fish assemblage over time was verified using 3.1
Evaluating EF violation drivers and characteristics The majority of basins face some kind of EF violation (either
in terms of severity or frequency or with higher probabili-
ties of shifting to and/or staying in a violated state) (Fig. 2). Between 1976 and 2005, 17 % and 45 % of basins, respec-
tively, experienced a violation frequency (F) of greater than
3 months per year and a severity (S) of greater than 20 %
from the EFE lower or upper bound (normalized violation in-
dex ≥0.25) (Fig. 2a and b). Additionally, 33 % of basins have
a higher chance of shifting (P.shift ≥0.5; i.e., 33 % of basins
have an over 50 % probability of shifting to a violated state) https://doi.org/10.5194/hess-26-6247-2022 Hydrol. Earth Syst. Sci., 26, 6247–6262, 2022 C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity 4
Discussion The findings from this study indicate that the EF–biodiversity
relationship is poorly correlated at global or ecoregion scales
with currently available data and methods. The most likely
explanation for the lack of correlation is the overwhelm-
ing heterogeneity of the freshwater ecosystems – e.g., with
some freshwater species being more susceptible to varia-
tions in flow than others (Poff and Zimmerman, 2010) –
which is not adequately represented in the spatial resolu-
tion used (level 5 HydroBASIN). Moreover, when it comes
to a larger-scale relationship, several other factors such as https://doi.org/10.5194/hess-26-6247-2022 Hydrol. Earth Syst. Sci., 26, 6247–6262, 2022 6254
C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity Figure 2. Four measures of environmental flow envelope (EFE) lower or upper bound violation estimated using the ensemble median of
four global hydrological models: (a) normalized frequency of violation, (b) normalized severity of violation, (c) probability of shifting to a
violated state from a non-violated state, and (d) probability of staying violated once shifted to a violated state. Figure 2. Four measures of environmental flow envelope (EFE) lower or upper bound violation estimated using the ensemble median
four global hydrological models: (a) normalized frequency of violation, (b) normalized severity of violation, (c) probability of shifting to
violated state from a non-violated state and (d) probability of staying violated once shifted to a violated state Figure 2. Four measures of environmental flow envelope (EFE) lower or upper bound violation estimated using the ensemble median of
four global hydrological models: (a) normalized frequency of violation, (b) normalized severity of violation, (c) probability of shifting to a
violated state from a non-violated state, and (d) probability of staying violated once shifted to a violated state. Figure 2. Four measures of environmental flow envelope (EFE) lower or upper bound violation estimated using the ensemble median of
four global hydrological models: (a) normalized frequency of violation, (b) normalized severity of violation, (c) probability of shifting to a
violated state from a non-violated state, and (d) probability of staying violated once shifted to a violated state. Figure 3. Scatter between EF violation indices and biodiversity indices (plots include linear fits) at a globally aggregated scale. Note: this
figure represents results from case 1 (level 5 HydroBASIN matched with biodiversity data). The results of other aggregation methods are
given in the Supplement (Figs. S5 and S6). C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity
6255 C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity
6255 g
y
Figure 4. (a) Spatial distribution of different G200 freshwater ecoregions and (b–h) the correlation between EF violation indices and fres
water biodiversity indicators for different G200 freshwater ecoregions. Note: the results for all the WWF freshwater ecoregions are giv
in the Supplement (Sect. S7). Abbreviations: F – frequency of violation; S – severity of violation; P.shift – probability of shifting to a vi
lated state; P.stay – probability of staying in a violated state; FiR – fish richness; TR – taxonomic richness; FR – functional richness; PR
phylogenetic richness; TD – taxonomic dissimilarity; FD – functional dissimilarity; PD – phylogenetic dissimilarity. Figure 4. (a) Spatial distribution of different G200 freshwater ecoregions and (b–h) the correlation between EF violation indices and fresh-
water biodiversity indicators for different G200 freshwater ecoregions. Note: the results for all the WWF freshwater ecoregions are given
in the Supplement (Sect. S7). Abbreviations: F – frequency of violation; S – severity of violation; P.shift – probability of shifting to a vio-
lated state; P.stay – probability of staying in a violated state; FiR – fish richness; TR – taxonomic richness; FR – functional richness; PR –
phylogenetic richness; TD – taxonomic dissimilarity; FD – functional dissimilarity; PD – phylogenetic dissimilarity. climate change (Davies, 2010; Poff et al., 2002), river frag-
mentation (Grill et al., 2015; Herrera-R et al., 2020), large-
scale habitat degradation (Moyle and Leidy, 1992), landscap-
ing/river scaping (Allan et al., 2005), alien species (Leprieur
et al., 2008, 2009; Villéger et al., 2011), and water pollution
(Brooks et al., 2016; Shesterin, 2010) can also impact the
freshwater ecosystem in multiple ways. Thus, at the Earth
system level, other interlinked factors potentially confound
the impact of EF violation on biodiversity degradation. ment. A generalized large-scale EF approach can underes-
timate the stress on the ecosystem at a smaller scale where
the actual action is taking place. It is undeniable that ad-
equate flow is essential for maintaining freshwater ecosys-
tems. Nonetheless, current generalized EF estimation meth-
ods need further refinement to adequately capture this im-
portance. The global hydrological EF methods are often val-
idated using locally calculated EF requirement values (Pas-
tor et al., 2014) with the assumption of adequate scalability
in the EF–biodiversity relationship. C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity
6255 However, more holistic
EF estimation methods combining hydrological, hydraulic,
and habitat simulation methods and expert knowledge (Poff
and Zimmerman, 2010; Shafroth et al., 2010) are essential 4.1
Implications for water management The lack of correlation between EF violation and freshwater
biodiversity has implications for large-scale water manage- 4
Discussion Abbreviations: F – frequency of violation; S – severity of violation; P.shift – probability of
shifting to a violated state; P.stay – probability of staying in a violated state; FiR – fish richness; TR – taxonomic richness; FR – functional
richness; PR – phylogenetic richness; TD – taxonomic dissimilarity; FD – functional dissimilarity; PD – phylogenetic dissimilarity. Figure 3. Scatter between EF violation indices and biodiversity indices (plots include linear fits) at a globally aggregated scale. Note: this
figure represents results from case 1 (level 5 HydroBASIN matched with biodiversity data). The results of other aggregation methods are
given in the Supplement (Figs. S5 and S6). Abbreviations: F – frequency of violation; S – severity of violation; P.shift – probability of
shifting to a violated state; P.stay – probability of staying in a violated state; FiR – fish richness; TR – taxonomic richness; FR – functional
richness; PR – phylogenetic richness; TD – taxonomic dissimilarity; FD – functional dissimilarity; PD – phylogenetic dissimilarity. https://doi.org/10.5194/hess-26-6247-2022 Hydrol. Earth Syst. Sci., 26, 6247–6262, 2022 Hydrol. Earth Syst. Sci., 26, 6247–6262, 2022 C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity
6255 4.2
Implications for a water planetary boundary The current rationale in using EF in the water planetary
boundary relationship is based on the assumption of its uni-
versal relationship with freshwater biodiversity. However,
with the currently available data and methods, the findings
for the EF–biodiversity relationship are inconclusive. More-
over, due to the heterogeneity of biodiversity response over
time and space, the trend at any aggregate scale is likely
to remain relatively constant instead of showing any dis-
cernible tipping point (Brook et al., 2013). We suggest that
the use of environmental flows in defining water planetary
boundaries should be reconsidered, given the higher de-
gree of heterogeneity and lack of strength of the ecosystem
function–biodiversity relationship. Some of the potential rea-
sons for this reconsideration are as follows. Firstly, freshwa-
ter biodiversity may not have pan-regional or “continental-
planetary”-scale threshold dynamics, and its link with EF vi-
olation might be inadequate to represent the finer-scale varia-
tions. Secondly, resource distribution and human impact het-
erogeneity suggest the need for regional boundaries, as pro-
posed by Steffen et al. (2015). Thirdly, the EF calculation
methods used in the current regional/planetary boundary def-
inition are highly restricted to hydrological methods, which
may not be adequate to capture the biodiversity status. A
regional boundary transgression can occur even well within
planetary-level safe limits (Brook et al., 2013; Nykvist et al.,
2017). Therefore, for an overly complex biophysical relation-
ship such as EF–biodiversity, where multiple shift states are
possible, it is difficult to prioritize and manage critical re-
gions without a regional/local boundary. Figure 5. Coefficients of correlation (R2) for multivariate regres-
sion between EF violation indicators and biodiversity indices. Each
row represents one biodiversity indicator and each column repre-
sents one G200 ecoregion. to ensure a healthy freshwater biodiversity. The policies and
decisions taken at various scales need a more dynamic frame-
work where different dominant drivers of ecosystem degra-
dation can be prioritized based on particular cases. For in-
stance, an integrated EF indicator which encompasses quan-
tity, quality, and timeliness of water in the streams will be a
better hydrologic indicator to evaluate freshwater ecosystem
health than an indicator which accounts only for quantity. Moreover, when making water management decisions, care
must be given to account for the temporal and spatial hetero-
geneity in the ecosystem dynamics. 4.2
Implications for a water planetary boundary Although there are some coordinated scientific efforts
such as ELOHA (Ecological Limits of Hydrologic Alter-
ations) (Poff et al., 2010) to provide a holistic framework
for EF estimation, its scientific complexity and high imple-
mentation cost constrains its use around the world (Richter
et al., 2012). For example, several European countries such
as Romania, Czech Republic, Serbia, and Luxembourg use
a national-level static method to define minimum environ-
mental flows (Linnansaari et al., 2012). Similarly, other juris-
dictions use the presumptive standards proposed by Richter
et al. (2012) to establish a legal basis for EF protection. These presumptive standards limit hydrologic modifications
to a percentage range of natural or historic flow variability. One example of such a case, North Carolina’s Environmental
Flow Science Advisory Board, uses a presumptive standard
of 80 %–90 % of the instantaneous modeled baseline flow
as the EF requirement (NCEFSAB, 2013). The limitation of
such a practice is the incorrect presumption of uniformity in
the EF needs over a larger region. Therefore, we recommend
the application of holistic indicators at these large scales
(covering all river stretches and tributaries) rather than using
simplified hydrologic-only metrics of EF (violation). How-
ever, the authors also acknowledge the limits in implemen- 6256
C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity Figure 5. Coefficients of correlation (R2) for multivariate regres-
sion between EF violation indicators and biodiversity indices. Each
row represents one biodiversity indicator and each column repre-
sents one G200 ecoregion. tation of a more dynamic EF framework in data-limited re-
gions. Programs for more monitoring and data collection and
improved, more holistic modeling methods using more/better
data need to be implemented in those regions. Thus, applying
a holistic framework such as ELOHA could be made possi-
ble and can capture the heterogeneity in the EF–biodiversity
relationship. https://doi.org/10.5194/hess-26-6247-2022 https://doi.org/10.5194/hess-26-6247-2022 Hydrol. Earth Syst. Sci., 26, 6247–6262, 2022 6256
C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity 6256
C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity
6257 2. Discrepancy in data resolution: the spatial and temporal
resolutions at which the EF violation is estimated here
and the biodiversity indicators measured/calculated are
inconsistent. The basic spatial measuring unit of the bio-
diversity is sometimes greater or less than the basin size
at which EF is measured. This discrepancy could have
some impact on the results. However, in this study, sev-
eral resolution-matching methods were used to account
for this uncertainty. Therefore, more detailed data with
better-matching scales are needed to overcome this lim-
itation. 2. Discrepancy in data resolution: the spatial and temporal
resolutions at which the EF violation is estimated here
and the biodiversity indicators measured/calculated are
inconsistent. The basic spatial measuring unit of the bio-
diversity is sometimes greater or less than the basin size
at which EF is measured. This discrepancy could have
some impact on the results. However, in this study, sev-
eral resolution-matching methods were used to account
for this uncertainty. Therefore, more detailed data with
better-matching scales are needed to overcome this lim-
itation. scales and could potentially be identified with more holistic
EF methods that include multiple factors (e.g., temperature,
water quality, intermittency, connectivity) and more exten-
sive freshwater biodiversity data. A single negative result is
not a final say, but it is a call to conduct more study on exist-
ing generalized and well-applied methods. The paper is not intended to be a definitive test on the rela-
tionship between EF and aquatic biodiversity, but more to be
an exploratory analysis that tests a widely used but rarely ver-
ified assumption of the relationship at the global and ecore-
gion scale. The lack of correlation in the EF–biodiversity re-
lationship found in this study suggests that particular care
should be taken when developing macro-scale EF policies
(regional and above), and further implies that the conceptual-
ization of a blue water planetary boundary ought to rest upon
a broader set of relationships between hydrological processes
and Earth system functioning. At larger scales, the enormous
spatial and temporal heterogeneity in the EF–biodiversity re-
lationship motivates a holistic estimation of EF grounded in
ecosystem dynamics. 3. Lack of multi-driver interaction: in this study, we con-
sider the impact of EF violations on biodiversity to be
an independent relationship. In reality, this might not be
the case. C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity
6257 Other drivers of ecosystem degradation, such
as land use change, habitat loss, stream modifications,
and geographical disconnection can influence the EF–
biodiversity relationship. These interactions were out-
side the scope of this study but should be taken into ac-
count in follow-up studies. 4. Simplified representation of human interference with
freshwater systems: the role of humans in impairing the
ecosystem balance is represented here based on how hu-
man water withdrawals violate the hydrologically de-
fined EF. Other human disturbances are thus not ac-
counted for, such as aquatic habitat degradation through
a change in land use, artificial introduction of nonna-
tive species, and non-point pollution from agriculture. Moreover, this study does not distinguish the climate-
driven impact on EF violation from the anthropogenic
impacts. Data availability. All data needed to reproduce the analysis in this
manuscript are available at https://doi.org/10.5683/SP3/2BYXZZ
(Mohan
et
al.,
2022a),
and
all
the
code
(Matlab)
used
is
available
at
https://github.com/ChinchuMohan/
Eflows-Biodiversity-Project (last access: 29 November 2022;
https://doi.org/10.5281/zenodo.7378494, Mohan et al., 2022b). Supplement. The supplement related to this article is available on-
line at: https://doi.org/10.5194/hess-26-6247-2022-supplement. Author contributions. CM, TG, and JSF devised the conceptual
and analysis framework of this study with inputs from MK, MP,
and VV. VV performed the EFE calculation with help from MK
and MP. CM performed the biodiversity data compilation and EF–
biodiversity analytical evaluation with help from TG, JSF, and XH. CM performed the final analysis and produced the results and visu-
alization shown in the study via discussions with TG, JSF, XH, MK,
MP, VV, and LWE. TG, JSF, MK, MP, VV, LWE, XH, DG, and
SCJ contributed to paper writing and the interpretation of the re-
sults. CM took the lead in writing the manuscript. All authors pro-
vided critical feedback and helped shape the research, analysis, and
manuscript. 5. Exclusion of the impact of dams: the dams are indeed
a large contributing factor to the uncertainty in the re-
sults. Dam-regulated rivers may have a significantly dif-
ferent effect on biodiversity compared to free-flowing
rivers. The ISIMIP data used to calculate EF violations
considers the effects of large dams on the streamflow. However, to explicitly isolate the effects of dams in this
analysis from other drivers, information on dam oper-
ation schemes for each sub-basin would be necessary,
and this would require a paper on its own. C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity
6257 Therefore, the
effects of the dams are incorporated in this study but are
not explicitly analyzed separately from other drivers. 5. Exclusion of the impact of dams: the dams are indeed
a large contributing factor to the uncertainty in the re-
sults. Dam-regulated rivers may have a significantly dif-
ferent effect on biodiversity compared to free-flowing
rivers. The ISIMIP data used to calculate EF violations
considers the effects of large dams on the streamflow. However, to explicitly isolate the effects of dams in this
analysis from other drivers, information on dam oper-
ation schemes for each sub-basin would be necessary,
and this would require a paper on its own. Therefore, the
effects of the dams are incorporated in this study but are
not explicitly analyzed separately from other drivers. Competing interests. The contact author has declared that none of
the authors has any competing interests. 4.3
Limitations and ways forward 1. Data scarcity: even though this study uses state of the
art global hydrological models and best-available global
estimates of EF requirements, freshwater ecological
data are limited to freshwater fish. Several other taxa,
such as crayfish and other benthic invertebrates, phyto-
plankton, or zooplankton, are also significant in deter-
mining the proper functioning of a freshwater ecosys-
tem (AL-Budeiri, 2021; Domisch et al., 2017; Nyström
et al., 1996). However, due to a lack of global data, these
taxa are not included in this study. To better examine the
relationship, global datasets for other freshwater biodi-
versity metrics are urgently needed. 1. Data scarcity: even though this study uses state of the
art global hydrological models and best-available global
estimates of EF requirements, freshwater ecological
data are limited to freshwater fish. Several other taxa,
such as crayfish and other benthic invertebrates, phyto-
plankton, or zooplankton, are also significant in deter-
mining the proper functioning of a freshwater ecosys-
tem (AL-Budeiri, 2021; Domisch et al., 2017; Nyström
et al., 1996). However, due to a lack of global data, these
taxa are not included in this study. To better examine the
relationship, global datasets for other freshwater biodi-
versity metrics are urgently needed. https://doi.org/10.5194/hess-26-6247-2022 Hydrol. Earth Syst. Sci., 26, 6247–6262, 2022 https://doi.org/10.5194/hess-26-6247-2022 6258
C. Mohan et al.: Poor correlation between large-scale environmental flow violations and freshwater biodiversity Acknowledgements. The authors acknowledge various funds that
made this research possible. Chinchu Mohan received funding from
the Canada First Research Excellence Fund (CFRE); Matti Kummu
received funding from the Academy of Finland funded project
WATVUL (grant no. 317320), the Academy of Finland funded
project TREFORM (grant no. 339834), and the European Research
Council (ERC) under the European Union’s Horizon 2020 research
and innovation program (grant agreement no. 819202). Vili Virkki
received funding from the Aalto University School of Engineering
Doctoral Program and the European Research Council (ERC) under
the European Union’s Horizon 2020 research and innovation pro-
gram (grant agreement no. 819202). Sonja C. Jähnig acknowledges
funding through the Leibniz Association for the project Freshwater
Megafauna Futures. Miina Porkka received funding from European
Research Council (ERC) under the European Union’s Horizon 2020
research and innovation program (grant agreement no. 819202). Lan Wang-Erlandsson was supported by the European Research
Council through the “Earth Resilience in the Anthropocene” project
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Summary and conclusion The relationship between EF violations and freshwater bio-
diversity was evaluated at globally aggregated levels in this
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freshwater biodiversity indicators was found at the global
or ecoregion scale using globally consistent methods and
currently available data. Relationships may exist at smaller Disclaimer. Publisher’s note: Copernicus Publications remains
neutral with regard to jurisdictional claims in published maps and
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Cost-Effectiveness Analyses on Various Models of The Red Light, Purple Light Self-Regulation Intervention for Young Children
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ORIGINAL RESEARCH
published: 14 October 2021
doi: 10.3389/fpsyg.2021.711578 Cost-Effectiveness Analyses on
Various Models of The Red Light,
Purple Light Self-Regulation
Intervention for Young Children Tao Li 1*, Megan M. McClelland 2, Shauna L. Tominey 3 and Alexis Tracy 2 1 Health Management and Policy, Oregon State University, Corvallis, OR, United States, 2 Human Development and Family
Sciences and the Hallie E. Ford Center for Healthy Children and Families, Oregon State University, Corvallis, OR, United
States, 3 Extension Family and Community Health and the Hallie E. Ford Center for Healthy Children and Families, Oregon
State University, Corvallis, OR, United States Early childhood interventions can improve self-regulation, but there are few economic
evaluations of such interventions. This study analyzed the cost-effectiveness of an early
childhood self-regulation intervention (Red Light Purple Light!; RLPL), comparing three
different models of implementation across stages of intervention development: (Model 1)
trained research assistants (RAs; graduate students) directly delivered the RLPL intervention
to children; (Model 2) RAs trained trainers (e.g., program coaches), who then trained
teachers to implement RLPL with children (e.g., train-the-trainer); and (Model 3) program
faculty trained teachers to deliver the RLPL intervention to children. We implemented a
cost-effectiveness analysis by calculating the incremental cost-effectiveness ratio. We also
conducted a series of sensitivity analyses to adjust for parameter uncertainty. Our base-
case analysis suggests that Model 2 was the most cost-effective strategy, in that a cost
of $23 per child was associated with a one-unit increase of effect size on self-regulation
scores. The “train-the-trainer” model remained the optimal strategy across scenarios in
our sensitivity analysis. This study fills an important gap in cost-effectiveness analyses on
early childhood self-regulation interventions. Our process and results can serve as a model
for future cost-effectiveness analyses of early childhood intervention programs and may
ultimately inform decisions related to intervention adoption that optimize resource allocation
and improve program design. Keywords: cost-effectiveness analysis, economic evaluations, red light purple light, early childhood intervention,
self-regulation, school readiness, executive function skills Edited by:
Jin Sun,
The Education University of Hong Kong,
Hong Kong, SAR China ,
The Education University of Hong Kong,
Hong Kong, SAR China Reviewed by:
Maria von Salisch,
Leuphana University, Germany
Marie Catherine White,
Gordon College, United States Reviewed by:
Maria von Salisch,
Leuphana University, Germany
Marie Catherine White,
Gordon College, United States Reviewed by:
Maria von Salisch,
Leuphana University, Germany
Marie Catherine White,
Gordon College, United States
*Correspondence:
Tao Li
tao.li2@oregonstate.edu Specialty section:
This article was submitted to
Developmental Psychology,
a section of the journal
Frontiers in Psychology Developmental Psychology,
a section of the journal
Frontiers in Psychology Keywords: cost-effectiveness analysis, economic evaluations, red light purple light, early childhood intervention,
self-regulation, school readiness, executive function skills Received: 18 May 2021
Accepted: 06 September 2021
Published: 14 October 2021 Connections Between Self-Regulation
and Academic Skills Self-regulation is a key factor supporting academic success
across the life span (McClelland et al., 2006, 2013; Blair and
Razza, 2007; Moffitt et al., 2011). Self-regulation predicts early
achievement (both math and literacy) in preschool, elementary
and middle school and even college (Duckworth et al., 2010;
McClelland et al., 2013, 2014). Especially important for school
readiness skills, self-regulation is related broadly to early aspects
of math and emergent literacy (McClelland et al., 2007a, 2014;
Schmitt et al., 2017), especially early numeracy skills (counting,
cardinality, numeral knowledge), which is most predictive of
later mathematics achievement (Nguyen et al., 2016). Moreover,
self-regulation interventions have shown significant effects on
children’s math and literacy (Blair and Raver, 2014; Schmitt
et al., 2015; Pandey et al., 2018) and may be especially predictive
of early math skills (Allan et al., 2014; McClelland et al., 2014;
Blair et al., 2015; Purpura et al., 2017).f Red Light Purple Light! (RLPL) is an easy-to-implement,
short-term intervention designed to promote self-regulation
and school readiness (Tominey and McClelland, 2011; McClelland
and Tominey, 2015). Although early childhood interventions
such as RLPL have been shown to improve self-regulation,
there are few economic evaluations of such interventions. This
study analyzed the cost-effectiveness of an early childhood
self-regulation intervention (RLPL) implemented through three
different models: 1) trained research assistants (RAs) implemented
the RLPL intervention with children; 2) RAs trained trainers
(e.g., early childhood coaches), who then trained teachers to
implement the RLPL intervention with children (e.g., train-
the-trainer); and 3) program faculty trained teachers to implement
RLPL with children. These three models used the same
intervention curriculum, but at different phases of testing and
iterative development of the implementation process, which
allowed us to explore and compare the cost-effectiveness of
each implementation model. p
In previous studies evaluating the RLPL program, effects
have been found for children’s improvements in self-regulation
(Schmitt et al., 2015; Duncan et al., 2018), especially those
with low baseline levels of self-regulation (Tominey and
McClelland, 2011), early math skills (Duncan et al., 2018),
particularly for low socioeconomic (McClelland et al., 2019)
and low-SES DLL children (Schmitt et al., 2015) and early
literacy skills (Tominey and McClelland, 2011; Duncan et al.,
2018). These effects are supported by other research showing
that classrooms characterized by consistent, organized classroom
practices lead to better academic outcomes for children (Cameron
et al., 2008; Cameron and Morrison, 2011). Connections Between Self-Regulation
and Academic Skills Previous research
on RLPL also indicates that participating children demonstrate
significantly stronger self-regulation on direct measures and
teacher ratings (Schmitt et al., 2015; Keown et al., 2020)
suggesting that children are demonstrating improvements in
directly assessed self-regulation and observations of children’s
behavior in classroom settings. These results are promising
but do not provide information related to the costs associated
with each model or the corresponding effects, indicating that
an evaluation of the cost-effectiveness of the RLPL program
is needed. INTRODUCTION Li T, McClelland MM, Tominey SL and
Tracy A (2021) Cost-Effectiveness
Analyses on Various Models of The
Red Light, Purple Light Self-
Regulation Intervention for Young
Children. Many children, especially those experiencing socio-demographic risks such as poverty, enter
formal schooling without key skills needed to thrive in a classroom environment (Blair and
Raver, 2015). These skills are included in a construct called school readiness and include
self-regulation and early academic skills (Snow, 2006). Unfortunately, children facing socio-
demographic risk who struggle with self-regulation and early academic skills are likely to Front. Psychol. 12:711578. doi: 10.3389/fpsyg.2021.711578 Front. Psychol. 12:711578. doi: 10.3389/fpsyg.2021.711578 October 2021 | Volume 12 | Article 711578 Frontiers in Psychology | www.frontiersin.org Cost-Effectiveness Analyses for Self-Regulation Intervention Li et al. face achievement gaps that persist and widen over time (Zelazo
et al., 2016). Self-regulation is a significant predictor of short-
and long-term academic, social, and life outcomes (Moffitt
et al., 2011; McClelland et al., 2013; Zelazo et al., 2016; Robson
et al., 2020). Importantly, self-regulation can be practiced and
improved (Diamond and Ling, 2016), and self-regulation
interventions may serve as a mechanism to protect children
at risk (Sasser et al., 2017; Pandey et al., 2018). Large-scale
classroom curricula that combine self-regulation and academic
skills have shown mixed effects (Farran et al., 2013; Blair and
Raver, 2014; Morris et al., 2014), but targeted self-regulation
interventions that can easily be implemented in the classroom
have shown positive effects across both self-regulation
and academic domains (Tominey and McClelland, 2011;
Schmitt et al., 2015; McClelland et al., 2019). school success (McClelland et al., 2007b, 2014; McClelland
and Cameron, 2012). We use the term self-regulation in
the present study to capture EF processes in real-world settings. Frontiers in Psychology | www.frontiersin.org The Importance of Self-Regulation
on School Success Self-regulation is a complex construct that includes aspects
of emotion, cognition, and behavior (McClelland et al., 2010). This paper focuses on the aspects of self-regulation most
relevant for children’s learning in school contexts, which
stem from three executive function (EF) processes: working
memory, attentional or cognitive flexibility, and inhibitory
control (Cameron Ponitz et al., 2009). Working memory
refers to the ability to hold and manipulate short-term
instructions in mind such as when children need to remember
the steps in an art activity (Gathercole, 2008; McClelland
et al., 2015); attentional or cognitive flexibility is ability to
focus on a task and switch to a new task when needed
such as when children need to stop what they are doing
and move to a new activity (Rothbart and Posner, 2005);
and inhibitory control is the ability to stop a dominant
response in favor of a more adaptive one (Blair, 2003). This
can be seen when children have to inhibit their impulse
to blurt out an answer and raise their hand instead. Although
each aspect of EF has been shown to predict academic
outcomes, their integration is particularly important for The Present Studyh The Present Study
The present study focuses on a cost-effectiveness analysis of
the Red Light, Purple Light (RLPL) self-regulation intervention,
which has been shown to have significant positive effects on
self-regulation (e.g., Tominey and McClelland, 2011; Schmitt
et al., 2015; Duncan et al., 2018; McClelland et al., 2019). The
RLPL program is an eight-week classroom-based self-regulation
intervention. The program consists of large group time music
and movement-based classroom games implemented twice a
week in 20–30-min sessions (McClelland and Tominey, 2015). Over the course of multiple randomized control trials, the
same RLPL curriculum was used, although the training and
implementation models varied. This variation allowed for an
exploratory cost-effectiveness analysis across each of these models. RLPL was developed through an iterative development process. The intervention was first piloted through a small randomized
trial where the sessions were led by a research assistant (RA)
who was also a former early childhood classroom teacher
(Model 1; Tominey and McClelland, 2011). The intervention
was replicated in another randomized trial with a larger sample
with RAs leading the games during classroom group time
activities (also Model 1; Schmitt et al., 2015). A “train-the-
trainer” model was then employed (Model 2; Duncan et al.,
2018) where two early childhood professional development
coaches were trained in the intervention by the RLPL development
team and then delivered the training to additional teachers in
their district as part of their existing role supporting professional
development efforts. Finally in Model 3, professional development
trainings were delivered to teachers by faculty members of the
original RLPL development team (McClelland et al., 2019).ff t
Interventions focused on the broader construct of social–
emotional skills (Diamond et al., 2007; Domitrovich et al.,
2007) have emerged in recent years, including programs
specifically targeting self-regulation (e.g., Tominey and
McClelland, 2011; Schmitt et al., 2015). Numerous programs
have demonstrated significant positive effects on children’s self-
regulation, although effects are typically modest (Bierman et al.,
2008; Raver et al., 2011; McClelland et al., 2017). In some
cases, program effects have even been inconsistent across studies
(Blair and Raver, 2014; Morris et al., 2014). The resources,
time, and skills required to implement early childhood
intervention programs to fidelity vary widely and are not often
well-documented. Few interventions have conducted cost-
effectiveness analyses to identify the costs associated with
implementation as well as associated outcomes. The Importance of Cost-Effectiveness
Analysis in Early Intervention Programsh Analysis in Early Intervention Programs
There is a growing interest among researchers, practitioners,
and policymakers in studying the economic impacts of early
childhood interventions to inform investment and policy
decisions (National Academies of Sciences, Engineering and
Medicine, 2016; Cannon et al., 2018; Jones et al., 2019). Although
few studies exist for school readiness interventions, researchers
have started to examine costs more closely. For example, Jones
and colleagues conducted cost analysis and examined the costs
of different versions of the Head Start Research-based,
Developmentally Informed intervention (Jones et al., 2019). Compared to cost analysis, we expect cost-effectiveness analysis
will be a more powerful tool and will provide more important
evidence to inform policymakers and stakeholders, because in
cost-effectiveness analysis we simultaneously take into account
the resources needed for program implementation as well as
the outcomes achieved. Thus, to echo this urgent need of
economic evaluation evidence, the purpose of this study is to
evaluate the cost-effectiveness of the RLPL program. Frontiers in Psychology | www.frontiersin.org The Potential of Early Interventions y
Early childhood is an important developmental period for self-
regulation growth because of the rapid development in brain
development, especially in the prefrontal cortex, an area that
is critical for self-regulation (Diamond, 2002; Montroy et al.,
2016). As such, early childhood settings provide an opportune
time for interventions aimed at strengthening these skills in
young children. Research shows that significant differences in
self-regulation are apparent in early childhood (McClelland
et al., 2015; Zelazo et al., 2016). Although some of these
differences in self-regulation are likely on account of natural October 2021 | Volume 12 | Article 711578 2 Cost-Effectiveness Analyses for Self-Regulation Intervention Li et al. Li et al. variability in children’s developmental ages and stages, these
differences relate to children’s abilities to engage in positive
classroom behaviors (Day et al., 2015; Zelazo et al., 2016). Research on the high rates of preschool suspensions and
expulsions indicates that early childhood teachers could benefit
from support managing challenging classroom behaviors likely
related to self-regulatory difficulties (Gilliam and Shabar, 2006). Early childhood educators enter the field with a range of
educational backgrounds and experiences, making in-service
trainings an especially valuable form of professional development
for the early childhood field. Professional development is seen
as a critical component of high-quality early childhood education
settings, and thus, significant time and funds are devoted to
supporting these efforts. It is through ongoing professional
development that interventions, including self-regulation
interventions, are often implemented. The Present Studyh Conducting
cost-effectiveness analyses is a critical next step in the field
of early childhood intervention to inform practice and policy
toward identifying programs that are the most feasible and
cost-effective. In the present cost-effectiveness study, cost and effectiveness
data are analyzed for each approach to implement and to
identify the model(s) that are most cost-effective. Although
the RLPL program was implemented across studies using various
models, each shared the common goal of improving children’s
self-regulation. Therefore, these data provide a unique opportunity
to compare cost-effectiveness among different models of the
RLPL intervention, as the cost-effectiveness analysis is particularly
helpful to compare among closely related interventions (Li et al.,
2017, 2019). We expect our findings will help identify the
optimal intervention design and strategy to improve children
self-regulation in the most cost-effective way, and will provide
critical evidence to inform decision making related to intervention
selection for schools, districts, and additional stakeholders. Model 1: Trained Research Assistants
(RAs; Graduate Students) Deliver the RLPL
Games to Children Tominey and McClelland (2011)
Sixty-five children (50% low-income as defined as participation
in Head Start) from nine classrooms in two preschool centers
participated in the study (children were randomized within
classrooms). Children were an average of 54.60 months (range:
44–60 months; 60% female). The average maternal education level
was about an Associate’s degree (M = 14.6 years) with a range
of 6–21 years. Three children spoke Spanish as a first language,
were assessed in Spanish, and received the intervention in Spanish. Participants For Model 2 dosage = (25 min per sess dels 1 and 3 dosage = (25 min per session * 16 sessions). For Model 2 the Institutional Review Board at Oregon State University,
United States. in the study with a final number of 99 children with data at
both time points. Children were an average age of 63.24 months
and did not have prior preschool experience. No additional
demographic information (i.e., parent education level) was
available from the school district. All children received the
RLPL intervention and self-regulation assessment in English. Procedure Across the four studies, the RLPL intervention was implemented
in three different models as outlined below. In all studies, teachers
reported that their normal curricula did not include games or
activities that were similar to the RLPL games or emphasized
self-regulation. Importantly, for all models in our study, participation
in the intervention (versus a delayed control) was randomized. Individuals involved in data collection were blind to the condition
of participating children and classrooms where randomization
occurred. Children in all delivery models received similar amounts
of the intervention. For Models 1 and 3, the duration was twice
a week over eight weeks for 16 sessions, and in the Model 2, Schmitt et al. (2015) Two hundred seventy-six children from 14 Head Start
classrooms across nine preschool centers participated (7
intervention and 7 control classrooms). Children averaged
51.69 months (range: 37.98 to 66.04 months; 51% female). The
average parent education level was less than a high school
degree (M = 11.15 years) with a range from 2 to 16 years of
education. There were 33% (N = 88) English language learners
(ELLs) who were assessed in Spanish and who received the
intervention in Spanish. Model 3: Program Faculty Train Teachers,
Who Then Deliver the RLPL Games to
Children McClelland et al. (2019)
One hundred fifty-seven children (52% female) from 13 Head
Start classrooms participated in the study (10 intervention, 3
control). Children were an average of 51 months (range of
38–62 months), and parents’ education level ranged from 2 to
17 years (M = 11.27). Fifty-eight percent of the sample identified
as Latino and 33% (N = 62) were ELLs. These children were
assessed in Spanish but received the intervention in English
following best practices for supporting Dual Language Learners
(DLL’s; Tominey and O'Bryon, 2018). Participants Data included in this study were collected as part of four
intervention studies examining the RLPL intervention in
young children. The four studies fit three models of RLPL
implementation as described below (see Table 1). Below,
we describe the original samples and in the analytic strategy
section we detail how sample sizes were analyzed to improve
comparability across models. The study was approved by October 2021 | Volume 12 | Article 711578 3 Cost-Effectiveness Analyses for Self-Regulation Intervention Li et al. Li et al. TABLE 1 | Parameters Sources and Values for Base-case Analysis on Different Models to Implement the RLPL Program. RLPL implementation modela
Model 1
Model 2
Model 3
Literature source
Tominey and McClelland, 2011;
Schmitt et al., 2015
Duncan et al., 2018
McClelland et al., 2019
N (children)
65; 276
125
157
Number of classrooms in interventionb
16
4
10
Child gender (% female)
60%/51%
N/Ac
52%
Child age (in months)
54.60/51.69
63.24
51
Low-income
50%/100%
N/A
100%
English language learner
4.6%/33%
N/A
33%
Average parent education level
14.6 years (Associate’s Degree
equivalent)/11.5 years (less than
High School)
N/A
11.27 years (less than High School
equivalent)
Total person-hours spent in RLPL implementation
62
6
6
Dosaged
16 sessions (M = 6.67 h)
15 sessions (M = 6.25 h)
16 sessions (M = 6.67 h)
Effect size of self-regulation scores
0.34/0.34
0.34
0.31
aModel 1 = trained research assistants (RAs) directly delivered the RLPL games to children; Model 2 = trained RAs trained trainers, who then trained teachers to deliver the RLPL
games to children; Model 3 = program faculty trained teachers, who then delivered the RLPL games to children. bThere were 16 classrooms that received RLPL intervention under Model 1 (9 in the Tominey and McClelland, 2011 study, and 7 in the Schmitt et al., 2015 study), and 10
classrooms in intervention under Model 3 (McClelland et al., 2019). In Model 2, RAs trained trainers, who then trained a total of 4 teachers to deliver the RLPL games, so
we analyzed cost for 4 classrooms. cN/A = Not available. TABLE 1 | Parameters Sources and Values for Base-case Analysis on Different Models to Implement the RLPL Program. dFor Models 1 and 3 dosage = (25 min per session * 16 sessions). For Model 2 dosage = (25 min per session * 15 sessions). dFor Models 1 and 3 dosage = (25 min per session * 16 sessions). Duncan et al. (2018) Duncan et al. (2018) In this “train-the-trainer” model, RAs trained trainers who then
trained teachers to deliver the RLPL games to children in a
summer program. Of the participating schools, three schools were
randomly assigned to receive the intervention (N = 67 children)
and one school was randomly assigned to serve as a control
group (receiving a summer program curricula, which was different
from RLPL; N = 32). Teacher fidelity and feasibility surveys indicated
that teachers implemented the games as intended and children
enjoyed playing them. Children’s self-regulation was directly assessed
at pre-program (July) and post-program (August) time points by
trained school district staff. Model 2: RAs Train Trainers, Who Then
Train Teachers to Deliver the RLPL Games
to Children (“Train-the-Trainer”) Duncan et al. (2018) In this implementation model, one hundred twenty-five children
from four schools in a large public school district participated October 2021 | Volume 12 | Article 711578 Frontiers in Psychology | www.frontiersin.org 4 Cost-Effectiveness Analyses for Self-Regulation Intervention Li et al. the duration was five days a week over three weeks for 15
sessions. Each session was 20–30 min across all delivery models. On average, the dosage was 6.67 h for Models 1 and 3 (average
of 25 min per session x 16 sessions), and 6.25 h for Model 2
(average of 25 min per session x 15 sessions; see Table 1). Schmitt et al. (2015) Fourteen classrooms were randomly assigned to an
intervention group (n = 7; 126 children) or a BAU control group
(n = 7; 150 children; N = 276 overall). Trained RAs directly
assessed children’s self-regulation in the fall (pre-test) and in
the spring (post-test) of the preschool year. The intervention
was implemented in the winter by RAs. Parents and teachers
completed demographic surveys. Analytic Strategy for the
Cost-Effectiveness Analysisf We conducted our cost-effectiveness analyses from the perspective
of RLPL program implementation (i.e., the costs associated with
conducting research were not included as these expenses were
supported by grants and would not be included in future
implementation and program costs). In doing so, we reduced
parameter uncertainty in our data collection; our analyses were
more directly relevant to program implementation; and our
findings could better support stakeholders’ decisions on replicating
and expanding our intervention. Based on this analytical perspective,
we included costs accrued from intervention implementation only,
excluding costs accrued for research and evaluation purposes. We applied the concept of opportunity cost when estimating
cost, which could better identify the trade-off value of cost items. As recommended by the Panel on Cost-Effectiveness in Health
and Medicine, “(a) change in the use of a resource caused by
a health intervention should be valued at its opportunity cost,
which is the value the resource could have produced if it were
spent in its best available alternative use” (Weinstein et al., 1996,
p. 1255). We considered program cost as the total cost to implement Outcomes
Self-Regulation g
To assess children’s self-regulation, the Head-Toes-Knees-
Shoulders (HTKS) measure was used in all studies. The HTKS
assesses aspects of executive function including attention, working
memory, and inhibitory control (McClelland et al., 2014) in
children ages 3–8. The task has three parts. In the first part,
children are asked to touch their head (or toes) when asked
to touch their toes (or head). In the next part, a new rule is
introduced where children touch their knees (or shoulders),
and then, both rules are included (head/toes opposite and
knees/shoulders opposite). In the third part, children are still
doing the opposite, but the rules are switched (head goes with
knees and shoulders goes with toes). Items are scored 0 for
an incorrect response, 1 for a self-corrected response, and 2
for a correct response. The overall scores range from 0 to 60
except in Tominey and McClelland (2011) when the task had
two parts and scores ranged from 0 to 40. Tominey and McClelland (2011) Tominey and McClelland (2011) Children within nine classrooms were randomly assigned to
receive the intervention (n = 28) or to a Business-As-Usual (BAU)
control group (n = 37; overall N = 65). Trained RAs assessed
children’s self-regulation individually, and parents and teachers
completed demographic surveys. Children were pretested in the
fall, the intervention was administered in the winter by RAs,
and children were post-tested in the spring of the preschool year. In the McClelland et al. (2019) study (Model 3 above), children
received a revised version of the HTKS, called the HTKS-Revised
(Gonzales et al., 2021). This version included a downward
extension with the same commands where children responded
to using verbal cues (e.g., “When I say Toes, you say Head”)
rather than gross motor responses. This section was administered
to children prior to receiving parts 1–3 of the original HTKS
measure and overall scores ranged from 0 to 116. The HTKS-R
and HTKS have both demonstrated strong reliability and validity
in preschool aged children (McClelland et al., 2014; Gonzales
et al., 2021). Previous research has demonstrated that the HTKS
is sensitive to intervention effects, where children receiving an
intervention showed significant improvement in self-regulation
when compared with children in a control group (Tominey and
McClelland, 2011; Schmitt et al., 2015; Duncan et al., 2018;
Landis et al., 2018; Upshur et al., 2019). Model 1: In this implementation model, RLPL was administered by
trained RAs who pulled children out of their classroom to
implement the RLPL games and activities. Frontiers in Psychology | www.frontiersin.org Model 3: McClelland et al. (2019) McClelland et al. (2019) In this model, program faculty trained teachers in the standard
three-hour RLPL training, who then delivered the RLPL games
to children. Classrooms were randomly assigned to two versions
of the RLPL intervention (N = 10 classrooms total; 120 children)
or a BAU control group (N = 3 classrooms; 37 children). In
the fall (pre-test) and spring (post-test) of the preschool year,
direct assessments of self-regulation were administered in English
or Spanish. The intervention was implemented in the winter
by teachers in either English or Spanish. Teacher fidelity and
feasibility surveys demonstrated that teachers implemented the
intervention with high fidelity. Parents and teachers completed
demographic questionnaires. October 2021 | Volume 12 | Article 711578 Frontiers in Psychology | www.frontiersin.org 5 Cost-Effectiveness Analyses for Self-Regulation Intervention Li et al. the RLPL program, which include personnel cost and material
cost. Regarding personnel cost, we excluded teachers’ time spent
in receiving trainings (e.g., trainers’ time in Model 2) and their
time spent in delivering the RLPL games in classroom (e.g.,
teachers’ time in Models 2 and 3) during their regular work
hours from our cost estimation, because these teachers would
have to spend such time on work even without the RLPL program. For faculty, staff, and graduate students in our RLPL implementation
team, we calculated their personnel costs as the product of their
wages and their time spent in either training the teachers (e.g.,
RAs’ and faculty’s time in Models 2 and 3) or directly delivering
the intervention (e.g., RA’s cost in Model 1). The RLPL program
coordinator recorded the wages and time spent information for
faculty, staff, and graduate students in each model. In addition
to personnel cost, the intervention cost also includes $100 per
classroom materials kit to deliver the RLPL games in each
classroom. Classroom material kits included items currently found
in early childhood classrooms for use during the intervention
games (e.g., different colored circles cut out of construction paper,
classroom drum and musical instrument egg shaker set). After
estimating total program cost equal to the sum of total personnel
cost and total material cost, we divided total program cost by
the number of students to get the average intervention cost per
student for each model. as the cost of additional investment in our intervention to gain
one additional unit of outcome improvement. In addition to base-case analysis, we conducted a series of
sensitivity analyses to adjust for parameter uncertainty. RESULTS g
Model (3) program faculty trained teachers, who then
delivered the RLPL games to children (McClelland et al., 2019). Model (3) program faculty trained teachers, who then
delivered the RLPL games to children (McClelland et al., 2019). We extracted data from previous research to assess both cost
and effectiveness for each model. There were 16 classrooms
that received RLPL intervention under Model 1 (9 in the Tominey
and McClelland, 2011 study, and 7 in the Schmitt et al., 2015
study), and 10 classrooms in intervention under Model 3
(McClelland et al., 2019). In the “train-the-trainer” model, RAs
trained trainers, who then trained a total of 4 teachers to deliver
the RLPL games, so we calculated cost of Model 2 for 4 classrooms
(Table 1). As the typical class size for early childhood education
settings is 15–20 children (specific to programs included in
these studies), in our base-case analysis we standardize our
estimates as 18 children in each classroom. This standardization
is important to improve comparability across models, and can
better support stakeholders’ decisions on replicating and expanding
the RLPL programs. We then calculated the average intervention
cost per student. Aligned with the goal of the RLPL program
to improve children self-regulation, we used the effect size of
self-regulation scores to measure effectiveness. Our base-case cost-effectiveness results are shown in Table 2. Although the program effectiveness was similar across the RLPL
models, the cost varied from $7.72 per child in Model 2 to
$11.15 per child in Model 1. Our base-case analysis suggested
that Model 2 was the most cost-effective strategy with an
ICER of 22.71. Other RLPL models were dominated, as they
were associated with lower or same effectiveness at a higher
cost compared to Model 2. Table 3 shows the results under various scenarios in our
sensitivity analysis. Model 2 was consistently the most cost-effective
strategy across Scenarios 1 to 3, while Models 1 and 3 were
dominated. Specifically, the ICER of Model 2 decreased from 27.25
to 20.44 when we adjusted the number of children per classroom
from 15 to 20 (Scenario 1), increased from 21.44 to 23.99 when
we adjusted the variance of person-hours from 80 to 120% (Scenario
2), and increased from 22.71 to 27.25 when we adjusted the total
implementation cost from 100 to 120% (Scenario 3). f
Figure 1 illustrates our cost-effectiveness analytical model. RESULTS We evaluated cost-effectiveness among the three models of the
RLPL program and compared that to no intervention. To conduct
the cost-effectiveness analysis, we calculated the incremental
cost-effectiveness ratio (ICER) associated with the RLPL program. Therefore, we would interpret our cost-effectiveness findings Frontiers in Psychology | www.frontiersin.org McClelland et al. (2019) This
would not only test the robustness of our main findings, but
also provide critical evidence to inform decision makings on
adopting and expanding our interventions at various scales
and under different scenarios. Specifically, we conducted
sensitivity analyses under three scenarios: Scenario (1) In our base-case analysis, we assumed 18
children in each classroom. The typical class size for early
childhood education settings included in the present study is
15–20 children. We ran a sensitivity analysis to adjust the
number of children per classroom from 15 to 20 for each model. Scenario (2) We ran a sensitivity analysis to adjust the
potential variance in workload to implement the intervention,
by increasing and reducing person-hours by 20% for each model. Scenario (3) In our base-case analysis we did not include
facility cost, because such cost would still exist even without our
RLPL program. Research suggests this cost could range from 10
to 20% of total cost (Jones et al., 2019). We ran a sensitivity
analysis to adjust for facility cost, and we increased the total cost
by the upper limit of 20% for each model. This allowed us to
draw a more conservative cost-effectiveness results, and could
comprehensively adjust for various uncertainties of increasing cost.t Table 1 presents the parameters sources and values used
in our base-case analysis. According to previous research, the
RLPL program had been implemented according to three models: We used TreeAge Pro software (TreeAge Pro 2021, Healthcare
R2, 2020) to implement our analyses. We adjusted all monetary
values to 2019 dollars by consumer price index (Bureau of
Labor Statistics, 2020). Model (1) RAs directly delivered the RLPL games to children
(Tominey and McClelland, 2011; Schmitt et al., 2015); Model (2) RAs trained trainers, who then trained teachers
to deliver the RLPL games to children (Duncan et al., 2018); and. Model (2) RAs trained trainers, who then trained teachers
to deliver the RLPL games to children (Duncan et al., 2018); and. The Importance of Cost-Effectiveness
Studies in School Readiness Interventions The Importance of Cost-Effectiveness
Studies in School Readiness Interventions
In the search to support children’s school readiness and success,
a plethora of early childhood interventions have been developed
and tested (Blair and Raver, 2014; Sasser et al., 2017; Diamond
et al., 2019; Welsh et al., 2020). Although research supports
the effectiveness of these interventions, it is unclear if they
are cost-effective for early childhood programs to implement
and few programs have been evaluated to examine this. Cost-
effectiveness analyses can provide important evidence to support
decision makings of investing in interventions and optimizing
program designs. However, the literature on cost-effectiveness
analyses of early childhood self-regulation intervention has
been sparse. Corso and colleagues (Corso et al., 2015) conducted
cost-effectiveness
analyses
on
a
group-based
parenting
intervention aimed at improving behavioral outcomes among
children living in poverty. Their intervention was associated
with a cost of $178,000 per child and a cost of $91,100 per
child, both in 2008 dollar, for severe behavioral problems
avoided and for attention-deficit hyperactivity disorder avoided,
respectively. These can be converted into 2019 dollar at around
$211,000 and $108,000, which are substantially higher than DISCUSSION The present study conducted cost-effectiveness analyses on three
implementation models of the Red Light, Purple Light (RLPL)
intervention, a self-regulation intervention for young children. October 2021 | Volume 12 | Article 711578 6 Cost-Effectiveness Analyses for Self-Regulation Intervention Li et al. FIGURE 1 | Cost-effectiveness Analytical Model. TABLE 2 | Cost-effectiveness Results in Base-case Analysis. Cost-effectiveness
rankinga
Cost ($ per child)b
Incremental cost
Effectiveness (effect size
of self-regulation scores)
Incremental
effectiveness
Incremental cost-
effectiveness ratio
No intervention
0
–
0
−
Model 2
7.72
7.72
0.34
0.34
22.71
Model 3
7.85
0.12
0.31
−0.03
dominated
Model 1
11.15
3.43
0.34
0
dominated
aCost-effectiveness analysis order is from the lowest-cost strategy to the highest-cost strategy. Model 1 = trained research assistants (RAs) directly delivered the RLPL games to
children; Model 2 = trained RAs trained trainers, who then trained teachers to deliver the RLPL games to children; Model 3 = program faculty trained teachers, who then delivered the
RLPL games to children. bMonetary values were adjusted to 2019 dollars by consumer price index. FIGURE 1 | Cost-effectiveness Analytical Model. TABLE 2 | Cost-effectiveness Results in Base-case Analysis. Cost-effectiveness
rankinga
Cost ($ per child)b
Incremental cost
Effectiveness (effect size
of self-regulation scores)
Incremental
effectiveness
Incremental cost-
effectiveness ratio
No intervention
0
–
0
−
Model 2
7.72
7.72
0.34
0.34
22.71
Model 3
7.85
0.12
0.31
−0.03
dominated
Model 1
11.15
3.43
0.34
0
dominated
aCost-effectiveness analysis order is from the lowest-cost strategy to the highest-cost strategy. Model 1 = trained research assistants (RAs) directly delivered the RLPL games to
children; Model 2 = trained RAs trained trainers, who then trained teachers to deliver the RLPL games to children; Model 3 = program faculty trained teachers, who then delivered the
RLPL games to children. bMonetary values were adjusted to 2019 dollars by consumer price index. FIGURE 1 | Cost-effectiveness Analytical Model. FIGURE 1 | Cost-effectiveness Analytical Model. FIGURE 1 | Cost-effectiveness Analytical Model. FIGURE 1 | Cost-effectiveness Analytical Model. TABLE 2 | Cost-effectiveness Results in Base-case Analysis. Cost-effectiveness
rankinga
Cost ($ per child)b
Incremental cost
Effectiveness (effect size
of self-regulation scores)
Incremental
effectiveness
Incremental cost-
effectiveness ratio
No intervention
0
–
0
−
Model 2
7.72
7.72
0.34
0.34
22.71
Model 3
7.85
0.12
0.31
−0.03
dominated
Model 1
11.15
3.43
0.34
0
dominated
aCost-effectiveness analysis order is from the lowest-cost strategy to the highest-cost strategy. Frontiers in Psychology | www.frontiersin.org DISCUSSION Model 1 = trained research assistants (RAs) directly delivered the RLPL games to
children; Model 2 = trained RAs trained trainers, who then trained teachers to deliver the RLPL games to children; Model 3 = program faculty trained teachers, who then delivered the
RLPL games to children. bMonetary values were adjusted to 2019 dollars by consumer price index. aCost-effectiveness analysis order is from the lowest-cost strategy to the highest-cost strategy. Model 1 = trained research assistants (RAs) directly delivered the RLPL games to
children; Model 2 = trained RAs trained trainers, who then trained teachers to deliver the RLPL games to children; Model 3 = program faculty trained teachers, who then delivered the
RLPL games to children The three implementation models included: (Model 1) trained
RAs directly delivered the RLPL intervention to children; (Model 2)
RAs trained trainers (e.g., program coaches), who then trained
teachers to implement RLPL with children (e.g., train-the-trainer);
and (Model 3) program faculty trained teachers to deliver the
RLPL intervention to children. Results of base-case analysis
indicated that Model 2 was the most cost-effective strategy. We estimated that by implementing the “train-the-trainer” model,
a cost of $23 per child was associated with a one-unit increase
of the effect size of self-regulation scores. Our base-case finding
remains robust to a series of sensitivity analyses, when we adjusted
the number of students per classroom, the variance of person-
hours, and the total cost to implement the RLPL program. The
“train-the-trainer” model remained the optimal strategy across
these scenarios, with ICER ranging from 20 to 27. the developer to other trained individuals (research assistants
and faculty members with experience in early childhood) and
finally to teachers. The second and third models (RAs training
trainers who then trained teachers and program faculty training
teachers) were only possible in later iterations of the intervention,
but also reflected a feasible method of intervention delivery
and implementation in current early childhood contexts. Iterative Intervention Developmenth p
The present study was unique in several respects. Notably,
having access to data from randomized control trials where
the same curriculum was tested using different models of
implementation allowed for comparison of cost-effectiveness
across these models. Each model was implemented at a different
point in time and different stage of the iterative development
process for the RLPL intervention. Although the intervention
curriculum remained the same across implementation methods,
the methods of implementation changed over time. In the
first study, the intervention was delivered by the curriculum
developers. This model is critical as an initial step in testing
a new intervention, but not feasible to support long-term scaling
efforts. The models that followed shifted the intervention from October 2021 | Volume 12 | Article 711578 Frontiers in Psychology | www.frontiersin.org 7 Cost-Effectiveness Analyses for Self-Regulation Intervention Li et al. Li et al. TABLE 3 | Cost and Incremental Cost-effectiveness Ratio (ICER) of Model 2
Under Various Scenarios in Sensitivity Analysis.a
Scenario 1: Adjust number of children per classroom
Number of Children
per classroom
Cost per child ($)
ICER
15
9.27
27.25
16
8.69
25.55
17
8.18
24.05
18 (Base case)
7.72
22.71
19
7.32
21.52
20
6.95
20.44
Scenario 2: Adjust person-hours to implement interventions
Percentage change of
person-hours
Cost per child ($)
ICER
−20%
7.29
21.44
−10%
7.51
22.08
Base case
7.72
22.71
+10%
7.94
23.35
+20%
8.16
23.99
Scenario 3: Adjust total cost to implement interventions
Percentage change of
total cost
Cost per child ($)
ICER
Base case
7.72
22.71
+5%
8.11
23.85
+10%
8.49
24.98
+15%
8.88
26.12
+20%
9.27
27.25
aOnly shows results of Model 2 (the “train-the-trainer” model) compared to no
intervention in sensitivity analysis, because other models were dominated. TABLE 3 | Cost and Incremental Cost-effectiveness Ratio (ICER) of Model 2
Under Various Scenarios in Sensitivity Analysis.a studies to compare with, which we see as the major contribution
of our current study. In addition, we collected data from previous
interventions of our program and conducted a retrospective
cost-effectiveness analysis in this study. In so doing, we could
only look back at how the intervention was already implemented
in each model, and assess the cost and effectiveness that were
already in place. As a result, we encountered some uncertainty
and variance in parameters. To address this limitation,
we conducted a series of sensitivity analyses aiming to adjust
for various possibilities. Conclusion Our economic evaluation of RLPL fills an important gap related
to the value of investment in early childhood self-regulation
interventions. These results can serve as a model for future
cost-effectiveness analyses of early childhood intervention
programs and inform policymakers and stakeholders on the
cost-effectiveness for the potential impact of an intervention
delivered by different models, so that resource allocation and
future program design can be optimized. the ICER in our RLPL program. It is worth noting that as
the two programs focused on different outcomes, exact
comparisons between our study and the one conducted by
Corso et al. (2015) cannot be made, but it may still help to
illustrate the magnitude of cost-effectiveness, and to aid
stakeholders’ investment decisions across programs. In addition,
other research with a similar school readiness intervention
(e.g., Head Start REDI) has documented the importance of
such evaluations along with how initial costs may pay off
years later (Bierman et al., 2018).h Limitations Although this study was unique in many respects, we recognize
several limitations. First, there is a lack of comparable evidence
in the literature that we can use as a reference case to assess
our findings. This is mainly because cost-effectiveness analyses
on early-childhood self-regulation have been sparse. However,
this provides us with a unique opportunity to address this critical
knowledge gap, and sets up a model for future cost-effectives DATA AVAILABILITY STATEMENT The original contributions presented in the study are included
in the article/supplementary material, further inquiries can
be directed to the corresponding author. The present study extended work in this area by examining
cost-effectiveness for the different implementation versions of
the RLPL intervention. Results found that costs were low across
all implementation methods. In particular, the program could
be more cost-effective by using a “train-the-trainer” model,
due to its great potential of reaching economies of scale. Future
research needs to further investigate the “train-the-trainer”
model of implementation as this model is expanded. Iterative Intervention Developmenth Our sensitivity analyses also provide
evidence to support stakeholders’ decisions on replicating and
expanding our interventions under different scenarios and at
various scales. Although fidelity data were collected across each
study, each model had different levels of resources to monitor
and support fidelity of implementation over time and thus, these
analyses were not included as part of the present study. Future
studies should consider the relation between cost-effectiveness
and fidelity data. Finally, we propose that future studies should
analyze cost-effectiveness on early childhood interventions
prospectively as part of the development and implementation
process. This approach will empower researchers to evaluate
interventions alongside intervention implementation, so that cost
and effectiveness data will be better aligned with study designs
and have greater potential to inform practice and policy. ETHICS STATEMENT The studies involving human participants were reviewed and
approved by Oregon State University Institutional Review Board. Written informed consent to participate in this study was
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in New Zealand. AUTHOR CONTRIBUTIONS All authors contributed to the conceptualization and design
of the study, data analysis and results, and the writing of
the manuscript. October 2021 | Volume 12 | Article 711578 Frontiers in Psychology | www.frontiersin.org 8 Cost-Effectiveness Analyses for Self-Regulation Intervention Li et al. Li et al. The content is the responsibility of the authors and does not
necessarily represent the official views of the Institute of
Education Sciences, or the U.S. Department of Education. This
research was supported by grants from the Ford Family
Foundation and Oregon State University. FUNDING The research reported here was supported by the U.S. Department
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Pediatr. 172, 566–575. doi: 10.1001/jamapediatrics.2018.0232 Copyright © 2021 Li, McClelland, Tominey and Tracy. This is an open-access
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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. Purpura, D. J., Schmitt, S. A., and Ganley, C. M. (2017). Foundations of
mathematics and literacy: The role of executive functioning components. J. Exp. Child Psychol. 153, 15–34. doi: 10.1016/j.jecp.2016.08.010 Raver, C. C., Jones, S. M., Li-Grining, C., Zhai, F., Bub, K., and Pressler, E. (2011). CSRP’s impact on low-income preschoolers’ preacademic skills: self- October 2021 | Volume 12 | Article 711578 Frontiers in Psychology | www.frontiersin.org 10
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Brain food: rethinking food-borne toxocariasis Healy, S. R., Morgan, E. R., Prada, J. M., & Betson, M. (2021). Brain food: rethinking food-borne toxocariasis. Parasitology. Advance online publication. https://doi.org/10.1017/S0031182021001591 Queen's University Belfast - Research Portal:
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unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. py g
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ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the
Research Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk. Toxocariasis and food safety Food safety is a major public health issue worldwide, and it is vitally important that any risks
to consumers are managed both to protect the population and maintain consumer confidence. One such risk is the food-borne transmission of parasites to humans, which can have severe
health implications for the global population. The most recently published assessment of the
burden of food-borne parasites to humans estimated that 6.64 million disability-adjusted life
years were lost due to the consumption of contaminated food in 2010 (Torgerson et al., 2015). Human toxocariasis affects an estimated 1.4 billion people worldwide (Ma et al., 2020). The
disease is caused by Toxocara canis and T. cati, common roundworm parasites of canines
and felines, respectively. Toxocara vitulorum affects bovine species and is generally believed
to have less zoonotic significance, although knowledge of its transmission biology is currently
lacking (Dewair and Bessat, 2020). Human toxocariasis is recognized as one of the most commonly reported zoonotic hel-
minth diseases worldwide (Magnaval et al., 2001; Nicoletti, 2013), having a significant impact
on global public health (Zibaei and Sadjjadi, 2017). Toxocariasis can lead to a number of dif-
ferent clinical manifestations in humans, including allergic, ophthalmic and neurological dis-
orders (Ma et al., 2018). Recent epidemiological studies and meta-analyses on cognitive
impairment, psychosis and epilepsy have associated Toxocara infection with these debilitating
neurological diseases (Walsh and Haseeb, 2012; Luna et al., 2018; Taghipour et al., 2021), and
a potential link to the development of degenerative conditions such as Alzheimer’s disease has
been hypothesized (Fan, 2020). Despite recognition of its clinical impact, toxocariasis remains
a neglected disease and major gaps in our understanding of the epidemiology of this parasite
remain. Large numbers of Toxocara eggs are excreted in the feces of infected dogs, cats and foxes
(Morgan et al., 2013). Once present in the environment, these eggs can develop to the infective
stage and persist in the soil for long periods (Fan et al., 2013). If infective eggs are consumed
by accidental or paratenic host species, including humans, the larvae that hatch out in the gut
subsequently migrate to several different organs in the body, becoming encapsulated in the
tissues where larval development ceases. Studies have suggested it is primarily the host’s
immune response to Toxocara which is responsible for much of the pathology seen in cases
of toxocariasis, rather than mechanical damage caused by migrating larvae (Epe et al.,
1994). Toxocariasis and food safety In cases of ocular infection, the response to a single larva can lead to vision loss
(Neafie and Connor, 1976). © The Author(s), 2021. Published by
Cambridge University Press. This is an Open
Access article, distributed under the terms of
the Creative Commons Attribution licence
(http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted re-use, distribution
and reproduction, provided the original article
is properly cited. Whilst it is widely accepted that contaminated soil acts as a source of Toxocara eggs for
human infections with potentially severe clinical consequences, little is known about food-
borne transmission of this parasite. With eggs contaminating grazing pastures and growing
crops, the pathway to food contamination appears wide open. However, this transmission
route has received relatively little attention in the literature. Toxocara larvae are able to persist
in the tissues of paratenic hosts, thus infected meat can act as a source of infection to humans
(Strube et al., 2013; Wu and Bowman, 2020), particularly if consumed undercooked (Fan et al.,
2015). A recently published review by Bowman (2021) discusses vegetable contamination with
both Ascaris spp. and Toxocara spp. eggs (Bowman, 2021), but given that Toxocara, unlike Brain food: rethinking food-borne toxocariasis Parasitology Sara R. Healy1
, Eric R. Morgan2, Joaquin M. Prada1 and Martha Betson1 1Faculty of Health and Medical Sciences, School of Veterinary Medicine, University of Surrey, Guilford, UK and
2Faculty of Health, Medicine and Life Sciences, School of Biological Sciences, Queen’s University Belfast,
Belfast, UK Open Access
Thi
h Open Access
This research has been made openly available by Queen's academics and its Open Research team. We would love t
this research benefits you. – Share your feedback with us: http://go.qub.ac.uk/oa-feedback Download date:24. Oct. 2024 Abstract Cite this article: Healy SR, Morgan ER, Prada
JM, Betson M (2021). Brain food: rethinking
food-borne toxocariasis. Parasitology 1–9. https://doi.org/10.1017/S0031182021001591 Cite this article: Healy SR, Morgan ER, Prada
JM, Betson M (2021). Brain food: rethinking
food-borne toxocariasis. Parasitology 1–9. https://doi.org/10.1017/S0031182021001591 Human toxocariasis is a neglected tropical disease, which is actually global in distribution and
has a significant impact on global public health. The infection can lead to several serious con-
ditions in humans, including allergic, ophthalmic and neurological disorders such as epilepsy. It is caused by the common roundworm species Toxocara canis and Toxocara cati, with
humans becoming accidentally infected via the ingestion of eggs or larvae. Toxocara eggs
are deposited on the ground when infected dogs, cats and foxes defecate, with the eggs con-
taminating crops, grazing pastures, and subsequently food animals. However, transmission of
Toxocara to humans via food consumption has received relatively little attention in the litera-
ture. To establish the risks that contaminated food poses to the public, a renewed research
focus is required. This review discusses what is currently known about food-borne
Toxocara transmission, highlighting the gaps in our understanding that require further atten-
tion, and outlining some potential preventative strategies which could be employed to safe-
guard consumer health. Received: 7 June 2021
Revised: 6 August 2021
Accepted: 2 September 2021 Received: 7 June 2021
Revised: 6 August 2021
Accepted: 2 September 2021 Keywords: Food safety; human toxocariasis; neglected
tropical disease; public health; zoonotic
helminth Author for correspondence:
Sara R. Healy, E-mail: s.r.healy@surrey.ac.uk Author for correspondence:
Sara R. Healy, E-mail: s.r.healy@surrey.ac.uk Toxocariasis and food safety een's University Belfast, on 10 Nov 2021 at 15:35:45, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms /10.1017/S0031182021001591
m https://www.cambridge.org/core. Queen's University Belfast, on 10 Nov 2021 at 15:35:45, subject to the Cambridge Core terms of use, available at h . https://doi.org/10.1017/S0031182021001591
Downloaded from https://www.cambridge.org/core. Queen's University Belfast, on 10 Nov 2021 at 15:35:45, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/te Natural infections in food-producing animals In addition to experimental studies, there are a limited number of
reports where larvae have been detected in naturally infected ani-
mals. For example, T. cati has been detected in pig tissues
intended for human consumption (Davidson et al., 2012) and
both T. canis and T. cati have been isolated from naturally
infected chickens (Zibaei et al., 2017; Okada et al., 2021). Indirect evidence of Toxocara infection in the form of serological
data has also been published, with anti-Toxocara antibodies
detected in several food-producing animals on farm and at
slaughter. Lloyd (2006) found that up to 47% of sheep in a
Welsh study had anti-Toxocara antibodies present in their
blood, with levels directly proportional to animals’ age. This find-
ing was supported by a 2011 Brazilian study, with 52.9% of female
sheep between 11 and 15 months reported to have anti-Toxocara
antibodies, compared with 5% sero-prevalence in lambs aged 0–6
months (Santarém et al., 2011). Serological investigations have also
been undertaken for chickens, with 58.5% (Campos-da-Silva et al.,
2015) and 67.7% (Oliveira et al., 2018) of birds in Brazil confirmed
to have anti-Toxocara antibodies. Whilst circulating antibodies do
not prove the presence of infective larvae in the tissues, they con-
firm exposure of animals to Toxocara spp. and indicate the pres-
ence of this parasite in farm environments and common
exposure of food-producing animals, presumably by ingestion of
infective eggs. Human case studies Given that larval migration to the liver is a common finding in
the published literature, it is easy to see why so many of the
human cases reported have been attributed to the consumption
of this particular tissue. Moreover, experimental studies provide
evidence of larval migration to other tissues consumed by
humans, including the musculature. Thus, the consumption of
raw or undercooked meat products, in particular liver tissue, is
best avoided to reduce the risk of developing toxocariasis. The reported cases of human toxocariasis attributed to food con-
sumption identified in the literature are shown in Table 1. In all
cases, the food product was reported to have been consumed
raw or only partially cooked. Some had missing information
about the patients’ age or the species of animal or type of tissue
consumed. In all studies listed, the diagnosis of human toxocaria-
sis was made by patient serology. When reviewing published toxocariasis case reports, one must
remain open to the possibility that we are only seeing the tip of
the iceberg. Toxocariasis can have non-specific symptoms, which
may be attributed to other aetiologies and not investigated, leading
to under diagnosis (Magnaval et al., 2001; Carlin and Tyungu,
2020). In many of the published case reports, details of the patients’
history were lacking: either no exposure history was taken by the
clinician, the patient was not questioned on their dietary habits,
or enquiries were only made regarding contact with animals. Cases were more commonly reported in countries where consump-
tion of raw meat products is more widespread, an association noted
elsewhere in the literature (Morimatsu et al., 2006). Larval distribution and persistence in animal tissues Previous experimental studies have investigated the larval migra-
tion patterns for Toxocara spp. in various paratenic host species. In mice and rats, both T. canis and T. cati are initially detected in
the liver and lungs during the so-called ‘hepato-pulmonary’ phase
and are later found to undergo a ‘myotropic-neurotropic phase’,
migrating to the skeletal tissues and brain (Abo-Shehada et al.,
1984). In mice, Toxocara cati is reported to be less likely than
T. canis to infect brain tissue, and more likely to be found in
the musculature (Havasiová-Reiterová et al., 1995). Once present
in these tissues, larvae have been found to persist for up to one
year in mice (Bardón et al., 1994). In rabbits experimentally infected with T. canis and T. cati, lar-
vae could be recovered from the liver, lungs, kidneys and brain,
with those in the liver being detected alive over 7 months post-
infection; the musculature was not examined in this study
(Pankavich, 1966). In piglets fed embryonated T. canis eggs, larvae
were recoverable from the lungs and the brain, as well as in tissues
more commonly consumed by humans including the kidneys,
liver and musculature. Larvae collected from the livers 30 days
post-infection and subsequently fed to recipient mice were able
to induce infection, confirming larval viability (Sasmal et al.,
2008). However, in a separate study, significant decreases in larval
numbers were observed up to 3 weeks post-infection, suggesting
that larvae seem to be less able to persist for long periods in
the tissues of pigs (Helwigh et al., 1999). In lambs fed infective
T. canis eggs, larvae were detected in the liver and lungs but Sara R. Healy et al. 2 Ascaris, cannot be transmitted by humans and has the potential to
contaminate meat as well as vegetable produce, the epidemiology
of this parasite warrants individual attention. muscles were not examined (Aldawek et al., 2002). An earlier
study detected larvae in the muscles of lambs in addition to the
pancreas, heart, kidney and brain, while larvae appeared to
remain in the liver in year-old animals, not migrating to other tis-
sues but remaining alive for the duration of the 12-week study
(Schaeffler, 1960). Toxocara larvae within chicken tissues have
been found to migrate to the lungs, liver, brain and muscles,
with an ability to remain infective for prolonged periods of
time. Toxocara canis larvae were found predominantly in chicken
liver for up to 3.5 years (Tsvetaeva et al., 1979), whereas T. cati
larvae appear to favour migration through the liver and lungs to
the musculature, remaining infective to mice 176 days post-
infection (Taira et al., 2011). Published studies in cattle are scarce,
but a study reported the usual larval migration sites: liver, lungs,
kidney and brain, in calves experimentally infected with T. canis. Muscle tissue was not examined for larvae in this study
(Fitzgerald and Mansfield, 1970). To establish the public health risks posed by Toxocara in food,
a new research focus is now required. This review highlights the
key knowledge gaps in our understanding of food-borne toxocar-
iasis, assessing the evidence for this route of transmission by
means of published human cases, serological studies and recovery
of Toxocara spp. eggs and larvae from foods. The ability of cur-
rent food safety measures to prevent toxocariasis is addressed,
alongside discussion into future research avenues to determine
appropriate prevention strategies for this overlooked, but poten-
tially significant issue. . https://doi.org/10.1017/S0031182021001591
Downloaded from https://www.cambridge.org/core. Queen's University Belfast, on 10 Nov 2021 at 15:35:45, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms een's University Belfast, on 10 Nov 2021 at 15:35:45, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms Contamination of Toxocara eggs on farms Several published studies have investigated the contamination of
agricultural environments with Toxocara eggs. In Poland, 34.6%
of soil samples from conventional farms and 21.3% from organic
farms were positive for Toxocara spp. eggs (Klapec and Borecka,
2012). In the Philippines, the overall prevalence of Toxocara in
soil samples was much lower at 4%, with no statistically significant
difference reported between conventional and organic farms
(Paller and Babia-Abion, 2019). Eggs may be transferred in con-
taminated soil to vegetable crops destined for human consump-
tion. In their study, Klapec and Borecka (2012) analysed
various vegetable produce harvested with the surrounding soil, 3 Parasitology 3 Table 1. Reported food-borne human toxocariasis cases in the literature Country
Food type
No. of
patients
Patient age/s
(years)
Clinical presentation
Year
Reference
Meat products
Turkey
Unspecified raw meat
1
36
Unilateral neuroretinitis
2016
Karaca et al. (2018)
Japan
Chicken meat and bovine liver
1
60
Myelitis
2015
Hiramatsu et al. (2017)
Korea
Meat, liver, blood
5
30–60
Optic neuropathy
2014
Yang et al. (2014)
Korea
Bovine liver
1
46
Optic disc cyst
2013
Kim et al. (2013)
Korea
Cow meat, omasum, liver
1
51
OLM and VLM
2012
Park et al. (2012)
Korea
Bovine liver
1
35
Lung and liver abscesses, urticaria
2010
Kim et al. (2010)
Chile
Goat meat
1
51
Neurotoxocariasis and
hepatopathy
2010
Finsterer et al. (2010)
Korea
Ostrich liver
1
17
Meningitis, lung and liver
pathology
2010
Noh et al. (2012)
Japan
Deer meat
1
19
Myocarditis
2009
Enko et al. (2009)
Japan
Bovine liver
1
30
Pulmonary nodules
2008
Hisamatsu et al. (2008)
Japan
Bovine liver
3
58, 57, 27
Nodules in liver/lungs
2007
Yoshikawa et al. (2008)
Germany
Duck liver
1
55
Cerebral toxocariasis
2006
Hoffmeister et al. (2007)
Japan
Chicken liver
2
45, 71
Eosinophilia and pulmonary
infiltrates
2006
Morimatsu et al. (2006)
Japan
Meat (species unknown)
1
26
Myocarditis
2002
Abe et al. (2002)
Korea
Dog liver and kidney
1
43
Pulmonary disease and
eosinophilia
2002
Kim et al. (2002)
Japan
Bovine liver
1
26
Lung, liver and skin nodules
1999
Aragane et al. (1999)
USA
Ovine liver
1
63
Abdominal pain, cough, facial
weakness
1992
Salem and Schantz
(1992)
Japan
Equine, porcine, avian ‘meat’
+ livers
2
52, 63
Liver granuloma
1990
Ishibashi et al. (1992)
Japan
Chicken liver and gizzard
2
22
Urticaria and hepatic enzyme
elevations
1988
Nagakura et al. Contamination of Toxocara eggs on farms (1989)
Japan
Chicken and cow liver
3
39, 57, 46
VLM
1986
Ito (1986)
Blood products
Korea
Roe deer blood
1
58
Liver mass, cough, skin rash
2018
Park et al. (2018)
Gastropods and earthworms
Switzerland
Slugs
1
71
Neurological, ocular, pulmonary
disease
2014
Fellrath and Magnaval
(2014)
Italy
Snails
1
54
Encephalitis, neurological disease
2012
Caldera et al. (2013)
USA
Earthworm
1
16
Pulmonary nodules
2004
Cianferoni et al. (2006)
Spain
Snails
1
34
VLM, pulmonary disease
1991
Romeu et al. (1991)
Vegetables
Turkey
Salad
1
29
Peritonitis
2019
Arslan et al. (2019)
Romania
Raw vegetables
1
17
Eosinophilic ascites
2005
Chira et al. (2005)
Abbreviations: OLM = Ocular larva migrans, VLM = Visceral larva migrans. Abbreviations: OLM = Ocular larva migrans, VLM = Visceral larva migrans. Toxocara eggs can also reach people from soil or feces via
invertebrates. There are reported human cases arising from
gastropod ingestion, and one case from earthworm ingestion
(Table 1) (Romeu et al., 1991; Cianferoni et al., 2006; Fellrath
and Magnaval, 2014). Whilst this situation is probably extremely
rare, one must bear in mind that contamination of vegetables with
common invertebrates such as these does occur and could pose a
risk to consumers if produce is consumed unwashed. Cases of
zoonotic disease from accidental ingestion of molluscs infected
with nematodes such as Angiostrongylus cantonensis (Slom
et al., 2002) attest to this possibility, while the possible growth detecting eggs on 10.8% of the sampled produce from organic
farms,
and
19.2%
from
conventional
farms
(Klapec
and
Borecka, 2012). An Iranian study found that overall, 3.97% of
sampled vegetables were contaminated with Toxocara eggs
(Fallah et al., 2016), slightly higher than the 1.68% prevalence
reported in another study from the same country (Rostami
et al., 2016) and the 1.5% prevalence reported in Turkey
(Kozan et al., 2005). It is difficult to compare the results of
these studies due to variations in sampling strategies and labora-
tory techniques, but these studies confirm the link between
Toxocara eggs in the soil and contamination of vegetable produce. detecting eggs on 10.8% of the sampled produce from organic
farms,
and
19.2%
from
conventional
farms
(Klapec
and
Borecka, 2012). een's University Belfast, on 10 Nov 2021 at 15:35:45, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms . https://doi.org/10.1017/S0031182021001591
Downloaded from https://www.cambridge.org/core. Queen's University Belfast, on 10 Nov 2021 at 15:35:45, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms Toxocara spp. larvae in meat tissues and animal serology In order to recover Toxocara larvae from meat products, the tissue
is usually subjected to chemical digestion treatment using an
HCl-pepsin solution with simultaneous incubation and mechan-
ical stirring, followed by sedimentation, filtration and identifica-
tion of larvae by microscopy. Subsequent molecular analysis of
the larvae obtained using PCR-based techniques is commonly
undertaken following isolation (Zibaei et al., 2017; Okada et al.,
2021). p
An Italian study which assessed the level of Toxocara eggs in
the soil of farms in the Marche region found that out of 60
farms sampled, around 50% had soil that tested positive for
Toxocara. In addition, the proportion of positive farms was
almost twice as high in the group which had more than three
dogs living on-site, compared to farms that had three dogs or
fewer (Habluetzel et al., 2003). Although scarce, other studies
have also found Toxocara spp. on livestock farms. These include
a 2002 study, which assessed a large-scale poultry unit and its sur-
rounding environment in Poland, detecting Toxocara spp. eggs in
soil within close proximity to the farm (Trawinska et al., 2002),
and a study in 2003 which assessed the environmental microbial
composition of two pig farms in Poland, detecting Toxocara
spp. eggs in the soil of both units sampled (Szostak and
Bekier-Jaworska, 2003). For the purpose of identifying animals with circulating
anti-Toxocara antibodies in their bloodstream, studies have uti-
lized enzyme-linked immunosorbent assay (ELISA) based detec-
tion using the excretory-secretory (TES) antigens of T. canis
larvae. To reduce cross-reactivity with Ascaris spp. and improve
test specificity, serum samples are typically pre-adsorbed with
the extract of an adult Ascaris worm prior to ELISA testing
(Santarém et al., 2011; Rassier et al., 2013; Campos-da-Silva
et al., 2015). No attempt is usually made to differentiate between
T. canis and T. cati infections in these assays, and cross-reactivity
between these species is likely (Santarém et al., 2011). Whilst the presence of circulating anti-Toxocara antibodies
does not definitively diagnose an active Toxocara infection, it is
suggestive of exposure to this parasite. In contrast, identification
of Toxocara spp. eggs in the feces of infected cats, dogs and
foxes is more suggestive of a patent infection, with adult worms
residing within the intestine of these definitive hosts (Fan,
2020). The risk of a false-positive diagnosis due to the ingestion
of faeces containing Toxocara spp. Control measures to minimize transmission risk The potential flow of Toxocara spp. from the farm to the con-
sumer, and a summary of the possible control measures at each
step of the production chain are summarized in Fig. 1. While
many food systems have steps in place to protect consumers
from other pathogens, their effectiveness against Toxocara spp. has not been systematically tested. Toxocara spp. larvae in meat tissues and animal serology eggs can be reduced with the
addition of ELISA-based coproantigen detection (Elsemore,
2020), or repeat sampling following the gut transit time. There are other studies in the literature which have focused on
determining the prevalence of Toxocara spp. in definitive hosts
that could access agricultural land. For example, some authors
have investigated Toxocara prevalence in free-roaming farm cats
by faecal analysis: T. cati was detected in 91% of sampled cats
in the UK (Yamaguchi et al., 1996), whereas an earlier study
obtained positive results in 63% of cats (Gethings et al., 1987). In farm dogs, a Portuguese study sampling canine feces on 165
small-ruminant farms detected a Toxocara prevalence of 8%
using microscopic evaluation (Cardoso et al., 2014), and studies
assessing T. canis infection in foxes found prevalence levels of
47.4% in Zurich, Switzerland (Hofer et al., 2000); and 55.9% in
the UK (Richards et al., 1995). It is always difficult to compare
the results of studies such as this directly, because of variations Farm biosecurity Toxocara spp. eggs are deposited in the soil via the feces of
infected dogs, cats and foxes, and once present in the soil can
remain infective for years due to their resistance to environmental
conditions (Parsons, 1987). Because organic fertilizer does not
typically incorporate the feces of these definitive hosts, its agricul-
tural use should not usually pose a risk of contaminating the
environment with the eggs of this parasite. No practical methods
exist to remove Toxocara eggs from the environment (Overgaauw
and van Knapen, 2013); therefore, controlling access of these
definitive hosts to agricultural land is a logical first intervention
point to explore for both meat and vegetable production
processes. Toxocara eggs in soil and vegetable produce Toxocara eggs in soil and vegetable produce Most of the published methods for the recovery of Toxocara sp. eggs from soil and vegetables are based on conventional techni-
ques, involving steps such as washing, sieving, sedimentation, fil-
tration and flotation prior to microscopic examination. In some
cases, egg viability determination is also undertaken, usually by
means of dye uptake differentiation (Dabrowska et al., 2014). Sample sizes vary widely between studies. In the case of soil, from
3 g up to 200 g of dried sample has been used, which is typically
extracted up to 5 cm from the soil surface (Paller and Babia-
Abion, 2019; Tyungu et al., 2020). For vegetable testing, preferred
weights of samples usually range from 100 to 250 g, with the pro-
cessing of sedimented washing solution and subsequent residue
concentration commonly undertaken prior to parasitological ana-
lysis via microscopy (Abougrain et al., 2010; Fallah et al., 2016;
Hajipour et al., 2021). Some studies have additionally utilized
PCR-based molecular assays, to differentiate between T. canis and
T. cati eggs in soil samples (Choobineh et al., 2019; Tyungu et al.,
2020) and vegetable produce (Guggisberg et al., 2020). Control measures to minimize transmission risk Contamination of Toxocara eggs on farms An Iranian study found that overall, 3.97% of
sampled vegetables were contaminated with Toxocara eggs
(Fallah et al., 2016), slightly higher than the 1.68% prevalence
reported in another study from the same country (Rostami
et al., 2016) and the 1.5% prevalence reported in Turkey
(Kozan et al., 2005). It is difficult to compare the results of
these studies due to variations in sampling strategies and labora-
tory techniques, but these studies confirm the link between
Toxocara eggs in the soil and contamination of vegetable produce. 1591
ge.org/core. Queen's University Belfast, on 10 Nov 2021 at 15:35:45, subject to the Cambridge Core terms of use, available at https://www.cambridge.o Sara R. Healy et al. 4 Sara R. Healy et al. of insects as human food in future (Babarinde et al., 2020;
Hawkey et al., 2021) demands consideration of their potential
as sources of zoonoses, including Toxocara. Insects are known
to transfer taeniid cestode eggs from feces to food (Benelli
et al., 2021), although this route has not yet been demonstrated
for Toxocara spp. is the case for animal serological testing, ELISA-based tests are
utilized to detect TES antibodies in the blood of human patients. As a confirmatory measure, following a positive ELISA result,
Western blotting techniques are recommended which improve
the sensitivity and specificity of diagnosis (Mazur-Melewska
et al., 2020). Research to develop improved serodiagnostic assays
for Toxocara sp. is ongoing. The use of recombinant antigens and
the detection of antibody subclasses are approaches currently
being explored for their potential to improve the reliability of test-
ing for toxocariasis in humans in the future (Nicoletti, 2020). Improvements in the diagnosis of Toxocara spp. infections in
humans would help to elucidate dietary risk factors by enabling
larger and more precise epidemiological studies. een's University Belfast, on 10 Nov 2021 at 15:35:45, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms . https://doi.org/10.1017/S0031182021001591
Downloaded from https://www.cambridge.org/core. Queen's University Belfast, on 10 Nov 2021 at 15:35:45, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms .org/10.1017/S0031182021001591
d from https://www.cambridge.org/core. Queen's University Belfast, on 10 Nov 2021 at 15:35:45, subject to the Cambridge Core terms of use, availab Meat inspection at slaughter In terms of reducing the risk of parasitic transmission to humans,
meat inspection is currently the principal means of controlling
Trichinella spp. and Taenia spp. infections in the food chain
(Dorny et al., 2009). At this time, the presence of Toxocara spp. larvae in the tissues of food animals is not assessed at slaughter
in any jurisdiction. Indeed, Toxocara larvae are frequently seen
as
a
‘contaminant’
when
isolated
during
routine
porcine
Trichinella testing by artificial tissue digestion (Marucci et al.,
2013), and their presence is often not reported. Some recent stud-
ies have advocated the use of molecular methods to improve the
detection of helminth contamination of meat and offal (Nguyen
et al., 2017; Wang et al., 2018; Karadjian et al., 2020). This is
an area which has the potential to greatly improve the detection
of Toxocara during the meat inspection process and warrants fur-
ther investigation. Regular treatment of farm cats and dogs with appropriate
anthelmintic drugs in accordance with guidelines such as those
of the European Counsel for Companion Animal Parasites
(ESCCAP) is one approach to reducing the contamination of
the agricultural environment with Toxocara eggs, particularly
for kittens, pups and nursing queens and bitches (Overgaauw
and van Knapen, 2013). A 2016 study by Nijsse reported that
fewer than a quarter of cat owners questioned were treating ani-
mals with an anthelmintic at the recommended frequency, with
a higher risk of Toxocara shedding in free roaming cats (Nijsse
et al., 2016). Treating owned animals is just one part of the pic-
ture. A meta-analysis showed the prevalence of Toxocara in
stray cats, working and rural dogs to be significantly higher
than that of pet cats and dogs (Rostami et al., 2020), and parasites
in foxes should also be considered (Deplazes et al., 2004). Diagnosing human toxocariasis In the case of human Toxocara infections, diagnosis is most com-
monly based on the results of clinical and serological findings. As 5 Parasitology Fig. 1. The potential flow of Toxocara spp. from the farm to the consumer, and a summary of the possible control measures at each step of the production chain. The potential flow of Toxocara spp. from the farm to the consumer, and (Defra, 2021). Farm size could impact the feasibility and eco-
nomic viability of biosecurity measures such as fencing. in sampling techniques and laboratory methods. In these studies,
the soil was not analysed, but infected animals potentially had
access to farmland, in particular land utilized for arable produc-
tion or livestock grazing. In the previously referenced study
reporting a 47% sero-prevalence of T. canis in Welsh sheep, all
participating farms had dogs resident on-site, and foxes were
also present (Lloyd, 2006). In addition, stray cats of unknown
health status were present on farms from which T. cati larvae
were isolated from pig meat destined for human consumption
in Norway (Davidson et al., 2012), and chicken meat from a com-
mercial unit in Japan (Okada et al., 2021). As well as defecating
onto agricultural land, there is the potential for definitive hosts,
mainly cats, to contaminate animal feed stores. This route of
infection has been seen in cases of toxoplasmosis in pigs, a para-
sitic infection which shares several features with toxocariasis
including a common definitive host (Li et al., 2010). . https://doi.org/10.1017/S0031182021001591
Downloaded from https://www.cambridge.org/core. Queen's University Belfast, on 10 Nov 2021 at 15:35:45, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms een's University Belfast, on 10 Nov 2021 at 15:35:45, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms Chilling and freezing of foods Refrigeration is used throughout the food chain, from the produc-
tion stage through to the consumer, to maximize food quality and
safety (Tassou et al., 2010). However, there are very few published
studies assessing the effect of chilling and freezing on the viability
of Toxocara spp. eggs and larvae, particularly in food products. A study by Taira et al. (2011) demonstrated that the infectivity
of T. cati larvae within chicken muscle tissue reduced substantially
following chilling at 4°C for 14 and 28 days, but larvae were still
viable and induced infections in recipient mice (Taira et al., 2011). In a similar study, chilling pig and poultry tissues at 4°C for 7 days
had a significant effect on larval infectivity, but again infections
could still be established in recipient pigs (Taira et al., 2004). Chilling infected mouse liver samples to between 0 and 4°C for
10 days was found to significantly reduce the intensity of infection
in recipient mice (Dutra et al., 2013). These studies suggest the
potential risk of Toxocara transmission by consumption of meat
products is decreased by refrigeration, but the risk of human
infection remains. Publications outlining the effect of the cooking process on
Toxocara spp. larvae in animal tissues and eggs on vegetable pro-
duce are currently lacking. A 2007 study assessed the infectivity of
T. canis larvae present in mouse liver following cooking for 5 min
in a household microwave, with the internal temperature of the tis-
sue reaching >70°C. Transmission of Toxocara to recipient mice
only occurred if infected liver was consumed raw (Cetinkaya
et al., 2007). To assess this control step further, it would be neces-
sary to determine precise thermal death curves (time vs tempera-
ture) for Toxocara spp. tissue larvae. This would be especially
useful for meat more commonly served ‘rare’, such as beef, and to
assess the risk of cooking methods more commonly associated
with under-cooking, such as barbequing. Freezing of meat tissues appears to have a more significant
effect on the viability of Toxocara larvae compared to refriger-
ation. Storing infected muscle tissue at −21°C for 12–48 h was
found to have a dramatic impact on larval viability, with no sub-
sequent infections detected in recipient mice. Larval motility was
reported as absent after 24 h freezing at −25°C, suggesting that the
thermal death point for the larvae had been reached (Taira et al.,
2011). Washing vegetable produce The contamination of vegetable produce at source and human
consumption without washing is a potential transmission route
for several parasites, including Toxocara spp. (Slifko et al., 2000;
Mosayebi et al., 2014). Several published studies have assessed
the degree of contamination of vegetable produce with the eggs
of Toxocara spp. worldwide, with leafy vegetables such as salad
leaves seemingly more susceptible compared to other vegetable
types. For example, in Iran, Hajipour et al. (2021) reported 40.2
and 33.0% of lettuce samples tested to contain the eggs of
T. cati and T. canis respectively, compared with radishes from
which only T. cati was isolated in 6.1% of samples (Hajipour
et al., 2021). In Libya, T. canis and T. cati eggs were detected in
37 and 48% of lettuces compared to 14 and 8% of cucumbers,
respectively (Abougrain et al., 2010). The higher levels of contam-
ination reported on leafy vegetables could be due to an increased
surface area in contact with the contaminated soil surface (Gupta In a recent study, Hajipour et al. (2021) assessed the impact of
fencing-off farmland on the contamination of vegetables with T. canis and T. cati in Iran and found unfenced cropping areas
had a vegetable contamination rate of 55.3% compared to 9.2%
in fenced areas (Hajipour et al., 2021). Whilst this control step
seems logical, the size of the farms is important to consider: in
this study, the largest site was 20 hectares, considerably smaller
than the average farm size of 87 hectares in England, UK 6 Sara R. Healy et al. Sara R. Healy et al. delayed once returned to higher temperatures (Azam et al.,
2012). Whilst consumers are unlikely to routinely store vegetables
for this length of time in domestic refrigerators, storing vegetable
produce at low temperatures could postpone the development of
eggs such that any consumed are less likely to be fully larvated
and thus infective. Freezing eggs was found to reduce the viability
of T. canis and T. cati, with longer treatment times correlating
with reduced viability (O’Lorcain, 1995). As is the case for meat
products, the process of freezing vegetable produce can lead to
structural changes and a reduction in product quality, with
some types of vegetable more negatively impacted by freezing
than others (Jeremiah, 2019). et al., 2009; Maikai et al., 2012). Studies have assessed the effect-
iveness of washing different vegetables in reducing parasitic con-
tamination. Avcioglu et al. Washing vegetable produce (2011) did not detect any helminth
eggs in vegetable samples following a washing step with clean
water alone (Avcioglu et al., 2011), a finding supported by other
studies in the literature (Fallah et al., 2012; Rostami et al.,
2016). Alternative washing methods for the removal of parasitic
species from vegetables have been investigated, with 0.95% cal-
cium hypochlorite solution found to be more effective at reducing
parasitic contamination than 1% lemon juice, 1% vinegar or a
diluted dishwashing detergent (Hajipour et al., 2021). However,
consumer safety and public acceptance of chemical food treat-
ments need to be considered. In some countries, for example,
the use of chlorine-based disinfectants for food produce is highly
restricted or not authorized (De Corato, 2020). Chilling and freezing of foods This finding was supported by Dutra et al. (2013), with a
100% reduction in larval viability following freezing of mouse
liver tissue at −20°C, with larvae showing detrimental morpho-
logical changes including ruptured cuticles and internal organ
degeneration (Dutra et al., 2013). In contrast, Sprent (1953)
reported finding motile larvae in mice carcasses subjected to
−20°C for 4 weeks, although the viability of these larvae was
not assessed (Sprent, 1953). Freezing meat products is a promis-
ing potential control point to investigate, and is utilized for some
other food-borne parasites such as Trichinella spiralis (Noeckler
et al., 2019). This would be especially important for meat
intended to be served in raw dishes, such as steak tartare. However, effects on food quality and palatability brought about
by the freezing process need to be considered if this control meas-
ure is implemented during processing (Zhang et al., 2019). Cooking foods Heat treatment remains one of the most reliable methods to
inactivate food contaminated with parasites, such as Taenia
spp., Toxoplasma gondii and some Trichinella spp., provided
the internal temperature is high enough (Franssen et al., 2019). For example, in the case of Taenia solium infected pork, cooking
for 10 min at a core temperature of 80°C, 20 min at 70°C or 30
min at 60°C has been found to kill cysticerci, but at core tempera-
tures of 40°C, metacestodes were still viable after up to 1 h of
cooking. This raises some concerns for faster cooking methods,
such as deep frying (Møller et al., 2020). In comparison, T. gondii
and T. spiralis appear to be more sensitive to heat treatment, with
T. gondii tissue cysts killed following exposure to temperatures
above 56°C for 10 min (Dubey et al., 1990), and a 15 min cooking
time at 55.6°C sufficient to inactivate T. spiralis larvae in pork
(Noeckler et al., 2019). . https://doi.org/10.1017/S0031182021001591
Downloaded from https://www.cambridge.org/core. Queen's University Belfast, on 10 Nov 2021 at 15:35:45, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms een's University Belfast, on 10 Nov 2021 at 15:35:45, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms Conclusions and future directions Human toxocariasis is a neglected disease which has a significant
negative impact on global public health. It has been known for
some time that people can acquire Toxocara by ingesting larvated
eggs from the environment, but there is also mounting evidence
for food-borne transmission of this parasite. Seropositivity and
viable larvae in food animals and Toxocara eggs on vegetable pro-
duce have been demonstrated and are of great concern, especially
as current food safety measures do not specifically take this para-
site into consideration. However, current knowledge of the true
importance of food consumption in the epidemiology of toxocar-
iasis is lacking. Further research is now required to quantify the
flow of Toxocara from the environment to the final food product
and evaluate the potential impact of any control interventions. A
multifaceted approach will be required to address the different
steps in the food production pathway, with molecular techniques
opening the doors to more sensitive detection methods for
Toxocara species to support both research and food safety. A
renewed research focus is urgently required to fill in the key
gaps, inform food safety policy and, ultimately, protect consumer
health. The environmental resistance of Toxocara spp. eggs is widely
reported (Mizgajska, 2001; Despommier, 2003), with studies dem-
onstrating the tolerance and development of eggs at a range of
ambient temperatures (Azam et al., 2012). A study by Azam
et al. (2012) assessed the impact of low temperatures on
Toxocara eggs. Toxocara canis eggs were stored at +1 and −2°C
for a 6-week period, during which time they did not embryonate
to become infective, and their subsequent development was Parasitology 7 Financial support. Sara Healy is funded by UK Research and Innovation,
Biotechnology and Biological Sciences Research Council, through the
FoodBioSystems Doctoral Training Programme. Dabrowska J, Zdybel J, Karamon J, Kochanowski M, Stojecki K, Cencek T
and Klapec T (2014) Assessment of viability of the nematode eggs
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English
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MicroRNA-137 inhibits BMP7 to enhance the epithelial-mesenchymal transition of breast cancer cells
|
Oncotarget
| 2,017
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cc-by
| 5,408
|
ABSTRACT Bone morphogenetic protein-7 (BMP7) is known to antagonize transforming
growth factor β 1 (TGFβ1)-mediated fibrosis through suppressing epithelial-
mesenchymal transition (EMT). We recently reported that BMP7 also antagonizes the
effects of TGFβ1 in breast cancer (BC) tumorigenesis-related EMT. Nevertheless, the
control of BMP7 expression in BC remains ill-defined. Here, we detected significantly
lower levels of BMP7 and significantly higher levels of microRNA-137 (miR-137) in the
BC specimens, relative to paired adjacent non-tumor breast tissue. BMP7 and miR-137
levels were correlated inversely. Additionally, the high miR-137 levels in BC specimens
were correlated with reduced patient survival. In vitro, overexpression of miR-137
significantly increased cell EMT and invasion, while depletion of miR-137 significantly
decreased cell EMT and invasion in BC cells. The increases in BC cell invasiveness by
miR-137 appeared to result from its suppression of BMP7, through direct binding of
miR-137 to the 3'-UTR of BMP7 mRNA, thereby blocking its protein translation in BC
cells. This study sheds light on miR-137 as a crucial factor that enhances BC cell EMT
and invasiveness, and points to miR-137 as a promising innovative therapeutic target
for BC treatment. MicroRNA-137 inhibits BMP7 to enhance the epithelial-
mesenchymal transition of breast cancer cells Xuexiang Ying1, Yunpo Sun1, Pingqing He1
1Department of General Surgery, Shanghai Jiaotong University Affiliated Sixth People's Hospital, 200233, China
Correspondence to: Pingqing He, email: hpq345@yeah.net
Keywords: breast cancer (BC), epithelial-mesenchymal transition (EMT), miR-137, bone morphogenetic protein-7 (BMP7)
Received: December 06, 2016 Accepted: January 11, 2017 Published: February 17, 2017 RESULTS BMP7 (Figure 2A-2B). Next, we transfected MCF7 cells
with miR-137 (Figure 2C), and transfected BT474 cells
with antisense for miR-137 (as-miR-137) (Figure 2D). The cells transfected with a null sequence were used as
a control (null). The levels of miR-137 in these modified
BC cells were assayed by RT-qPCR. The alterations of
miR-137 levels in these cells were confirmed (Figure
2C-2D). These miR-137-modified BC cells were used
to examine the functional binding of miR-137 to BMP7
mRNA predicted by bioinformatics algorithms (Figure
2E, Table 2). The intact 3'-UTR of BMP7 mRNA (BMP7
3'-UTR) and a 3'-UTR with mutant at miR-137-binding
site of BMP7 mRNA (BMP7 3'-UTR mut) were prepared
and then cloned into luciferase reporter plasmids. First,
BT474 cells were co-transfected with 1μg as-miR-137/
null plasmids and 1μg BMP7 3'-UTR or BMP7 3'-UTR
mut plasmids (Figure 2F). Next, MCF7 cells were co-
transfected with 1μg miR-137/null plasmids and 1μg
BMP7 3'-UTR or BMP7 3'-UTR mut plasmids (Figure
2G). The results show that miR-137 specifically targets
the 3’-UTR of BMP7 mRNA to inhibit its translation in
BC cells. BMP7 (Figure 2A-2B). Next, we transfected MCF7 cells
with miR-137 (Figure 2C), and transfected BT474 cells
with antisense for miR-137 (as-miR-137) (Figure 2D). The cells transfected with a null sequence were used as
a control (null). The levels of miR-137 in these modified
BC cells were assayed by RT-qPCR. The alterations of
miR-137 levels in these cells were confirmed (Figure
2C-2D). These miR-137-modified BC cells were used
to examine the functional binding of miR-137 to BMP7
mRNA predicted by bioinformatics algorithms (Figure
2E, Table 2). The intact 3'-UTR of BMP7 mRNA (BMP7
3'-UTR) and a 3'-UTR with mutant at miR-137-binding
site of BMP7 mRNA (BMP7 3'-UTR mut) were prepared
and then cloned into luciferase reporter plasmids. First,
BT474 cells were co-transfected with 1μg as-miR-137/
null plasmids and 1μg BMP7 3'-UTR or BMP7 3'-UTR
mut plasmids (Figure 2F). Next, MCF7 cells were co-
transfected with 1μg miR-137/null plasmids and 1μg
BMP7 3'-UTR or BMP7 3'-UTR mut plasmids (Figure
2G). The results show that miR-137 specifically targets
the 3’-UTR of BMP7 mRNA to inhibit its translation in
BC cells. MiR-137 decreases BMP7 protein but not mRNA
in BC cells The effects of miR-137 on BMP7 were then
examined in BC cells. The BMP7 mRNA did not alter
(Figure 3A), but the BMP7 protein was significantly
decreased in miR-137-overexpressing MCF7 cells (Figure
3B). Moreover, the BMP7 mRNA did not alter (Figure
3C), but the BMP7 protein was significantly increased in
miR-137-depleted BT474 cells (Figure 3D). Association of high BC miR-137 levels with poor
patient prognosis The levels of BMP7 and miR-137 in 40 pairs of
resected BC tissues (Stage IV) and adjacent non-tumor
breast tissues (NT) were measured by Western blot
and RT-qPCR, respectively (Table 1). BC specimens
contained significantly lower levels of BMP7 (Figure
1A), and significantly higher levels of miR-137 (Figure
1B). We then performed a correlation test using these 40
BC specimens, and detected a strong inverse correlation
between BMP7 and miR-137 (Figure 1C, ɤ=-0.72,
p<0.0001, N=40), indicating a possible regulatory
relationship between miR-137 and BMP7 in BC. These
patients were followed up for 60 months to assess overall
survival. The relationship of miR-137 or BMP7 levels and
clinicopathological characteristics was evaluated using
multivariate Cox regression analysis, showing that both
were significantly associated with survival of the BC
patients (Table 2). Next, the median value for miR-137 in
these patients was used as the cutoff point for separating
miR-137-high cases (n=20) from miR-137-low cases
(n=20). Kaplan-Meier curves showed that patients with
high miR-137 levels in BC tissue had a significantly lower
5-year survival than those with low miR-137 levels in BC
tissue (Figure 1D). These data suggest that high miR-137
levels in BC specimens may be associated with reduced
patient survival. INTRODUCTION but significantly inhibited activation of EMT-related
genes by TGFβ1 in BC cells, thereby reducing TGFβ1-
mediated cell growth and metastasis [16]. However, the
regulation of BMP7 in BC remains unclear. Breast cancer (BC) is a common malignant tumor
in women worldwide [1]. The transforming growth
factor β (TGFβ) superfamily receptor signaling pathway
plays a key role in the tumorigenesis of BC [2–5], in
which action of TGFβ receptor signaling by its ligand
TGFβ1 promotes a biological process called Epithelial-
Mesenchymal Transition, which cancer cells use to favor
an invasive and metastatic phenotype (EMT) [6–9]. In
this process, cancer cells begin to secrete proteinases
in order to traverse collagenous extracellular matrix
proteins. Bone morphogenetic protein-7 (BMP7)
is a well-described matrix proteinase that breaks
down collagen type IV, a constituent of the basement
membrane. Down-regulation of BMP7 facilitates the
metastatic spread of BC cells [10–15]. TGFβ1 and
BMP7 are two central members in the TGFβ superfamily
that each have different effects on EMT regulation. We
recently reported that BMP7 does not modify TGFβ1-
stimulated phosphorylation of the TGFβ receptor, MicroRNAs (miRNAs) are non-coding small
RNAs that regulate gene expression at a post-
transcriptional level, through specific binding to the
3′-untranslated region (3′-UTR) of target mRNA [17–
19]. Specifically, miRNAs have been shown to play an
important role in the tumorigenesis of BC [20–28], and
in the control of BMP7 activation [29–32]. However,
previous studies on miR-137 never addressed BMP7 as
a potential target [33–37]. Here, we studied the expression of BMP7 and
miR-137 in BC tissues, and investigated the association
of miR-137 levels with patient prognosis. We used
bioinformatics analyses to elucidate the interaction
between miR-137 and BMP7. We then overexpressed
miR-137 or inhibited miR-137 in 2 established BC
cell lines in vitro, and examined their effects on BMP7
activation and BC cell invasion. www.impactjournals.com/oncotarget Oncotarget 18348 Table 1: Clinical-pathological characteristics (total) Table 1: Clinical-pathological characteristics (total) Table 1: Clinical-pathological characteristics (total)
Patients (n; %)
p
BC tissue/ Normal tumor-adjacent
tissue
40 (100%) /40 (100%)
Age (<60/≥60 years old)
12 (30%) /28 (70%)
0.62
Gender (male/female)
0 (0%) /40 (100%)
Tumor site (breast)
40 (100%)
Tumor grade (well or moderate/poor)
0 (0%) /16 (40%) /24 (60%)
0.008
Tumor stage (I/II/III/IV)
0 (0%) /0 (0%) /20 (50%) /20 (50%)
0.005
Lymph node metastasis (no/yes)
0 (0%) /40 (100%)
0.003
Distal metastasis at diagnosis (no/yes)
40 (0%) /0 (0%)
0.003 MiR-137 inhibits BMP7 protein translation in
BC cells Next, we examined the levels of miR-137 and
BMP7 levels in diffirent BC cell lines. We found that
BT474 expressed relatively high level of miR-137 and
relatively low level of BMP7, while MCF7 expressed
relatively low level of miR-137 and relatively high level of www.impactjournals.com/oncotarget MiR-137 reduces BC cell EMT and invasion The effects of miR-137 modification on the EMT
and invasion of cultured BC cells were then investigated. We found that miR-137 overexpression in MCF7 cells www.impactjournals.com/oncotarget Oncotarget 18349 Table 2: Analysis of the prognostic values of miR-137 and BMP7 in BC patients by Cox regression model
HR
95% Cl
P value
miR-137 (high vs low)
5.11
3.13-9.92
0.005
BMP7 (low vs high)
4.42
2.21-7.97
0.004 Table 2: Analysis of the prognostic values of miR-137 and BMP7 in BC patients by Cox regression model
HR
95% Cl
P value
miR-137 (high vs low)
5.11
3.13-9.92
0.005
BMP7 (low vs high)
4.42
2.21-7.97
0.004 e prognostic values of miR-137 and BMP7 in BC patients by Cox regression model HR
95% Cl
P value
miR-137 (high vs low)
5.11
3.13-9.92
0.005
BMP7 (low vs high)
4.42
2.21-7.97
0.004 tj
l
/
t
t
ure 1: High miR-137 levels in BC specimens is associated with poor prognosis. A-C. The levels of BMP7 and miR-137
airs of BC tissues and adjacent non-tumor breast tissues (NT) were measured by Western blot (A) and RT-qPCR (B). C. A correlati
was performed between BMP7 and miR-137, using the 40 BC specimens. D. The 40 BC patients were followed-up for 60 months. T
ian value of all 40 cases was chosen as the cutoff point for separating miR-137-high cases (n=20) from miR-137-low cases (n=2
lan-Meier curves were performed to compare 5-year survival between two groups. *p<0.05. **p<0.01. N=40. Figure 1: High miR-137 levels in BC specimens is associated with poor prognosis. A-C. The levels of BMP7 and miR-137 in
40 pairs of BC tissues and adjacent non-tumor breast tissues (NT) were measured by Western blot (A) and RT-qPCR (B). C. A correlation
test was performed between BMP7 and miR-137, using the 40 BC specimens. D. The 40 BC patients were followed-up for 60 months. The
median value of all 40 cases was chosen as the cutoff point for separating miR-137-high cases (n=20) from miR-137-low cases (n=20). Kaplan-Meier curves were performed to compare 5-year survival between two groups. *p<0.05. **p<0.01. N=40. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 18350 18351
ctjournals com/oncotarget
2: MiR-137 targets BMP7 to inhibit its protein translation in BC cells. A-B. The levels of miR-137 by
7 by Western blot (B) in BC cell lines BT474 and MCF7, compared to BC tissue from patients. C. MCF7 cells w
-137 mimics (miR-137) or null as a control and examined for miR-137 levels. D. BT474 cells were transfected wit
(as-miR-137) or null as a control and examined for miR-137 levels. E. Prediction of miR-137-binding sites on BM
matics algorithms. F-G. The intact 3'-UTR of BMP7 mRNA (BMP7 3'-UTR), together with a 3'-UTR with muta
ite of BMP7 mRNA (BMP7 3'-UTR mut), was then cloned into luciferase reporter plasmids. Luciferase activity w
cells, which were co-transfected with 1μg as-miR-137/null plasmids and 1μg BMP7 3'-UTR or BMP7 3'-UTR mu
CF7 cells, which were co-transfected with 1μg miR-137/null plasmids and 1μg BMP7 3'-UTR or BMP7 3'-UTR
0.05. N=5. Figure 2: MiR-137 targets BMP7 to inhibit its protein translation in BC cells. A-B. The levels of miR-137 by RT-qPCR (A)
and BMP7 by Western blot (B) in BC cell lines BT474 and MCF7, compared to BC tissue from patients. C. MCF7 cells were transfected
with miR-137 mimics (miR-137) or null as a control and examined for miR-137 levels. D. BT474 cells were transfected with antisense for
miR-137 (as-miR-137) or null as a control and examined for miR-137 levels. E. Prediction of miR-137-binding sites on BMP7 mRNA by
bioinformatics algorithms. F-G. The intact 3'-UTR of BMP7 mRNA (BMP7 3'-UTR), together with a 3'-UTR with mutant at miR-137-
binding site of BMP7 mRNA (BMP7 3'-UTR mut), was then cloned into luciferase reporter plasmids. Luciferase activity was determined
in BT474 cells, which were co-transfected with 1μg as-miR-137/null plasmids and 1μg BMP7 3'-UTR or BMP7 3'-UTR mut plasmids (F),
and in MCF7 cells, which were co-transfected with 1μg miR-137/null plasmids and 1μg BMP7 3'-UTR or BMP7 3'-UTR mut plasmids
(G). *p<0.05. N=5. Figure 2: MiR-137 targets BMP7 to inhibit its protein translation in BC cells. A-B. The level Figure 2: MiR-137 targets BMP7 to inhibit its protein translation in BC cells. A-B. The levels of miR-137 by RT-qPCR (A)
and BMP7 by Western blot (B) in BC cell lines BT474 and MCF7, compared to BC tissue from patients. C. MCF7 cells were transfected
with miR-137 mimics (miR-137) or null as a control and examined for miR-137 levels. D. www.impactjournals.com/oncotarget BT474 cells were transfected with antisense for
miR-137 (as-miR-137) or null as a control and examined for miR-137 levels. E. Prediction of miR-137-binding sites on BMP7 mRNA by
bioinformatics algorithms. F-G. The intact 3'-UTR of BMP7 mRNA (BMP7 3'-UTR), together with a 3'-UTR with mutant at miR-137-
binding site of BMP7 mRNA (BMP7 3'-UTR mut), was then cloned into luciferase reporter plasmids. Luciferase activity was determined
in BT474 cells, which were co-transfected with 1μg as-miR-137/null plasmids and 1μg BMP7 3'-UTR or BMP7 3'-UTR mut plasmids (F),
and in MCF7 cells, which were co-transfected with 1μg miR-137/null plasmids and 1μg BMP7 3'-UTR or BMP7 3'-UTR mut plasmids
(G). *p<0.05. N=5. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 18351 Figure 3: MiR-137 decreases BMP7 protein but not mRNA in BC cells. A-B. The BMP7 levels in miR-137-overexpressing (an
BMP7-overexpressing) MCF7 cells by RT-qPCR (A) and by Western blot (B). C-D. The BMP7 levels in miR-137-depleted (and BMP
depleted) BT474 cells by RT-qPCR (C) and by Western blot (D). *p<0.05. NS: non-significant. N=5. Figure 3: MiR-137 decreases BMP7 protein but not mRNA in BC cells. A-B. The BMP7 levels in miR-137-overexpressing (and
BMP7-overexpressing) MCF7 cells by RT-qPCR (A) and by Western blot (B). C-D. The BMP7 levels in miR-137-depleted (and BMP7-
depleted) BT474 cells by RT-qPCR (C) and by Western blot (D). *p<0.05. NS: non-significant. N=5. Figure 3: MiR-137 decreases BMP7 protein but not mRNA in BC cells. A-B. The BMP7 levels in miR-137-overexpressing (and
BMP7-overexpressing) MCF7 cells by RT-qPCR (A) and by Western blot (B). C-D. The BMP7 levels in miR-137-depleted (and BMP7-
depleted) BT474 cells by RT-qPCR (C) and by Western blot (D). *p<0.05. NS: non-significant. N=5. www.impactjournals.com/oncotarget Oncotarget 18352 Overexpression of BMP7 abrogated the promoting effects
of miR-137 on the EMT and cell invasion in MCF7 cells
(Figure 4B-4C), without affected cell growth (Figure 4A). Next, BT474-as-miR-137 was further transfected with
shBMP7, resulting in decreases in BMP7 mRNA (Figure
3C) and protein (Figure 3D) in these cells. We found that
the effects of as-miR-137 on BMP7 protein compromised
the effects of shBMP7 on BMP7 protein, which explained
the findings in BC cells transfected with both as-miR-137
and shBMP7. We found that BMP7 suppression abolished
the inhibitory effects of as-miR-137 on EMT and cell
invasion in BT474 cells (Figure 5B-5C), without affected
cell growth (Figure 5A). Thus, miR-137 may enhance BC
cell invasion by suppressing BMP7 (Figure 6). did not alter cell growth in an MTT assay (Figure 4A),
but significantly increased the potential of EMT and
cell invasion in a transwell cell migration assay (Figure
4B-4C). Moreover, miR-137 depletion in BT474 cells
did not alter cell growth in an MTT assay (Figure 5A),
but significantly decreased the potential of EMT and
cell invasion in a transwell cell migration assay (Figure
5B-5C). Thus, MiR-137 decreases BC cell EMT and
invasion. MiR-137 enhances BC cell invasion by
suppressing BMP7 In order to ascertain whether miR-137 promotes
BC cell invasion through suppressing BMP7, we
prepared plasmids for BMP7 overexpression (BMP7) and
depletion (shBMP7). First, MCF7-miR-137 cells were
further transfected with BMP7, which increased BMP7
mRNA (Figure 3A) and protein (Figure 3B) in these cells. Cell growth assay An MTT Kit (MTT, Roche, USA) was used for
analyzing cell growth. MiRNA target prediction and 3'-UTR luciferase-
reporter assay To summarize, the current study may provide
evidence for using miR-137 as a specific target for future
BC therapies. MiRNAs targets were predicted using the
algorithms from TargetScan [40]. The data were analyzed
as previously described [41]. The candidate miRNAs
were analyzed for context+ score (Supplementary Table
1). The BMP7 3'-UTR reporter plasmid (pRL-BMP7)
and the BMP7 3'-UTR reporter plasmid with a mutant
at miR-137 binding site (pRL-BMP7-mut) were both
purchased from Creative Biogene (Shirley, NY, USA). Dual-luciferase reporter assay (Promega, Fitchburg, WI,
USA) was performed according to the instructions from
manufacturer. Transwell cell invasion assay Transwell cell invasion assay was performed as has
been described previously [16]. In addition to regulation of BMP7 by miRNAs,
BMP7 protein levels are modulated at the level of
degradation, such as through protein ubiquitination. Moreover, miR-137 may have targets other than BMP7,
and these targets should be analyzed to have an overview
of the effects of miR-137 in the carcinogenesis of BC. Besides, future studies may also address the regulation of
miR-137 in BC and confirm this model in vivo. DISCUSSION The inhibitory role of BMP7 in BC EMT and
invasion has been well documented in the past studies. Figure 4: Overexpression of miR-137 increases MCF7 EMT and cell invasion through suppressing BMP7. A-C. MCF7
cell invasion by miR-137 overexpression (and BMP7 overexpression) in a transwell cell invasion assay, shown by quantification (A-B), and
by representative images (C). *p<0.05. N=5. Figure 4: Overexpression of miR-137 increases MCF7 EMT and cell invasion through suppressing BMP7. A-C. MCF7
cell invasion by miR-137 overexpression (and BMP7 overexpression) in a transwell cell invasion assay, shown by quantification (A-B), and
by representative images (C). *p<0.05. N=5. Figure 4: Overexpression of miR-137 increases MCF7 EMT and cell invasion through suppressing BMP7. A-C. MCF7
cell invasion by miR-137 overexpression (and BMP7 overexpression) in a transwell cell invasion assay, shown by quantification (A-B), and
by representative images (C). *p<0.05. N=5. www.impactjournals.com/oncotarget Oncotarget 18353 Figure 5: Depletion of miR-137 decreases BT474 EMT and cell invasion through augmentation of BMP7. A-C. BT474
cell invasion by miR-137 depletion (and BMP7 depletion) in a transwell cell invasion assay, shown by quantification (A-B), and by
representative images (C). *p<0.05. N=5. Figure 5: Depletion of miR-137 decreases BT474 EMT and cell invasion through augmentation of BMP7. A-C. BT474
cell invasion by miR-137 depletion (and BMP7 depletion) in a transwell cell invasion assay, shown by quantification (A-B), and by
representative images (C). *p<0.05. N=5. representative images (C). *p<0.05. N=5. Figure 6: Schematic of the model. MiR-137 enhances BC cell EMT and invasion, through translational suppression of BMP7. Figure 6: Schematic of the model. MiR-137 enhances BC cell EMT and invasion, through translational suppression of BMP7. Figure 6: Schematic of the model. MiR-137 enhances BC cell EMT and invasion, through translational su www.impactjournals.com/oncotarget Oncotarget 18354 Culturing and transfection of BC cells Human BC cell lines MCF7 [38] and BT474
[39] were originated from adenocarcinoma and ductal
carcinoma, respectively. Both lines were purchased from
ATCC (American Type Culture Collection, Manassas,
VA, USA), and cultured in in RPMI1640 medium
(Invitrogen, Carlsbad, CA, USA) supplemented with 15%
fetal bovine serum (FBS; Sigma-Aldrich, St Louis, MO,
USA) in a humidified chamber with 5% CO2 at 37 °C. All
constructs were purchased from Origene (Beijing, China). Transfection was performed with 50nmol/l plasmids,
using Lipofectamine 2000 (Invitrogen). The transfection
efficiency (>95%) was determined based on expression of
GFP in the transfected cells. Here, we used bioinformatics analyses to screen
all miRNAs that target BMP7, and we focused on the
expression levels of those which were altered in BC
specimens compared to normal tissue control. We found
miR-137 to be one such microRNA. To the best of our
knowledge, this follow-up study of our previous work [16]
is the first study showing that BMP7 protein levels could
be regulated by a miRNA in BC. High level of miR-137
in BC tissues was associated with poor prognosis in BC
patients. We thus designed in vitro experiments to show
a regulatory relationship between miR-137 and BMP7 in
BC cells, which was consistence with the clinic findings
showing an inverse correlation of these two factors in BC
specimens. Patient specimens However, the regulation of BMP7 by miRNAs was
only recently reported in lung cancer [42]. Yang et al. reported that miR-137 was significantly down-regulated
in NSCLC tissues and cell lines. An In vitro functional
assay demonstrated that over-expression of miR-137
inhibited lung cancer cell proliferation, migration and
invasion, suggesting that miR-137 could act as a tumor
suppressor in lung cancer progression. In addition, they
identified BMP7 as a target of miR-137 in lung cancer
cells, and used a luciferase reporter assay to show that
miR-137 directly targeted 3'-UTR of BMP7. Furthermore,
they showed that re-expression of BMP7 substantially
reversed the tumor suppressive effects of miR-137 on lung
cancer cell proliferation, migration, and invasion [42]. This study is interesting but also suggests that the role of
a molecular could be very different from cancer to cancer,
since BMP7 is believed to be a tumor suppressor in many
cancers [43–46]. Surgical BC resected specimens were obtained from
40 BC patients (all Stage III or IV) and paired adjacent
non-tumor breast tissues (NT) in Shanghai Jiao Tong
University Affiliated Sixth People's Hospital from 2008
to 2010 (Table 1). All patients were followed-up for 60
months, before which they obtained Informed consent and
provided signed agreement about this study. The histology
of the resected tissue were examined and determined
independently by 2 senior pathologists. Experimental protocol approval All experimental protocols were approved by
the Research Bureau of Shanghai Jiao Tong University
Affiliated Sixth People's Hospital. All mouse experiments
were approved by the Institutional Animal Care and Use
Committee at Shanghai Jiao Tong University Affiliated
Sixth People's Hospital (Animal Welfare Assurance). Animal and human specimens were handled according to
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Approaches to Integrated Parasite Management (IPM) for Theileria orientalis with an Emphasis on Immunity
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Pathogens
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pathogens pathogens Review David Lyall Emery Sydney school of Veterinary Science, University of Sydney, Sydney, NSW 2006, Australia;
david.emery@sydney.edu.au Abstract: Integrated parasite management (IPM) for pests, pathogens and parasites involves reducing
or breaking transmission to reduce the impact of infection or infestation. For Theileria orientalis, the
critical impact of infection is the first wave of parasitaemia from the virulent genotypes, Ikeda and
Chitose, associated with the sequelae from the development of anaemia. Therefore, current control
measures for T. orientalis advocate excluding the movement of naïve stock from non-endemic regions
into infected areas and controlling the tick Haemaphysalis longicornis, the final host. In Australia,
treatment of established infection is limited to supportive therapy. To update and expand these
options, this review examines progress towards prevention and therapy for T. orientalis, which are
key elements for inclusion in IPM measures to control this parasite. Keywords: Theileria orientalis; Haemaphysalis; parasitaemia; integrated parasite control
Citation: Emery, D.L. Approaches to
Integrated Parasite Management
(IPM) for Theileria orientalis with an
Emphasis on Immunity. Pathogens
2021, 10, 1153. https://doi.org/
10.3390/pathogens10091153
Academic Editor: Geoff Hide
Received: 17 August 2021
Accepted: 5 September 2021
Published: 7 September 2021 pathogens pathogens
The fundamental philosophy underpinning the success of integrated parasite manage-
ment (IPM) for pests, pathogens and parasites involves reducing or breaking transmission
to reduce the impact of infection or infestation. For parasites, prerequisites for the ra-
tional formulation of comprehensive control measures are: 1, a thorough understanding
of the parasite life cycle and mode(s) of transmission; 2, knowledge of the pathogene-
sis of clinical disease arising from the host–pathogen interaction; and, 3, awareness of
the seasonal epidemiology of the parasite–environment interaction that determines fluc-
tuations in parasite populations. For nematode parasites, the various IPM programs
comprise 5 major interactive components to reduce parasite availability and infection,
thereby reducing pathogenic sequelae and prolonging the effective life of parasiticides by
decreasing the need for treatments. From a typical IPM program, “Drenchplan” (https:
//www.dpi.nsw.gov.au/__data/assets/pdf_file/0004/38551/drenchplan-2005.pdf, (ac-
cessed on 27 May 2021)), these components include; Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. •
the effective use of treatments to reduce pathology and parasite reproduction (drenches)
•
grazing management to reduce parasite numbers on pasture or prevent host access
•
differential management of resistant and susceptible populations (weaning and introductions)
•
breeding for parasite resistance to limit parasite numbers in the host
•
regular testing to ensure treatments remain effective. g
p
p
•
regular testing to ensure treatments remain effective. For those parasites with intermediate hosts or vectors, additional control measures
target the vector to interrupt or reduce transmission. Copyright: © 2021 by the author. 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/). Given that Theileria buffeli, causing “benign theileriosis” had been present in Queens-
land, Australia since 1912 [1,2], it was long considered a benign parasite [3–7]. Historically,
there was widespread taxonomic confusion regarding various Asian/Australasian theile-
rial parasites, as T. sergenti caused clinical disease Japan and Korea [8]. However, based
on morphological and serological data and results from transmission experiments, all
members of the T. sergenti, T. buffeli, and T. orientalis group were classified as a single
species, T. orientalis [2,5,8]. Phylogenetic analyses using major piroplasm surface protein https://www.mdpi.com/journal/pathogens Pathogens 2021, 10, 1153. https://doi.org/10.3390/pathogens10091153 Pathogens 2021, 10, 1153 2 of 14 (MPSP) and p32/34 piroplasmic gene sequences have revealed the genetic diversity of
T.
orientalis in Japan [9,10], Korea [11], Kenya, and Australia [6,12]. Currently, 11 genotypes
of T. orientalis (type 1 or Chitose, type 2 or Ikeda, type 3 or buffeli, types 4–8, and N1-N3)
have been identified based on MPSP gene sequences [10,12]. Of these genotypes, 1 and 2
cause the majority of clinical disease in cattle in Australia [13,14], and elsewhere [5]. These
pathogenic genotypes are recognised in many countries including Australia [15], New
Zealand [16], Japan [17], Korea [18], and USA [19]. The disease has been estimated to cost
the livestock industries in Japan and Korea around USD $100 million annually [17,18]. In
Australia, NSW-DPI have estimated an average cost of AUD 59K for dairy producers and
AUD 11.6K for beef producers, which equates to AUD 131/head for dairy cattle and AUD
67/head for beef cattle for farms impacted by the parasite, in all, costing around AUD 20m
per annum nationally [20]. Mortality rates vary from 1 to 30% in naive stock [20,21]. p
y
y
y
In the bovine intermediate host, the vast majority of the pathology, clinical disease
and deaths results from the anaemia caused by the high levels of parasitaemia associated
with the “first wave” of asexual reproduction reaching its maximum around 2–3 months
after infection [5,21]. Recovered cattle remain asymptomatic carriers with low levels of par-
asitaemia (as detected by PCR) for at least 30 months and likely for life [22]. This situation
maintains the risk of ongoing tick infestation. Interestingly, several studies from Australia
and New Zealand have indicated that the carrier state arising in recovered dairy cattle
does not compromise subsequent productivity [23,24]. However, recrudescence of clinical
disease may be induced by transport stress, but carrier cattle calve successfully on their
home farms. Although mortality remains relatively low in endemic regions, unexposed
animals, including calves and introduced stock develop disease around 5–6 weeks after
birth or entry [13,22,25]. These cohorts are the focus of theilerial control measures. Current control measures for T. orientalis advocate excluding the movement of naïve
stock (from non-endemic regions) into endemic regions while current treatment of clinical
theileriosis in Australia is limited to supportive therapy. To update and expand these
options, this review examines progress towards prevention and therapy for T. orientalis,
which are key elements for inclusion in IPM measures to control this parasite. 2. Features of the Transmission and Pathogenesis of T. orientalis
2.1. Life Cycle of T. orientalis The life cycle of T. orientalis begins with the inoculation of sporozoites from the salivary
gland of the tick vector (species) as it feeds. After infection as larvae, both nymphal and
adult ticks of Rhipicephalus appendiculatus and Haemaphysalis longicornis are capable of
injecting sporozoites of Theileria parva and T. orientalis, respectively [8,26,27]. Sporozoites
attach and invade host leucocytes and, following division, become schizonts. These
were documented transiently in (parotid) lymph nodes draining tick attachment sites
around 7–10 days after infection with Korean isolates of T. orientalis in 3/7 calves [8] and
4–8 days after inoculation of tick-derived stabilate [28]. However the schizont stage is not
responsible for the pathology associated with the infection, unlike with T. parva [29,30]. Asexual development results in uninucleate merozoites, which then escape from parasitised
leucocytes and invade erythrocytes where the parasite multiplies into piroplasms. The
invasion of red blood cells by merozoites takes place about 10 days post inoculation, and is
responsible for the febrile episodes and the clinical manifestations of T. orientalis, including
the signs associated with anaemia (pale mucous membranes, tachycardia, tachypnoea,
weakness) [5,31]. The analogous period with T. parva infection is also associated with the
most severe inflammatory reactions as leucocytes rupture in submucosal tissues to release
microschizonts. Although the mechanism of attachment and invasion of erythrocytes is
poorly understood, high parasitaemias can result in severe anaemia and death of cattle [31]. The prepatent period (infection to clinical signs, usually fever) ranges from 7–10 days in
Korea [18] and, in Australia, has been variously estimated at 14–47 days [32], 12–16 days
after tick application [26,33] to around 20 days after ticks were seen [21]. Therefore, clinical
disease occurs well after ticks have engorged (5–7 days) [21], and the prepatent period most Pathogens 2021, 10, 1153 3 of 14 3 of 14 likely reflects the quantum of infection as occurs with dose-dependent responses to T. parva
stabilate [34] and to graded blood volumes of T. orientalis Ikeda [35]. In newborn calves
and in naïve cattle arriving into endemic zones in Australia, clinical disease associated
with anaemia is readily apparent 4–8 weeks after birth or introduction [22,25]. Reports indicating that the productivity of carrier cattle appears “normal” [23,24]
would suggest that a “steady-state” host–parasite relationship is established after recovery,
preventing the recurrence of clinical disease following ongoing, seasonal, tick challenges in
endemic regions [6,14,21]. 2.2.1. Vector Competency, Mechanical and Biological Transmission The distributions of parasites with indirect life cycles are restricted by the availability
of either the intermediate or final host. So the distribution of T. orientalis is limited by
cattle (the obligate intermediate host) or the definitive host. The full spectrum of biological
vectors (or definitive) hosts for T. orientalis has not been resolved. Amongst arthropods,
the 3-host ixodid tick Haemaphysalis longicornis (Neumann, 1901) [38] has been confirmed
in transmission trials as one biological vector for T. orientalis in early studies [8] in Aus-
tralia [26,39] and the USA [19]. H. longicornis has a predicted, widespread distribution
in countries with temperate climates [40], including Australia, New Zealand, Fiji, New
Caledonia, China, former USSR, Korea and Japan [41,42], the USA, and several other Pacific
Islands, including Hawaii [43]. The vector competency of various 3-host ticks varies across locations, likely related to
regional host–parasite adaptations. Uilenberg et al. [8] reported that most global stocks of
T. orientalis could be transmitted transtadially by H. longicornis and H. punctata, but not by
3 Amblyomma species or by Dermacantor reticularis. However, a USA isolate of T. orientalis
buffeli was not transmitted by H. longicornis ticks from Korea or by H. punctata [8,44]. In
Japanese investigations, Australian H. longicornis could transmit only T. sergenti (T. orientalis
Ikeda/Chitose) and could not transmit T. orientalis buffeli, whereas as Japanese H. longicor-
nis could transmit both [45]. Early vector studies in Australia indicated that H. bancrofti
and H. humerosa were likely vectors for T. orientalis buffeli in northern Australia [33,46]. While H. longicornis nymphs and adults readily transmitted T. orientalis Ikeda [39,47], these
failed to transmit T. orientalis buffeli to naïve calves in Sydney trials on 2 occasions [27,47]. This is perhaps not surprising as the definitive distribution of H. longicornis occurs in the
coastal areas of Victoria and New South Wales and extends northwards as far as Gympie in
Queensland but is absent from large areas of northern Australia where Theileria sp. (T. ori-
entalis buffeli) is present [32]. The 1-host tick Rhipicephalus microplus was proposed as an
alternative vector in India, but while eggs from ticks feeding on infected cattle were positive
for T. orientalis by PCR, hatched larvae were not tested for any successful transmission [48]. Clinical infection with T. parva and T. 2.2. Pathogenesis of Infection with Multiple Genotypes of T. orientalis In endemic regions with multiple theilerial genotypes present, infections of virulent
genotypes Ikeda and Chitose clearly outpace the development of T. orientalis buffeli in
susceptible cattle [27,37]. In 30 calves sampled from each of 2 Dorrigo farms in 2017
and 2019 where birth dates were available, results confirmed that calves were readily
and heavily infected with T. orientalis Ikeda and Chitose genotypes within 4–5 weeks of
birth [22], similar to results from unexposed cattle introduced into another endemic area
in Australia [25]. Weaner cattle introduced to Dorrigo in late summer were PCR-positive
within 3 weeks after introduction and exhibited clinical theileriosis within 5–6 weeks after
arrival, with an estimated weight loss around 20 kg over the first 3 months [22]. 2.2.1. Vector Competency, Mechanical and Biological Transmission 2. Features of the Transmission and Pathogenesis of T. orientalis
2.1. Life Cycle of T. orientalis Queensland, the widespread presence of cattle harbouring infec-
tions with the “benign” T. orientalis buffeli genotype, has been deemed partly responsible
for preventing infections with the virulent genotypes in that state [36]. 2.2.1. Vector Competency, Mechanical and Biological Transmission orientalis can be established by tick feeding or
h
h h
d
d
l
f
k d
d
b l
“G
S” (
d Clinical infection with T. parva and T. orientalis can be established by tick feeding or
through the production and inoculation of a tick-derived stabilate or “GUTS” (ground-up
tick supernate) [38,49–51]. For the transmission of protozoa, several days of tick feeding
is needed to mature sporozoites prior to inoculation into the intermediate host [52]. For Pathogens 2021, 10, 1153 4 of 14 the maturation of T. parva sporozoites, infected R. appendiculatus are fed for 4 days on
rabbits [53] to prevent clinical disease if fed on cattle. For T. orientalis stabilate production,
GUTS produced from newly moulted, adult H. longicornis (infected with T. orientalis Ikeda
as nymphs), were only marginally positive by PCR. Therefore around 3000 of the infected
adult ticks were fed for 3 days on an uninfected, splenectomised (SplX) Murray-Grey cross
(not Bos indicus) before removal and produced strongly positive results in the PCR [39]. Interestingly, while this animal ultimately developed detectable theileriosis it did not
develop clinical theileriosis over the subsequent 2.5 months before it was sold. Anecdotal
experience at the Tick Fever Centre in Queensland notes that SplX animals can control the
initial peak of parasitaemia (up to 10% in blood smears) without reductions in haematocrit
and subsequent mortality (P. Rolls, unpublished). The mechanism for these differences
with intact cattle is not known. Haematophagous insect vectors have also been suggested as involved in mechani-
cal/horizontal transmission, but only the cattle louse, Linognathus vituli, has been success-
fully harvested, transferred, and produced infection in unexposed cattle [54]. Biting flies in
large numbers may be another mechanical vector as transfer of a little as 0.1 mL blood was
able to establish an infection detectable by PCR [34]. In the field, transmission by lice would
appear minimal as the entire life cycle occurs on the host. Mechanical transfer is likely
irrelevant to the epidemiology of clinical theileriosis but is only pertinent to the spread of
the parasite. As discussed in Section 2.2.2, and for reasons not entirely clear, the mechanical
transfer of blood does not appear to cause clinical disease [34]; sexual reproduction in the
definitive host (tick) appears necessary to maintain the virulence that is also associated
with sporozoite infection. p
Cattle in endemic zones often harbour multiple theilerial genotypes [22,25,55]. H. 2.2.1. Vector Competency, Mechanical and Biological Transmission longi-
cornis populations feeding on these carriers are also positive by PCR for these genotypes,
and multiple genotypes can be detected in tick saliva and stabilate [55]. This indicates
that ticks are competent vectors to transmit multiple genotypes to susceptible stock [22]. The intra-genotypic interactions during sexual reproduction in H. longicornis leading to
cooperation or competition have not been studied. However, recent trials have indicated
that H. longicornis nymphs, infected as larvae with T. orientalis Ikeda, can infect naïve calves
and, after moulting, infect additional naïve calves as adult ticks, thereby retaining the
original infection across 2 moults [47]. This finding has implications for the control of
3-host ticks and the spread of infection through movement by sylvatic second hosts, such
as kangaroos. 3.1. Chemical Control of H. longicornis 3.1. Chemical Control of H. longicornis With confirmation of the definitive host, chemical trials targeting H. longicornis are
underway, but most results remain unpublished. In Australia, historical effort has focused
on treatments for the 1-host ticks, R. microplus and R. australis, to control bovine babesiosis,
while similar regimes have been developed to control the 3-host tick R. appendiculatus [63]. Flumethrin pour-ons have reduced numbers of H. longicornis and theilerial infections in
Korea, with tick control remaining the main focus for control of the infection [64]. Macro-
cyclic lactones (MLs; moxidectin) have been reported to provide partial reduction in tick
numbers on cattle, but infection is still readily transmitted in endemic regions (C. Shirley,
unpublished). Three-host ixodid ticks are more difficult to control than 1-host ticks, as these
only feed 5–7 days to engorge and are not host-specific [39,65]. However, since around
3 days are required to mature sporozoites, the rapid 12 h knockdown (“speed of kill”) pro-
vided by isoxazoline acaricides against ixodid ticks, including H. longicornis, on companion
animals [66,67] would be ideal to prevent transmission if developed for livestock (provided
that the residue limits are acceptable) [65]. As noted in Korea and observations in Australia
where reduced tick numbers result in decreased clinical disease [64,68], vector control
may reduce the number of sporozoites inoculated, enabling infected cattle to control the
intensity of the ensuing parasitism. To limit the spread of ticks on purchased cattle, acaricide treatment prior to transport
would be required and noted on vendor declarations. However, the movement of ticks
outside of their current distributions may not guarantee their continued survival. 2.2.2. Infection with Blood Stages of T. orientalis Successful infection with T. orientalis, as detected by smear and PCR, has been consis-
tently achieved by the inoculation of around 108 infected bovine erythrocytes containing
several theilerial genotypes [35,47,56–59]. Both intravenous (iv) and subcutaneous (sc)
inocula infected with around 108 T. orientalis buffeli or T. orientalis Ikeda each produced para-
sitosis detectable by PCR within 4 weeks [27,47], consistent with previous reports [2,35,59]. In each case, the parasitosis appeared to peak around 6–8 weeks before stabilising at
2000–10,000 gene copies uL−1, irrespective of genotype. Infections with 1.25 × 107 para-
sites had a longer prepatent period [35]. The results from these combined studies indicate that the parasitaemias induced by
blood inoculation of single or multiple benign and virulent genotypes of T. orientalis do not
reach “clinical” levels and do not produce clinical disease in adult cattle or calves >4 months
of age [2,35,59]. A similar situation is apparent in calves infected by intrauterine or colostral
transmission in endemic zones; these animals remain asymptomatic carriers [47,60,61]. Around 10% of calves born to infected dams were PCR positive at 3 months of age in Japan,
but whether clinical disease occurred was not reported [62]. The exact reasons for the
differences in pathogenesis between the high parasitaemias generated by tick or stabilate
infection and those induced by infected blood remain unresolved. Pathogens 2021, 10, 1153 5 of 14 Since the levels of parasitaemia generated by the inoculation of parasitised blood
remain relatively and persistently low and comparable to those in recovered and carrier
cattle [22], the method could be examined for immunization against tick challenge (see
Section 5.3 below). 3.2. Vaccination against H. longcornis During blood feeding on immunized animals, haematophagous parasites also ingest
antibodies which may target their gut antigens, digestive enzymes, or microflora. Success-
ful vaccines against R. australis (formerly Boophilus microplus) with Tickguard [69] and the
nematodes Haemonchus contortus (Barberva) [70,71] and hookworms [72] have prompted
ongoing developments and vaccination strategies using gut antigens. There are no successful vaccines currently available for H. longicornis in cattle despite
the isolation of several candidate antigens, including proteases, ferritins [73], and sub-
olesin [74]. New technological advances in tick genomics, transcriptomics, and microbiome
analysis offer the possibilities to target endosymbionts [75,76] or crucial genera in the gut
microbiome of H. longicornis to reduce reproductive capability or to block the maturation
and transmission of parasites [77,78]. Given that H. longicornis is parthenogenic, it is not
known whether doxycycline or tetracycline targeting endosymbionts might compromise
tick development and reproductive fitness [79]. 4.2. Chemotherapy of the Carrier State Another means to break the transmission of theilerial parasites in endemic regions is to
cure the carrier state. Experimentally, this develops after the mechanical transfer of infected
blood and in the field, after the first wave of parasitaemia. Several compounds were utilized
for a chemotherapeutic trial with selections based on the premise that clinical disease from
T. orientalis coincided with the appearance of piroplasms, fever, and parasitaemia, and that
these merozoite stages multipled in erythrocytes like babesial and malarial parasites. So
drugs with activity against other haemoprotozoa with important erythrocytic stages for
asexual development could be effective against T. orientalis. In Queensland, T. orientalis
buffeli could be cured by the administration of primaquine and halofuginone or primiquin
and buparvaquone (BPQ) [84,85]. Previously, oxytetracycline and imidocarb (Imadox) have been used for the “treat-
ment” of clinical cases of T. orientalis. The napthoquinones, parvaquone and BPQ, and the
febrifuginone, halofuginone lactate, will cure clinical disease associated with T. annulata or
T. parva [86,87] but are not registered for clinical use in Australia. BPQ targets the schizont
stage of the parasite, which is associated with the clinical signs in East Coast fever (ECF). BPQ also reduces number of T. orientalis Ikeda piroplasms in blood within 4 days [88,89],
while the addition of chloroquine, quinine, or pyrimethamine to bovine blood cultures
in vitro inhibited the proliferation of T. orientalis [sergenti] [90]. p
g
When 4 of these potential therapeutic compounds were examined for effects on blood-
induced infections with T. orientalis Ikeda, only BPQ suppressed parasitosis; imidocarb
(Imadox), tulathromycin (Draxxin) and oxytetracycline had no effect ([27]; S. de Burgh,
unpublished). However, BPQ did not cure the infection as recipients still were PCR-positive
2 months later. Further field work would be needed to confirm BPQ’s effectiveness in
clinical outbreaks, but it would not contribute to IPM programs. This study discounted the
use of oxytetracycline and Imadox for the treatment of T. orientalis, while Imadox also failed
to reduce parasitaemias (detected in blood smears) in 3 calves infected with T. orientalis
(Ikeda (P. Carter, unpublished). p
It is possible that further testing may reveal more effective compounds, but the low
mortality rates from T. orientalis may not justify investment. Drugs used for the treatment
of canine babesiosis or human malaria (not registered for use in cattle anywhere) are expen-
sive, lack residue depletion data, and likely to have the same lack of efficacy. 4.1. Prevention of Parasite Development in the Intermediate Host 4.1. Prevention of Parasite Development in the Intermediate Host Toltrazuril (Baycox) is known to be active against the schizont stages of Eimeria and
Isospora spp., which are related to Theileria [80]. Baycox remains at therapeutic levels
in calves for around 8 days [81], which would “cover” the early schizont development. However, when administered at 15 mg/kg to 20 dairy calves, 4 weeks after turnout in New
Zealand, Baycox did not prevent or ameliorate theilerial parasitaemias significantly [82]. Similarly in Australia, Baycox was given at 15mg/kg, to 15 calves, 4 days following chal-
lenge with 50 unfed adult H. longicornis that had been infected as nymphs with T. orientalis
Ikeda. The timing was specifically aimed to coincide with the early schizont stages of the
parasite and included the 3 days of feeding required to mature sporozoites in H. longicornis
prior to inoculation [39]. In comparison with infected but untreated calves, toltrazuril had
no significant effect on developing parasitaemia [83]. At this time, oxytetracycline has not
been examined for “infect and treat” regimens for T. orientalis. 4. IPM through Chemotherapy to Prevent Development and Persistence of
T. orientalis While effective acaracides prevent the development of pathogenic theilerial genera
in the final host, the control of theilerial species in the intermediate can target schizont
development or remove blood stages that could be ingested by the final tick host. In
prevention or retarding the development of T. parva in the “infect and treat” protocol,
oxytetracycline and stabilate are administered concurrently [50,51]. Due to the difficulties in
producing tick-derived stabilate, this has not been attempted for T. orientalis, but anecdotal
evidence suggested that toltrazuril may attenuate schizont development in calves (C. Shirley, unpublished). Pathogens 2021, 10, 1153 6 of 14 4.2. Chemotherapy of the Carrier State Diminazene
(Berenil) and primiquin [91] are still possibilities to examine, the latter (pamaquin and
primaquine) being active against the piroplasms of T. annulata [92,93] but are unable to
cure T. orientalis buffeli parasitosis [2]. However, due to residues and withholding periods,
these appear less important in the overall integrated management of clinical theileriosis,
and the parasiticide is often administered too late if clinical signs are already apparent. Parenthetically, this “timing issue” gives rise to anecdotal “cures” for T. orientalis, when
compounds are administered after animals have passed the first peak of parasitaemia and
have entered the recovery phase towards carrier status. Pathogens 2021, 10, 1153 7 of 14 7 of 14 In endemic regions with ongoing seasonal tick challenge, the carrier state appears to
prevent the recurrence of clinical theileriosis without reducing productivity, so that curing
the carrier state may not be beneficial. 5. IPM to Produce Resistant Hosts: Immunisation against Infection The fourth significant component for theilerial IPM is the generation of resistant
livestock. Apart from tick resistance between cattle breeds [94], innate genetic resistance
to theilerial parasites appears to be lacking, and vaccination provides an alternative ap-
proach. The precise mechanism providing protective immunity against T. orientalis remains
unresolved. Recovered animals enter a persistent carrier state after the “first wave” of
parasitosis with the virulent genotypes of T. orientalis around 2–3 months after infection,
whether from tick infestation or stabilate containing single or multiple theilerial geno-
types [22,23,35,37]. This situation also reflects field experience wherein recovered cattle
may harbour multiple theilerial genotypes in the carrier state [13,22,49]. In recovered
cattle, some type and level of immunity exists in carrier cattle which resembles a “pre-
munity” [95], interfering with the severity of subsequent challenge infestations [2]. The
use of premunity has a long history in early “vaccinations” against Leishmania, malaria,
East Coast fever, babesiosis and poultry coccidiosis (“precocious strains”) see [96,97]. In
Australia, pre-existing infections with T. orientalis buffeli actually suppressed infections
with B. bovis and Anaplasma marginale but not B. bigemina in SPLx calves [98] and could do
so for T. orientalis Ikeda in an experimental trial [47]. p
Passive infection during gestation or through colostral antibodies do not appear
to provide reliable protection against T. orientalis. In Korea, intra-uterine infection with
T. orientalis [sergenti] occurred readily, but did not protect against field challenge after
birth [60,61]. The same situation occurs in endemic regions of T. orientalis in Australia [22]. Calves born of infected dams are not fully protected against tick challenge with T. orientalis,
indicating that colostral antibodies are not protective [60,61]. The effect may be due to
genetic diversity in genotypes of T. orientalis or in their MPSP genes (and epitopes) [20,99]. The lack of protection may also reflect the low “immunizing dose” in utero or from
colostrum, or the level of tick challenge, as parasitaemia following infection with T. orientalis
Ikeda by 200 H. longicornis nymphs was significantly reduced in two calves thT were
presumably infected during gestation or at birth with the Ikeda genotype [47]. 5.2. Immunisation against T. orientalis with Inactivated or Subunit Vaccines 5.2. Immunisation against T. orientalis with Inactivated or Subunit Vaccines To avoid the possible transfer of pathogens using whole blood vaccines, the search
for protective antigens and vaccine formulations from the blood stages of T. orientalis have
been based on three principal research outcomes. Firstly, these stages of the parasites are
deemed responsible for the clinical sequalae of the infection, and, secondly, the molec-
ular typing of the MPSP has clearly identified the virulent genotypes [5,10]. Moreover,
the temporal kinetics for genotypes of T. orientalis during ongoing infections has been
attributed to “escape” from protective, MPSP-specific antibodies [37,97]. This resembles
the sequential production of neutralizing antibodies to variant-specific surface antigens
(VSSA) of trypanosomes, although in this infection, the gene splicing by the parasites
produces novel VSSAs ahead of the host response [106]. Further support for subunit vaccines had been encouraged from several trials attempt-
ing to identify candidate antigens, especially involving MPSP, to generate neutralizing
antibodies for genotype-specific protection. The passive transfer of ascitic fluid from
hybridomas recognising the P32 protein from T. orientalis [sergenti] prevented the develop-
ment of parasitaemia in 3 SplX calves challenged with T. orientalis Chitose merozoites from
infected blood [107]. However, this was not tested against tick challenge or sporozoite-
based stabilate. In immunization trials using dissociated parasites, calves were inoculated
with 2 × 100 mg doses of sonicated T. orientalis [sergenti] merozoites in complete Freund’s
adjuvant subcutaneously. When subjected to field challenge 2–5 months after the initial vac-
cination, their parasite burdens were significantly reduced at 3 months post-challenge, [59]. Unfortunately, this trial was terminated 2 months later as all controls and 20% (4/20) of
vaccinates required treatment with diminazene (Berenil) for anaemia [59]. The outcome
would indicate that, while the vaccine had induced substantial protection, either the field
challenge overwhelmed (a waning) immunity or field strains contained virulent genotypes
that were not present in the vaccine. p
Two studies also investigated the efficacy of recombinant MPSP in vaccines. Calves
immunized with recombinant MPSP from “I” (Ikeda) and “C” (Chitose) genotypes in
Freund’s adjuvant or liposomes showed “vaccine effects” after challenge with a stabilate
containing both “variants” (genotypes) [57]. This study was the first to indicate that cross-
protective immunity could be generated against genotypes of T. orientalis, but the levels of
parasitaemia were not reported. A recombinant MPSP vaccine for T. 5.2. Immunisation against T. orientalis with Inactivated or Subunit Vaccines orientalis [sergenti]
utilised three vaccinations at 3-week intervals, producing an antibody response but no
protection against challenge [108]. p
g
g
From the lack of consistent generation of protective immunity, these recombinant
vaccines have not been pursued to date. 5.1. Immunisation against Theilerial Parasites (T. parva, T. annulata) Since the principal protective immune response against T. parva is genetically re-
stricted CD8+ -mediated lympho-cytotoxicity (CML) against the macroschizont-infected
lymphocyte [100,101], protection requires the live parasite for induction but is restricted
to the parasite genotype in the vaccine. This underscores the effectiveness of stabilates
containing sporozoites of T. parva to generate protection against challenge when adminis-
tered with long-acting oxytetracycline in the “infect and treat” method [50,51]. However, as
predicted, limited cross-protection is produced, particularly against buffalo genotypes, and
multivalent stabilates can lead to the recombination of T. parva genotypes during the immu-
nization period [101]. A similar restricted protection is induced by the administration of
around 108 allogenic cultured lymphoblasts infected with macroschizonts of T. parva [100]
or 5 × 106 cells infected with T. annulata [102]; reinforcing the importance of this parasitic
stage to the pathology and protective immunity against both parasites [103]. The lack
of cross-protection and complications from the exquisite specificity of CML has fostered
research into other antigens and modes of delivery [101,104]. As discussed above, the merozoites/piroplasmic stages of T. orientalis appear more
important than the schizont to the pathogenesis and anaemia of clinical theileriosis [5]. In field trials, GUTS stabilate for T. parva immunizations equate to around 10 ticks per
dose [105], whereas stabilate used for T. orientalis infection was around 30 ticks per dose [39]. The lower infection rates in H. longicornis and reduced mortalities compared to T. parva do Pathogens 2021, 10, 1153 8 of 14 not justify pursuit of an “infect and treat” protocol for T. orientalis, so alternative strategies
have been investigated. not justify pursuit of an “infect and treat” protocol for T. orientalis, so alternative strategies
have been investigated. 5.3. Immunisation against T. orientalis with Blood Stabilates Reports from field infections in endemic regions of T. orientalis consistently indicate
that recovered carrier cattle resist seasonal reinfection from ticks [20], and ongoing pro-
ductivity appears to be unaffected [23,25]. Since the blood stages of T. orientalis cause the
clinical pathology and any untoward deaths, it is not surprising that blood-based vaccines
have been examined, with variable success [5,36]. Historically, a blood vaccine containing
2 × 108 infected red blood cells per dose “had an inhibitory effect on the clinical manifesta-
tion of T. orientalis [sergenti]” with a need for “proliferation of the inoculum” [56,60] but
this was not continued. A whole blood vaccine against T. orientalis [sergenti] was tested
in Korea but outcomes were not reported and challenge appeared to use blood stabilate
(see [58]). Inocula of blood containing live T. sergenti induced variable levels of protection
against tick challenge in Japan, an effect attributed to genetic variations in the MPSP p32
protein across the country [57]. Pathogens 2021, 10, 1153 9 of 14 9 of 14 Dose-response studies on blood vaccines have not been completed due to the rather
ad hoc history of these trials [36], but several results emphasise that dose may influence
the levels of protection generated. Both the passive infection in utero and the transfer
of antibody does not appear to provide protective immunity after birth [60,61], and this
may be related to low dose infection and the transient time-frame of maternal antibody
under consistent tick challenge (Section 4 above). However, the interval between (passive)
infection and tick challenge, the intensity of the tick challenge, and nutrition may affect the
outcome, with calves consistently parasitized by 3–4 weeks of age [22]. Another complica-
tion for vaccines against T. orientalis is possible genetic diversity within genotypes [10,109],
a problem already appreciated for T. parva [101]. p
y pp
p
Whole blood vaccines against T. orientalis were not pursued due to the possible
transfer of viruses [58]. More recently, we have revisited the blood vaccine approach with
the “benign” buffeli genotype [36], as few cases of clinical theileriosis occur in Queensland
where this genotype has been present since 1910 [3]. However, as mentioned previously,
competent tick vectors for T. orientalis buffeli and the virulent genotypes are different
in Australia [33,46]. Calves inoculated intravenously (IV) or subcutaneously (SC) with
blood infected with T. orientalis buffeli became PCR-positive within 4 weeks. 5.3. Immunisation against T. orientalis with Blood Stabilates The infection
was allowed to “mature” for another 2–10 weeks. When challenged with 200 nymphal
H. longicornis infected with the Ikeda genotype of T. orientalis, the first peak of parasitemia
was significantly reduced by up to 80% between 6 and 9 weeks after challenge [27,47]. g
y
y
p
g
This mode of protection would not work for calves in endemic zones as the virulent
genotypes appear first [22,25]. It would be possible for proposed introductions that could
be “immunised” before movement into endemic areas. Consequently, groups of 12 cattle
aged 8–10 months were inoculated SC with either T. orientalis buffeli or Ikeda and were
positive 4 weeks later; a control group remained negative. Six weeks after “immunization”,
35 animals were transported 700 km to an endemic region with “heavy” tick challenge. Inoculated animals did not recrudesce with clinical theileriosis, and no animals died during
the first 6 months after arrival in spring. Those given T. orientalis Ikeda had significantly
reduced parasitaemia during the first wave 6–9 weeks after arrival. In contrast to the
previous study, prior immunization with T. orientalis buffeli was not significantly protective
(D. Emery, unpublished). PCV and weight gains showed a similar effect, but with adequate
feed available over summer, the initial weight losses had been recouped by compensatory
growth within 6 months after introduction (D. Emery, unpublished). The different outcomes
with T. orientalis buffeli was likely due to the heavier tick challenge, and studies are ongoing. y
g
g
g
Field trials in endemic regions with high levels of tick infestation carrying multiple
genotypes is vital to determine the robustness of this method to reduce the impact of
T. orientalis on survival and productivity. Despite the difficulties of experimental tick
infestation, these may be required to determine dose rates and genotypic combinations
for establishing any reliable immunisation protocols. There may also be some synergy for
a combination of “immunisation” before movement and the application of long-acting
effective acaricides on arrival to attenuate the tick challenge by also reducing the quantum
of ticks infesting immigrant cattle. However, once entering the carrier state, productivity
is expected to attain normal benchmarks [23,25]. Currently, restricting the levels of tick
infestation is the most viable option for the prevention of clinical theileriosis in newborn
calves in endemic regions. 6. Conclusions and Further Research The cattle industries in regions of endemic T. orientalis would benefit substantially
from measures to reduce the impact of the initial infection either by means to control the
vector or the early stages of the pathogenesis of the infection. If this can be managed into
the carrier state, then animals under conventional husbandry appear to be protected from
further clinical disease. For protection of cattle moving into endemic zones, the deliberate pre-infection of
cattle prior to movement requires further field trials in endemic regions with high levels Pathogens 2021, 10, 1153 10 of 14 10 of 14 of tick infestation carrying multiple genotypes are needed to determine the robustness
of the procedure. Despite the difficulties of experimental tick infestation, these may
be required to determine dose rates and genotypic combinations for establishing any
reliable immunisation protocols. There may also be some synergy for a combination of
pre-infection before movement and the application of long-acting effective acaricides on
arrival to attenuate the tick challenge by also reducing the quantum of ticks infesting
immigrant cattle [110,111], combined with rotational grazing after arrival [112]. However, pre-infection with merozoites does not address the other at-risk cohort;
neonatal calves born in endemic regions of T. orientalis. For these animals, tick attachment
and infection with T. orientalis Ikeda and Chitose occurs within the first week after birth. Limiting tick numbers could be approached by the location of calving paddocks well
removed from bushland to avoid H. longicornis carried by wildlife or possible movement of
calving times to avoid the seasonal appearances of adult ticks around spring. Since calving
intervals are usually 6–8 weeks duration, producers are reluctant to muster animals for
acaricide treatment of neonatal calves. However, the judicious use of effective existing and
new acaricides offer additional control options to reduce tick numbers and limit the dose
of theilerial genotypes transmitted for neonatal calves and introduced cattle. thor Contributions: Compiled and written by the author. Author Contributions: Compiled and written by the author. Funding: No direct funding; much of the research by the author was supported by Meat and
Livestock Australia. Acknowledgments: The author is especially grateful for a range of collaborations with researchers
from the University of Sydney, the Tick Fever Centre (TFC), Brisbane, and the Elisabeth Macarthur
Agricultural Institute, Menangle. Support for studies by the author was provided by Meat & Livestock
Australia. 6. Conclusions and Further Research Comments from reviewers were appreciated to enhance the quality of the manuscript. Conflicts of Interest: The author declares no conflict of interest. Conflicts of Interest: The author declares no conflict of interest. References 1. Stewart, N.P.; Standfast, N.H.; Baldock, F.C.; Reid, D.J.; de Vos, A.J. The distribution and prevalence of Theileria buffeli in cattle in
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Computational Modelling of Materials for Wind Turbine Blades: Selected DTU Wind Energy Activities
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Citation (APA):
Mikkelsen, L. P., & Mishnaevsky, L. (2017). Computational Modelling of Materials for Wind Turbine Blades:
Selected DTUWind Energy Activities. Materials, 10(11), Article 1278. https://doi.org/10.3390/ma10111278 General rights
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U
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h Computational Modelling of Materials for Wind Turbine Blades: Selected DTUWind
Energy Activities Mikkelsen, Lars Pilgaard; Mishnaevsky, Leon Mikkelsen, Lars Pilgaard; Mishnaevsky, Leon Document Version
Publisher's PDF, also known as Version of record Review
Computational Modelling of Materials for Wind
Turbine Blades: Selected DTU Wind Energy Activities
Lars Pilgaard Mikkelsen *
ID and Leon Mishnaevsky Jr. *
ID
Department of Wind Energy, Technical University of Denmark, DK-4000 Roskilde, Denmark
* Correspondence: lemi@dtu.dk (L.M.J.); lapm@dtu.dk (L.P.M.); Tel.: +45-4677-5729 (L.M.J.)
Received: 30 August 2017; Accepted: 24 October 2017; Published: 8 November 2017 Abstract: Computational and analytical studies of degradation of wind turbine blade materials at
the macro-, micro-, and nanoscale carried out by the modelling team of the Section Composites and
Materials Mechanics, Department of Wind Energy, DTU, are reviewed. Examples of the analysis of
the microstructural effects on the strength and fatigue life of composites are shown. Computational
studies of degradation mechanisms of wind blade composites under tensile and compressive loading
are presented. The effect of hybrid and nanoengineered structures on the performance of the
composite was studied in computational experiments as well. Keywords: composite materials; wind energy; modeling; micromechanics; finite elements 1. Introduction The perspectives of utilization and expansion of wind energy technology depend on the reliability
and lifetime of wind turbines. Degradation processes in wind turbine blades are controlled by
microscale processes in the materials [1]. The mechanical behavior and strength of materials can be
studied as a function of their microstructures using numerical models based on the computational
micro- and mesomechanics of the materials. In this paper, methods and approaches of computational
analysis of wind blade composites are discussed. The main objectives of these studies are: #
Understanding the degradation mechanisms of wind blades as a function of their structures,
and the prediction of the lifetime and performance of wind blades. For this, we estimate the
loads on the wind blades (Section 2), and develop the micromechanical models of wind blade
composite degradation at the level of fibers and bundles under tensile (Section 3) and compressive
(Section 4) loading. #
Exploring the promising ways to optimize the wind blade performance by tailoring the materials’
structures. Here, we develop computational models of hybrid and nanoengineered composites
(Section 5) and estimate the effect of these structures on the composite performance and lifetime. #
Exploring the promising ways to optimize the wind blade performance by tailoring the materials’
structures. Here, we develop computational models of hybrid and nanoengineered composites
(Section 5) and estimate the effect of these structures on the composite performance and lifetime. Mainly, the works carried out at the Modelling team of the Section Composites and Materials
Mechanics, Department of Wind Energy, DTU, are summarized. Link back to DTU Orbit Link back to DTU Orbit Citation (APA):
Mikkelsen, L. P., & Mishnaevsky, L. (2017). Computational Modelling of Materials for Wind Turbine Blades:
Selected DTUWind Energy Activities. Materials, 10(11), Article 1278. https://doi.org/10.3390/ma10111278 this document breaches copyright please contact us providing details, and we will remove access to the work immediate
ur claim. materials materials materials 2.1. Loads on a Wind Blade
A wind turbine blade A wind turbine blade is a long, slender structure where the dominating loads are given by the
aerodynamics and the gravitation. Seen on the large scale, a wind turbine blade is a simple beam
structure with well-defined load and boundary conditions. The beam theory describing the blade can
be made at different levels of complexity depending on its use [6]. An example of a more complex
version is the BECAS model (BEam Cross-section Analysis Software) (see, e.g., [7]). A simplified 1D
momentum theory (see, e.g., [6]), the maximum aerodynamic load on a blade in the flap-wise direction
can be simplified as a linear varying distributed load with vanishing load intensity at the root to a load
intensity of:
aerodynamics and the gravitation. Seen on the large scale, a wind turbine blade is a simple beam
structure with well-defined load and boundary conditions. The beam theory describing the blade
can be made at different levels of complexity depending on its use [6]. An example of a more
complex version is the BECAS model (BEam Cross-section Analysis Software) (see, e.g., [7]). A simplified 1D momentum theory (see, e.g., [6]), the maximum aerodynamic load on a blade in the
flap-wise direction can be simplified as a linear varying distributed load with vanishing load
intensity at the root to a load intensity of:
2
8
q
u
L
ρ
π
(1) qL = 8
27ρairu2
RπL
(1)
27
L
air
R
q
u
L
ρ
π
=
(1)
h
3
1 2
/
k
i
h d
i
f h
i
d
i
h (1)
(1) at the blade tip. Here, L is the blade length, ρair = 1.2 kg/m3 is the density of the air, and uR is the
so-called rated wind speed for the turbine which corresponds to the wind speed when the turbine
production is reaching the generator size. In the edge-wise direction, the load has, in [6], been assumed
to be constant. at the blade tip. Here, L is the blade length,
3
1.2
/
air
kg
m
ρ
=
is the density of the air, and
R
u is the
so-called rated wind speed for the turbine which corresponds to the wind speed when the turbine
production is reaching the generator size. In the edge-wise direction, the load has, in [6], been
assumed to be constant. 2. Loads on the Wind Blades and Stresses in the Material In this section, a simple method to estimate loads on the wind blades is presented. There exist
a number of studies estimating the loads on wind blades from aerodynamic, physical loads [2–5]. However, in this work, we seek to develop a simple, beam model-based approach, which can be
introduced in the complex materials model as a boundary condition. Materials 2017, 10, 1278; doi:10.3390/ma10111278 www.mdpi.com/journal/materials Materials 2017, 10, 1278
Materials 2017, 10, 1278 2 of 15
2 of 15 2.1. Loads on a Wind Blade
A wind turbine blade 2(
)
(
)
(
)
2
2
3
flap
flap
flap
flap
L
x
x
x
q
q
L
x
L
x
M
q
L
x
L
x
−
−
−
=
−
+
−
(2)
(
)
2
edge
x
blade
blade
L
x
M
A
g L
x
ρ
−
=
−
(3)
M f lap
x
= q f lap
x
(L −x) L −x
2
+ q f lap
L
−q f lap
x
2
(L −x)2(L −x)
3
(2)
Medge
x
= ρbladeAbladeg(L −x) L −x
2
(3) (2)
(2) (3)
(3) (
)
2
x
blade
bladeg
ρ
h
ft
b tit ti
f th l
d i t
it
b
itt
i
t
where, after substitution of the load intensity, can be written in stresses as: (
)
2
x
blade
bladeg
ρ
h
f
b i
i
f h l
d i
i
b
i
i
where, after substitution of the load intensity, can be written in stresses as: the load intensity, can be written in stresses as:
2
2
3
8
1
1
81
2
flap
air
R
x
flap
flap
x
u
L
x
x
A
h
L
L
ρ
π
σ
=
+
−
(4)
2
2
1
1
2
edge
blade
blade
x
edge
A
gL
x
A
h
L
ρ
σ
=
−
(5)
σ f lap
x
= 8
81
ρairu2
RπL3
A f laph f lap
x
1 + x
2L
1 −x
L
2
(4)
σedge
x
= 1
2
ρbladeAbladegL2
Aedgehedge
x
1 −x
L
2
(5) (4) flap
x
2
2
1
1
2
edge
blade
blade
x
edge
A
gL
x
A
h
L
ρ
σ
=
−
(5)
σedge
x
= 1
2
ρbladeAbladegL2
Aedgehedge
x
1 −x
L
2
(5) (5) 2
edge
x
A
h
L
where
2(
)
blade
flap
edge
A
A
A
=
+
. In order to achieve a constant stress level,
0
flap
edge
x
x
σ
σ
σ
=
=
in the
blade material, it can be seen from Equations (4) and (5) that the blade height,
flap
xh
, and blade
where Ablade = 2(A f lap + Aedge). 2.1. Loads on a Wind Blade
A wind turbine blade U i
th
i
lifi d
ti
h
i
Fi
1 th
fl
d
d
i
t i
th at the blade tip. Here, L is the blade length, ρair = 1.2 kg/m3 is the density of the air, and uR is the
so-called rated wind speed for the turbine which corresponds to the wind speed when the turbine
production is reaching the generator size. In the edge-wise direction, the load has, in [6], been assumed
to be constant. at the blade tip. Here, L is the blade length,
1.2
/
air
kg
m
ρ
=
is the density of the air, and
R
u is the
so-called rated wind speed for the turbine which corresponds to the wind speed when the turbine
production is reaching the generator size. In the edge-wise direction, the load has, in [6], been
assumed to be constant. U
h
l f d
h
h
fl
d
d
h Using the simplified cross-section shown in Figure 1, the flap and edge-wise moment in the blade
(see, e.g., [5]) can be found as follows:
Using the simplified cross-section shown in Figure 1, the flap and edge-wise moment in the
blade (see, e.g., [5]) can be found as follows: Figure 1. The simplified wind turbine blade cross-section used in the study. Upstream
Figure 1. The simplified wind turbine blade cross-section used in the study. Upstream Figure 1. The simplified wind turbine blade cross-section used in the study. Figure 1. The simplified wind turbine blade cross-section used in the study. Figure 1. The simplified wind turbine blade cross-section used in the study. Figure 1. The simplified wind turbine blade cross-section used in the study. 2.2. Stresses in a Wind Blade
where the case
86
L
m
=
, w
f
b
f k
bl Following Equation (6) will result in blade geometries which are solely based on material strength. In reality, the blade geometry is given by a combination of structural and aerodynamic requirements. In Figure 2, Equation (6) is compared with typical blade cross-section variations, where the case
L = 86m, while the other two cases are representative blade geometries averaged from a number of
known blade geometries. The blade height h f lap
0
and width hedge
0
are specified such that Equation (6)
is crossing the representative blade geometry in the largest value points which, for the thickness
direction, is at the blade root while, for the width, it is at a point located away from that (see Figure 2). During this, it turns out that h f lap
0
and hedge
0
, as a good approximation, can be assumed to follow linear
functions, with L as the blade length:
from a number of known blade geometries. The blade height
0h
and width
0h
are specified
such that Equation (6) is crossing the representative blade geometry in the largest value points
which, for the thickness direction, is at the blade root while, for the width, it is at a point located
away from that (see Figure 2). During this, it turns out that
0
flap
h
and
0
edge
h
, as a good
approximation, can be assumed to follow linear functions, with L as the blade length:
0
0
0.066
0.369
;
0.130
0.082
flap
edge
h
L
m
h
L
m
=
−
=
+
(7)
Therefore, based on the parameters given in [6], the mass of a specific wind turbine blade can be
estimated. Keeping in mind the assumption of a constant cross-section along the blade, the height h f lap
0
= 0.066L −0.369m; hedge
0
= 0.130L + 0.082m
(7)
e obtained aiming for a constant stress value throughout the blade. In
gn given by the material strength, the blade should also be stiff enough in
hitting the tower In [8] the distance from the blade tip to the tower is given (7)
In
h in Therefore, based on the parameters given in [6], the mass of a specific wind turbine blade can be
estimated. Keeping in mind the assumption of a constant cross-section along the blade, the height and
width variation are obtained aiming for a constant stress value throughout the blade. 2.1. Loads on a Wind Blade
A wind turbine blade In order to achieve a constant stress level, σ f lap
x
= σedge
x
= σ0 in the
blade material, it can be seen from Equations (4) and (5) that the blade height, h f lap
x
, and blade width,
hedge
x
, should vary in the following manner: 2
edge
x
A
h
L
where
2(
)
blade
flap
edge
A
A
A
=
+
. In order to achieve a constant stress level,
0
flap
edge
x
x
σ
σ
σ
=
=
in the
blade material, it can be seen from Equations (4) and (5) that the blade height,
flap
xh
, and blade
where Ablade = 2(A f lap + Aedge). In order to achieve a constant stress level, σ f lap
x
= σedge
x
= σ0 in the
blade material, it can be seen from Equations (4) and (5) that the blade height, h f lap
x
, and blade width,
hedge
x
, should vary in the following manner: y in the following manner:
2
2
1
1
;
1
flap
edge
x
x
h
h
x
x
x
=
+
−
=
−
(6)
h f lap
x
h f lap
0
=
1 + x
2L
1 −x
L
2
; hedge
x
hedge
0
=
1 −x
L
2
(6) (6) Materials 2017, 10, 1278
Following Equa
strength. In reality, 3 of 15
aterial
namic 2.2. Stresses in a Wind Blade
where the case
86
L
m
=
, w
f
b
f k
bl In addition to
a blade design given by the material strength, the blade should also be stiff enough in order to avoid
the blade hitting the tower. In [8], the distance from the blade tip to the tower is given as 18 m; this is,
therefore, the maximum allowable deflection wmax = 18m for a L = 86m turbine blade. Solving the
Bernoulli-Euler equations numerically, one obtains the rotation and deflection at the root section:
order to avoid the blade hitting the tower. In [8], the distance from the blade tip to the tower is given
as 18 m; this is, therefore, the maximum allowable deflection
max
18m
w
=
for a
86m
L =
turbine
blade. Solving the Bernoulli-Euler equations numerically, one obtains the rotation and deflection at
the root section:
(
)
1
1
1
1
1
;
1,.... 2
i
i
i
i
i
i
i
i
M
M
x
x
i
n
EI
EI
θ
θ
θ
θ
−
−
−
−
=
+
+
−
=
+
(8) θi = θi−1 + 1
2
Mi
EIi + Mi−1
EIi−1
(xi −xi−1)
;
i = 1, . . . n
wi = wi−1 + θi+θi−1
2
(xi −xi−1)
;
i = 1, . . . n
(8)
(
)
1
1
;
1,.... 2
i
i
i
i
w
w
x
x
i
n
−
−
=
+
−
=
erse deflection,
x
M is the moment in the beam at location x, and E is the
M
1 (8)
the where w is the transverse deflection, Mx is the moment in the beam at location x, and E is the material
stiffness, d2w
∂x2 = Mx
EIx withIx = 1
2 A f lap
h f lap
x
2
. material stiffness,
2
x
x
M
d w
dx
EI
=
with
2
1
(
)
2
flap
flap
x
x
I
A
h
=
. Figure 2. Typical blade geometries compared with the strength-determined geometries defined in
Equation (6). Figure 2. Typical blade geometries compared with the strength-determined geometries defined in
Equation (6). Figure 2. Typical blade geometries compared with the strength-determined geometries defined in
Equation (6). Figure 2. Typical blade geometries compared with the strength-determined geometries defined in
Equation (6). 3.1. Computational Modelling of Micromechanisms of Degradation of Wind Blade Composites In order to analyze the micromechanisms of damage in wind energy composites, micromechanical
finite element simulations have been used [29]. A series of programs has been developed, which
should automate the steps of the generation of 3D microstructural models of composites. After a 3D
microstructural model of a material with a complex microstructure is generated, the numerical testing
of the microstructure is carried out with the use of commercial finite element software, Abaqus 6.13
(Dassault Systèmes, Vélizy-Villacoublay, France). In order to model the fiber cracking, we introduced
potential fracture planes (in theform of damageable cohesive elements) in random sections of fibers,
suggested by González and LLorca [30]. The random arrangement of the potential failure planes
in this case reflects the statistical variability of the fiber properties. A similar concept was used to
simulate the interface cracking of composites. Given that surfaces of fibers can be rather rough, and
the interface regions in many composites contain interphases, the interface debonding was considered
not as a two-dimensional opening of two contacting plane surfaces, but rather as a three-dimensional
process in a thin layer. Thus, the interface was represented as a “third (interphase) material layer”
between the homogeneous fiber and matrix materials. The thickness of the interface layer can be varied
in simulations. The damage evolution in the damageable layers, placed in random sections of fibers, as
well as in the matrix and interphase layers, was modeled using the finite element weakening method. The idea of this approach is that the stiffness of finite elements is reduced if a stress or a damage
parameter in the element or a nodal point exceeds some critical level. This approach has been realized
in the Abaqus subroutine User Defined Field. In this subroutine, the phase to which a given finite
element in the model is assigned is defined through the field variable of the element. Figure 3a shows an example of the generated FE models with 20 fibers, and removed layers
of potential fracturing. A number of three-dimensional multifiber unit cells have been generated
automatically with the use of these programs and were subject to a uniaxial tensile displacement
loading along the axis of the fibers. As output results, the stress-strain curves and the damage strain
curves were obtained, as well as the stress and strain, and damaged element distributions in the unit
cells (see [29]). 3. Three Dimensional Modelling of Composite Degradation under Tensile Loading
3. Three Dimensional Modelling of Composite Degradation under Tensile Loading An important task of the analysis of wind blade materials is the prediction of service properties
and lifetime of the wind blades. This information can be obtained from computational modelling,
which can be carried out at several scale levels: mesolevel (laminates, sandwiches of the blade shells,
fiber bundles), microlevel (fibers and matrix), and nanolevel (interfaces, nanoparticles). In order to
simulate the deformation and strength of fiber reinforced composites, a number of approaches can
An important task of the analysis of wind blade materials is the prediction of service properties and
lifetime of the wind blades. This information can be obtained from computational modelling, which
can be carried out at several scale levels: mesolevel (laminates, sandwiches of the blade shells, fiber
bundles), microlevel (fibers and matrix), and nanolevel (interfaces, nanoparticles). In order to simulate
the deformation and strength of fiber reinforced composites, a number of approaches can be used, e.g.,
analytical models (often based on the shear lag model [9], among these models, one can mention the
break influence superposition (BIS) technique [10], Green function model [11,12], etc.); the fiber bundle Materials 2017, 10, 1278 4 of 15 model (FBM) developed initially by Daniels [13], and further improved and generalized by many
groups (see, for example, [14]), fracture mechanics-based models (often applied to the cases of brittle
matrix and fiber bridging [15–17]), continuum damage mechanics-based models [18,19], and numerical
continuum mechanical (often, finite element-based) models [20–27], and multiscale models [28]. A detailed overview of models of fiber-reinforced composites can be found elsewhere [25,26]. p
Below, the micromechanical unit cell models of composites at the level of the fiber/matrix and
bundles are reviewed. 3.2. Fiber Bundle Modelling with an Experimentally-Determined Fiber Distribution
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3.2. Fiber Bundle Modelling with an Experimentally-Determined Fiber Distribution 3.2. Fiber Bundle Modelling with an Experimentally-Determined Fiber Distribution
3.2. Fiber Bundle Modelling with an Experimentally-Determined Fiber Distribution The model of the Section 3.1 above was applied to an ideal unidirectional structure. In order to
simulate real structures of uni-directional non-crimp fabric-reinforced epoxy composites, an
approach based on 3D X-ray tomography scanning is used [31]. The bundle and fiber/matrix
structure of the composite is segmented as follows: A complete 0° load-carrying bundle, as well as
complete 90° and 45° bundles, are contained in the 3 mm × 3 mm × 3 mm FoV scan. This makes it
possible to choose three cut planes. Each cut plane is orthogonal to one of the bundles and contains
its complete cross-section, as shown in Figure 4a. All subsequent analysis is performed in those three
2D images, separately for each of the fiber bundles. The bundles are manually segmented, and the
example of a 45° bundle is shown in Figure 4. For detecting the individual fibers, the automatic
approach of Emerson et al. [32] is applied for estimating fiber centers. Fibers are then modeled as
circles with the diameters calculated so that no fibers overlap, but all fibers touch at least one
neighboring fiber. An example of the segmentation results for the fibers from a 45° bundle is shown
in Figure 3 right
The model of the Section 3.1 above was applied to an ideal unidirectional structure. In order to
simulate real structures of uni-directional non-crimp fabric-reinforced epoxy composites, an approach
based on 3D X-ray tomography scanning is used [31]. The bundle and fiber/matrix structure of the
composite is segmented as follows: A complete 0◦load-carrying bundle, as well as complete 90◦
and 45◦bundles, are contained in the 3 mm × 3 mm × 3 mm FoV scan. This makes it possible to
choose three cut planes. Each cut plane is orthogonal to one of the bundles and contains its complete
cross-section, as shown in Figure 4a. All subsequent analysis is performed in those three 2D images,
separately for each of the fiber bundles. The bundles are manually segmented, and the example of
a 45◦bundle is shown in Figure 4. For detecting the individual fibers, the automatic approach of
Emerson et al. [32] is applied for estimating fiber centers. Fibers are then modeled as circles with
the diameters calculated so that no fibers overlap, but all fibers touch at least one neighboring fiber. 3.1. Computational Modelling of Micromechanisms of Degradation of Wind Blade Composites On the basis of the simulations, it was concluded that homogeneous fibers ensure
a higher strength of a composite at the pre-critical load. However, the fibers with randomly-distributed
strengths lead to the higher strength at post-critical loads, i.e., after the fiber cracking started. Let us consider now the interaction between all three damage modes in composites (matrix cracks,
interface damage, and fiber fracture). Figure 3b,c show the results of simulations in the unit cell with
20 fibers: damage formation in the fibers, interface and matrix. The damage evolution begins by
formation of a crack in a fiber and (in another, rather far site) in the matrix (ε = 0.01). Then, the interface
crack forms near the fiber crack, and the large matrix crack is formed (ε = 0.015). It is of interest that, in the case when all the three damage mechanisms are possible, the competition
between the matrix cracking and the interface debonding is observed. In the area where the interface
is damaged, no matrix crack forms; vice versa, in the area where the long matrix cracks are formed,
the fiber cracking does not lead to the interface damage. One can conclude that local weak places
in composite interfaces can be rather beneficial for the composite strength and toughness: they can
prevent the matrix failure (by channeling the fracture energy into interface defects), and even delay the
fiber failure. Practically, it means that a heterogeneous interface (interface with both weak and strong
regions) can prevent the matrix failure and, therefore, ensure the integrity of the material. 5 of 15
ensure Materials 2017, 10, 1278
(interface with both (a)
(b)
(c)
Figure 3. 3D FE micromechanical model: (a) example of a 3D unit cell; (b) competition of damage
modes: one failed fiber and a few microcracks in the matrix (red); and (c) two fibers have failed, the
interface crack is formed in the vicinity of a fiber crack and the matrix crack is formed (ε = 0.015)
(reprinted from [29] with kind permission from Elsevier). Figure 3. 3D FE micromechanical model: (a) example of a 3D unit cell; (b) competition of damage
modes: one failed fiber and a few microcracks in the matrix (red); and (c) two fibers have failed,
the interface crack is formed in the vicinity of a fiber crack and the matrix crack is formed (ε = 0.015)
(reprinted from [29] with kind permission from Elsevier). 3.2. Fiber Bundle Modelling with an Experimentally-Determined Fiber Distribution
Th
d l
f h S
i
3 1 b
li d
id
l
idi
i
3.2. Fiber Bundle Modelling with an Experimentally-Determined Fiber Distribution An example of the segmentation results for the fibers from a 45◦bundle is shown in Figure 3, right. in Figure 3, right. Compared with the expected fiber diameter of 17 µm and 16 µm for the load carrying 0° and
the backing bundles (45°, 90°), the segmentation is found to underestimate the diameters somehow. As it can be seen in the zoomed image in Figure 4, right, it is difficult to distinguish individual fibers,
so some uncertainty may also be expected for the chosen resolution. The current segmentation is
performed for 2D cross-sections and a full 3D based segmentation is expected to give an improved
precision. Additionally, a scan with a higher zoom and a better resolution would improve the
precision significantly. The last column in Table 1 shows the estimated tex-values based on the
segmentated data. Compared with the tex-values reported for the fabric, the 0° load-carrying bundle
is found to be overestimated, while the backing bundles are found to be underestimated. Based on
the under-estimation of the fiber diameters, an under-estimation of the tex-values was expected for
all the bundles. Nevertheless, the boundary between the load carrying 0° bundles can be hard to
identify on a 2D slice so the segmented results may include some part of a neighboring bundle
Compared with the expected fiber diameter of 17 µm and 16 µm for the load carrying 0◦and
the backing bundles (45◦, 90◦), the segmentation is found to underestimate the diameters somehow. As it can be seen in the zoomed image in Figure 4, right, it is difficult to distinguish individual fibers,
so some uncertainty may also be expected for the chosen resolution. The current segmentation is
performed for 2D cross-sections and a full 3D based segmentation is expected to give an improved
precision. Additionally, a scan with a higher zoom and a better resolution would improve the
precision significantly. The last column in Table 1 shows the estimated tex-values based on the
segmentated data. Compared with the tex-values reported for the fabric, the 0◦load-carrying bundle
is found to be overestimated, while the backing bundles are found to be underestimated. Based on the
under-estimation of the fiber diameters, an under-estimation of the tex-values was expected for all the
bundles. Nevertheless, the boundary between the load carrying 0◦bundles can be hard to identify on
a 2D slice, so the segmented results may include some part of a neighboring bundle. 3.1. Computational Modelling of Micromechanisms of Degradation of Wind Blade Composites (b) (c) (a) (b) (a) (c) Figure 3. 3D FE micromechanical model: (a) example of a 3D unit cell; (b) competition of damage
modes: one failed fiber and a few microcracks in the matrix (red); and (c) two fibers have failed, the
interface crack is formed in the vicinity of a fiber crack and the matrix crack is formed (ε = 0.015)
(reprinted from [29] with kind permission from Elsevier). Figure 3. 3D FE micromechanical model: (a) example of a 3D unit cell; (b) competition of damage
modes: one failed fiber and a few microcracks in the matrix (red); and (c) two fibers have failed,
the interface crack is formed in the vicinity of a fiber crack and the matrix crack is formed (ε = 0.015)
(reprinted from [29] with kind permission from Elsevier). 3.2. Fiber Bundle Modelling with an Experimentally-Determined Fiber Distribution
Th
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3.2. Fiber Bundle Modelling with an Experimentally-Determined Fiber Distribution Table 1. Quantification from the fiber segmentation inside the bundles. Bundle
Number of
Fibers
Average Fiber
Diameter
Total Bundle
Area
Total Fiber
Area
Local Fiber
Volume Fraction
Estimate
Tex-Value
0◦
5954
15.8 µm
1.926 mm2
1.197 mm2
0.62
3113
45◦
794
14.9 µm
0.245 mm2
0.142 mm2
0.58
369
90◦
375
15.4 µm
0.125 mm2
0.071 mm2
0.57
185 Table 1. Quantification from the fiber segmentation inside the bundles. Materials 2017, 10, 1278 6 of 15 Based on the segmented fiber architecture, a two-dimensional micromechanical model oriented
orthogonally to the bundle orientation is built (see Figure 5a). In practice, the fiber architecture is
segmented as fiber center points and diameters. Based on this, a representative rectangular part of
the bundle area is selected for the finite element model and subsequently loaded in the transverse
direction. As the much stiffer fibers will constrain the matrix deformation in the fiber direction, a plane
strain linear triangle element is used. For the 45◦bundle, a 1.0 × 1.0 mm representative box with
443 fibers is selected. Only the solution for a very small part of the simulated representative box is
shown in Figure 5a. The local fiber volume fraction inside the representative volume is found to be
Vf = 0.56. Based on the micromechanical finite element model, the transverse stiffness and Poisson’s
ratio are found to be EFEM = 13.8 GPa and νFEM = 0.49, whereas the inverse rules of mixture will give
EInvRoM = 6.4 GPa and the Halpin-Tsai estimate will give EHalpin−Tsai = 11.8 GPa. When the full 3D
constitutive relation of the bundle structure is obtained, this will be applied on the segmented bundle
structure, which is sketched in Figure 5. Materials 2017, 10, 1278
6 of 15 (a)
(b)
(c)
Figure 4. Segmentation of 0°, magenta: 45°, and green: 90° bundles, where the 0° is in the loading
direction. (a) Cut planes, (b) Bundle segmentation, (c) Fibre segmentation. Figure 4. Segmentation of 0◦, magenta: 45◦, and green: 90◦bundles, where the 0◦is in the loading
direction. (a) Cut planes, (b) Bundle segmentation, (c) Fibre segmentation. Materials 2017, 10, 1278
6 of 15
(a)
(b)
(c)
Figure 4. 3.2. Fiber Bundle Modelling with an Experimentally-Determined Fiber Distribution
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3.2. Fiber Bundle Modelling with an Experimentally-Determined Fiber Distribution Segmentation of 0°, magenta: 45°, and green: 90° bundles, where the 0° is in the loading
di e tio
(a) Cut pla e
(b) Bu dle e
e tatio
(c) Fib e e
e tatio (c)
6 of 15 (b) (a)
Materials 2017, 10, 1278 (b) (c) (a) Figure 4. Segmentation of 0°, magenta: 45°, and green: 90° bundles, where the 0° is in the loading
direction. (a) Cut planes, (b) Bundle segmentation, (c) Fibre segmentation. Figure 4. Segmentation of 0◦, magenta: 45◦, and green: 90◦bundles, where the 0◦is in the loading
direction. (a) Cut planes, (b) Bundle segmentation, (c) Fibre segmentation. (a)
(b)
(c)
Figure 4. Segmentation of 0°, magenta: 45°, and green: 90° bundles, where the 0° is in the loading (a)
(b)
Figure 5 (a) The normal horizontal stress contour plot of a transverse-loaded 45° backing bundle and
(b) the 3D structure where the resulting constitutive law will be implemented. Based on the segmented fiber architecture, a two-dimensional micromechanical model oriented
(a)
(b)
Figure 5 (a) The normal horizontal stress contour plot of a transverse-loaded 45° backing bundle and
(b) the 3D structure where the resulting constitutive law will be implemented. Figure 5. (a)The normal horizontal stress contour plot of a transverse-loaded 45◦backing bundle and
(b) the 3D structure where the resulting constitutive law will be implemented. (b)
contour plot of a transverse-loaded 45° backing bundle and
(b) (a)
Figure 5 (a) The normal horizontal stress
(a) (b)
of a transverse-loaded
(b) oaded
(b) norm
(a) (b) the 3D structure where the resulting constitutive law will be implemented. Based on the segmented fiber architecture, a two-dimensional micromechanical model orien
Figure 5 (a) The normal horizontal stress contour plot of a transverse-loaded 45° backing bundle and
(b) the 3D structure where the resulting constitutive law will be implemented. Figure 5. (a)The normal horizontal stress contour plot of a transverse-loaded 45◦backing bundle and
(b) the 3D structure where the resulting constitutive law will be implemented. 4.1. Statistical Model of Compressive Damage Evolution
applied load, but rather by the load redistribution aft
This model can be applied, among others, to a In this section, we investigate the effect of microstructures and the statistical distribution of
microstructural parameters of the composites on the damage, and the compressive and fatigue strength
of carbon fiber-reinforced composites under compressive and cyclic loading. A computational model
of a composite with a number of randomly-distributed and randomly-misaligned fibers has been
developed [34]. The model is based on the Monte-Carlo method and the Budiansky-Fleck fiber kinking
condition. The fibers are randomly arranged in the cell, using the RSA (random sequential absorption)
algorithm. The misalignment angles are assigned to each fiber, using random normal number generator
(with truncated Gaussian probability distribution, from −3◦to 3◦). Then, the unit cells were subject to
axial loading (or repeated axial loadings). For each fiber, the kinking condition is checked according to
the Budiansky-Fleck formula. If one or several fibers kink, the stress is redistributed over the remaining
fibers, thus, increasing the load on the remaining fibers and the likelihood of their kinking. The load
sharing on the remaining fibers after kinking of some fibers was calculated using the “effective stress
concept” of damage mechanics and the power load sharing law. The schema of the multifiber unit cell
with random misalignments is given in Figure 6. compressive strength of the composites. In order to make the clustering effect better visible, we
considered the composite with 20% volume content of fibers. The unit cell models with random and
clustered arrangements of fibers were subject to loadings (“quick” loading scheme). In the
simulations, it was observed that the fiber clustering has no effect on the damage at the first “quick”
loading. However, at the second “quick” loading, the composites with clustered fiber arrangements
demonstrated sufficiently higher damage. Figure 6 shows the distributions of failed fibers in the
cases of clustered and random homogeneous fiber arrangements, for the N = 100 (five clusters), after
the second loading cycle. One can see that the clustered fiber arrangement leads to the quicker
failure of the composite, due to the effect of the load redistribution. While there might be no
difference between the clustered and homogeneous fiber arrangement if the material is not
pre-damaged, the clustered arrangement leads to the much quicker failure of fibers at the second
loading (or if the material is pre-damaged). 4.1. Statistical Model of Compressive Damage Evolution
applied load, but rather by the load redistribution aft
This model can be applied, among others, to a For instance, at the compressive stress is 1500 MPa, the
damage in the composite with clustered fibers is 32.5% higher than in the composite with
homogeneously-arranged fibers. Figure 5. A schema of the multifiber unit cell with random misalignments and fiber misalignments. Figure 6. A schema of the multifiber unit cell with random misalignments and fiber misalignments. Figure 5. A schema of the multifiber unit cell with random misalignments and fiber misalignments. Figure 6. A schema of the multifiber unit cell with random misalignments and fiber misalignments. Two scenarios of the load redistribution after the fiber kinking were considered, depending on the
“rate” of the load redistribution: “quick” loading (when the fibers are loaded, and fail independently,
and the load redistribution takes place only at the next loading), and “slow” loading (after a fiber is
failed, the stress on the remaining fibers increases instantly according to the “effective stress concept”
and “load sharing rule”, and so on for all the fibers which fail successively one after another). In the first
case, a j-th fiber does not “know” that the i-th fiber failed, until the next cycle of loading (the “quick”
loading term reflect non-sharing of the load, as if it was quicker than the load transfer from failed
to intact fibers). Only in the next cycle, the load is redistributed over remaining fibers. In the latter
case, the fibers kink one after another, depending on the misalignment of each fiber. Thus, the “slow”
loading leads to autocatalytic fiber kinking, caused not by the increase of the applied load, but rather
by the load redistribution after the fiber begins to kink. This model can be applied, among others, to analyze the effect of the fiber clustering on the
compressive strength of the composites. In order to make the clustering effect better visible, we
considered the composite with 20% volume content of fibers. The unit cell models with random
and clustered arrangements of fibers were subject to loadings (“quick” loading scheme). In the
simulations, it was observed that the fiber clustering has no effect on the damage at the first “quick”
loading. However, at the second “quick” loading, the composites with clustered fiber arrangements
demonstrated sufficiently higher damage. Figure 7 shows the distributions of failed fibers in the cases
of clustered and random homogeneous fiber arrangements, for the N = 100 (five clusters), after the
second loading cycle. orthogonally to the bundle orientation is built (see Figu
segmented as fiber center points and diameters. Based o
Based on the segmented fiber architecture, a two-dim
orthogonally to the bundle orientation is built (see Figu
4. Compressive Strength of Wind Turbine Composites the bundle area is selected for the finite element model and subsequently loaded in the transverse
direction. As the much stiffer fibers will constrain the matrix deformation in the fiber direction,
a plane strain linear triangle element is used. For the 45° bundle, a 1.0 × 1.0 mm representative box
with 443 fibers is selected. Only the solution for a very small part of the simulated representative box
is shown in Figure 5a. The local fiber volume fraction inside the representative volume is found to be
g
y
(
g
)
p
segmented as fiber center points and diameters. Based on this, a representative rectangular part of
the bundle area is selected for the finite element model and subsequently loaded in the transverse
direction. As the much stiffer fibers will constrain the matrix deformation in the fiber direction,
a plane strain linear triangle element is used. For the 45° bundle, a 1.0 × 1.0 mm representative box
with 443 fibers is selected. Only the solution for a very small part of the simulated representative box
The downwind side of the blade and the spar of the blade are subject to compressive loading. The failure mechanisms of unidirectional composites under compressive loading differ strongly from
those under tensile loading [33]. The compressive strength of composites is often sufficiently lower
than their tensile strength, especially for carbon fiber composites where it can be 50–60% of the
tensile strength. 0.56
f
V =
. Based on the micromechanical finite element model, the transverse stiffness and
Poisson’s ratio are found to be
13.8GPa
FEM
E
=
and
0.49
FEM
ν
=
, whereas the inverse rules of
y
y
p
p
is shown in Figure 5a. The local fiber volume fraction inside the representative volume is found to be
0.56
f
V =
. Based on the micromechanical finite element model, the transverse stiffness and
In this section, two approaches to simulate the compressive strength of composites are considered:
a statistical, fiber bundle-based model [34], and a smeared-out compressive approach [34]. The first Materials 2017, 10, 1278
another). In the first
loading (the “quick 7 of 15
ycle of
e load analytical approach can be easily extended to more complex loading (fatigue, complex structures),
while the smeared out model is applicable for larger amounts of fibers. transfer from failed to intact fibers). Only in the next cycle, the load is redistributed over remaining
fibers. orthogonally to the bundle orientation is built (see Figu
segmented as fiber center points and diameters. Based o
Based on the segmented fiber architecture, a two-dim
orthogonally to the bundle orientation is built (see Figu
4. Compressive Strength of Wind Turbine Composites In the latter case, the fibers kink one after another, depending on the misalignment of each
fiber. Thus, the “slow” loading leads to autocatalytic fiber kinking, caused not by the increase of the 4.1. Statistical Model of Compressive Damage Evolution
applied load, but rather by the load redistribution aft
This model can be applied, among others, to a Effect of Local Fiber Misalignment and Wrinkles on Compression Strength of the Composite As an alternative to modelling the individual fibers, which is far too detailed an approach when
we are talking about wind turbine blades which consist of millions of fibers, a smeared out approach
can be used where both the fiber and matrix constituents can be modelled as a non-linear
elastic-plastic model, but without modelling them individually. Such an approach is used in [35] and
the example on predictions for a specific material combination is shown in Figure 7. It has been
found that the kink-band formation is highly dependent on the plasticity of the matrix material
(the soft phase) in interaction with the fiber misalignment. Therefore, it has, e.g., been demonstrated
that temperature change from 20 °C to 50 °C can easily reduce the compression strength by 30%. In [36], the individual fiber orientation was segmented in a filament-wound carbon fiber and a glass
fiber composite. Thereby, rather good agreement between experimental measurement and
numerical predictions were obtained. S
d
t
d l
t li
it d t
d lli
th
i
f il
t th
fib
t i
As an alternative to modelling the individual fibers, which is far too detailed an approach
when we are talking about wind turbine blades which consist of millions of fibers, a smeared out
approach can be used where both the fiber and matrix constituents can be modelled as a non-linear
elastic-plastic model, but without modelling them individually. Such an approach is used in [35]
and the example on predictions for a specific material combination is shown in Figure 8. It has been
found that the kink-band formation is highly dependent on the plasticity of the matrix material (the
soft phase) in interaction with the fiber misalignment. Therefore, it has, e.g., been demonstrated
that temperature change from 20 ◦C to 50 ◦C can easily reduce the compression strength by 30%. In [36], the individual fiber orientation was segmented in a filament-wound carbon fiber and a glass
fiber composite. Thereby, rather good agreement between experimental measurement and numerical
predictions were obtained. Smeared out models are not limited to modelling the compression failure at the fiber matrix
level. In [35], a 2D FE model of alternating layers of elastic-plastic matrix and elastic orthotropic fiber
laminae was modelled. 4.1. Statistical Model of Compressive Damage Evolution
applied load, but rather by the load redistribution aft
This model can be applied, among others, to a One can see that the clustered fiber arrangement leads to the quicker failure of 8 of 15 Materials 2017, 10, 1278 the composite, due to the effect of the load redistribution. While there might be no difference between
the clustered and homogeneous fiber arrangement if the material is not pre-damaged, the clustered
arrangement leads to the much quicker failure of fibers at the second loading (or if the material is
pre-damaged). For instance, at the compressive stress is 1500 MPa, the damage in the composite with
clustered fibers is 32.5% higher than in the composite with homogeneously-arranged fibers. Materials 2017, 10, 1278
8 of 15 Figure 6. Distribution of failed fibers in the case of clustered and random homogeneous fiber
arrangements. The case of 100 fibers, five clusters, vc = 20%, damage parameter of 0.37 in random
structures, and 0.43 in clustered structures (reprinted from [34] with kind permission from Elsevier). Figure 7. Distribution of failed fibers in the case of clustered and random homogeneous fiber
arrangements. The case of 100 fibers, five clusters, vc = 20%, damage parameter of 0.37 in random
structures, and 0.43 in clustered structures (reprinted from [34] with kind permission from Elsevier). Figure 6. Distribution of failed fibers in the case of clustered and random homogeneous fiber
arrangements. The case of 100 fibers, five clusters, vc = 20%, damage parameter of 0.37 in random
structures, and 0.43 in clustered structures (reprinted from [34] with kind permission from Elsevier). Figure 7. Distribution of failed fibers in the case of clustered and random homogeneous fiber
arrangements. The case of 100 fibers, five clusters, vc = 20%, damage parameter of 0.37 in random
structures, and 0.43 in clustered structures (reprinted from [34] with kind permission from Elsevier). Figure 6. Distribution of failed fibers in the case of clustered and random homogeneous fiber
arrangements. The case of 100 fibers, five clusters, vc = 20%, damage parameter of 0.37 in random
structures, and 0.43 in clustered structures (reprinted from [34] with kind permission from Elsevier). Figure 7. Distribution of failed fibers in the case of clustered and random homogeneous fiber
arrangements. The case of 100 fibers, five clusters, vc = 20%, damage parameter of 0.37 in random
structures, and 0.43 in clustered structures (reprinted from [34] with kind permission from Elsevier). 4.2. Effect of Local Fiber Misalignment and Wrinkles on Compression Strength of the Composite
4.2. 4.1. Statistical Model of Compressive Damage Evolution
applied load, but rather by the load redistribution aft
This model can be applied, among others, to a Additionally, at this larger scale, the compression strength of a wrinkled
laminate was found to be strongly dependent on the maximum initial lamina misalignment angle
and the plasticity of the matrix material. Finally, it is found that in hybrid wrinkles, an increasing
amount of carbon increases the compression strength. I
additio
to th ou h thi k e
i kle
e
o
ide ed
a tial
i kle [37] It
a
ho
Smeared out models are not limited to modelling the compression failure at the fiber matrix level. In [35], a 2D FE model of alternating layers of elastic-plastic matrix and elastic orthotropic fiber laminae
was modelled. Additionally, at this larger scale, the compression strength of a wrinkled laminate was
found to be strongly dependent on the maximum initial lamina misalignment angle and the plasticity
of the matrix material. Finally, it is found that in hybrid wrinkles, an increasing amount of carbon
increases the compression strength. In addition to through-thickness wrinkles we considered partial wrinkles [37]. It was shown
that the undisturbed laminate next to the wrinkle along the length reduces the kinking strength
which, afterwards, increases when the undisturbed laminate is on top of that added below and/or
above the waviness. The delamination study demonstrates that the global wrinkle model actually
fails by delamination for an initial misorientation equal to, or greater than, 4°. In addition to through-thickness wrinkles we considered partial wrinkles [37]. It was shown that
the undisturbed laminate next to the wrinkle along the length reduces the kinking strength which,
afterwards, increases when the undisturbed laminate is on top of that added below and/or above
the waviness. The delamination study demonstrates that the global wrinkle model actually fails by
delamination for an initial misorientation equal to, or greater than, 4◦. 9 of 15
ctually Materials 2017, 10, 1278
above the waviness
fails by delamination (a)
(b)
Figure 7. Smeared out finite element modeling of the fibre reinforced polymer matrix composite. (a) Smeared out material behavior; (b) Kink-band formation with yielding zone. Figure 8. Smeared out finite element modeling of the fibre reinforced polymer matrix composite. (a) Smeared out material behavior; (b) Kink-band formation with yielding zone. (a) (b) (b) (a) Figure 7. Smeared out finite element modeling of the fibre reinforced polymer matrix composite. (a) Smeared out material behavior; (b) Kink-band formation with yielding zone. Figure 8. 4.1. Statistical Model of Compressive Damage Evolution
applied load, but rather by the load redistribution aft
This model can be applied, among others, to a Smeared out finite element modeling of the fibre reinforced polymer matrix composite. (a) Smeared out material behavior; (b) Kink-band formation with yielding zone. 5. Computational Modelling of Hybrid and Hierarchical Composites Recently, several new promising directions of wind blade composites development has attracted
the attention of researchers, namely, hybrid and nanoengineered materials. In a number of works, the strength and damage mechanisms of hybrid composites were
studied [38–43]. It was shown that the incorporation of glass fibers in carbon fiber-reinforced
composites allows the improvement of their impact properties and tensile strain to failure of
the carbon fibers. For the analysis of nanocomposites, a number of different micromechanical
models have been developed; for instance, approaches based on composite models (Halpin-Tsai
model, Mori–Tanaka theory, Eshelby model, and shear lag models [44–46]) and molecular
mechanics/molecular dynamics [47–52]. A number of special nanostructure-based models were
developed, e.g., the effective particle model [53], dilute suspension of clusters model [54], effective clay
platelet (effective nanofiller) [55], effective cluster material [56], etc. In this section, the computational
finite element of composites, developed in Sections 3 and 4, are applied to the analysis of the hybrid
and hierarchical composites. 5.1. Modellling of Hybrid Composites While the effect of the hybrid structures of unidirectional composites on the stiffness and weight
is apparent, and can be well described by the rule-of-mixture (higher fraction of lightweight and stiff
fibers means a proportional increase of the composite stiffness and reduction of the weight), the effect
of the hybridization on the damage and fatigue resistance is more complex. For the analysis of
structure-strength/damage resistance relationships of hybrid composites, computational experiments
have been carried out with the use of the probabilistic fiber bundle model and micromechanical
multifiber FE unit cell models [33,57–59]. Figure 9a,b show examples of the unit cell FE models of
glass/fiber hybrid composites, with fiber misalignment. Figure 9c shows the simulated cracking
in fibers. Further, the statistical model of composite cracking described in the previous section was applied
to the hybrid glass and hybrid composites. Figure 10 shows the critical stress (corresponding to the
load at which 50% of fibers fail), plotted versus the fraction of carbon fibers in hybrid glass/carbon
fiber composites [57], obtained using this model. One can see that the strength of hybrid composite decreases when the fibers are mixed,
reaches lowest point at the fraction of carbon fibers 40–50% (of all fibers) and then begins to grow
sharply, to achieve the highest point for the pure carbon composite. Similar results were obtained
experimentally [60]. 10 of 15
p y
btained Materials 2017, 10, 1278
achieve the highe perimentally [60]. (a)
(b)
(c)
Figure 8. Examples of unit cell FE models of glass/fiber hybrid composites (a) aligned;
and (b) misaligned fibers, and simulated cracking in fibers (c) (reprinted from [57] with kind
permission from Elsevier). Figure 9. Examples of unit cell FE models of glass/fiber hybrid composites (a) aligned; and
(b) misaligned fibers, and simulated cracking in fibers (c) (reprinted from [57] with kind permission
from Elsevier). terials 2017, 10, 1278
10 o (b) (c) (a) (b) (a) (c) Figure 8. Examples of unit cell FE models of glass/fiber hybrid composites (a) aligned;
and (b) misaligned fibers, and simulated cracking in fibers (c) (reprinted from [57] with kind
permission from Elsevier). Figure 9. Examples of unit cell FE models of glass/fiber hybrid composites (a) aligned; and
(b) misaligned fibers, and simulated cracking in fibers (c) (reprinted from [57] with kind permission
from Elsevier). ials 2017, 10, 1278
10 o Figure 9. 5.2. Nanoengineered Composites
5.2. Nanoengineered Composites It has been demonstrated [61–67] that nanoparticles distributed in the polymer matrix or at the
fiber/matrix interface of the composites should enhance the compressive strength and fatigue
lifetime of the composites. In order to analyze the effect of the secondary nanoparticles on the
mechanical behavior and strength of composites, computational multiscale models were developed,
which include the fiber/matrix interaction at the higher-scale level (microlevel) and
nanoparticles/epoxy matrix interaction at the nanolevel. h
h
f
ll
d l I
h
d l
l
h
l
It has been demonstrated [61–67] that nanoparticles distributed in the polymer matrix or at the
fiber/matrix interface of the composites should enhance the compressive strength and fatigue lifetime
of the composites. In order to analyze the effect of the secondary nanoparticles on the mechanical
behavior and strength of composites, computational multiscale models were developed, which include
the fiber/matrix interaction at the higher-scale level (microlevel) and nanoparticles/epoxy matrix
interaction at the nanolevel. Figure 10 shows a schema of a unit cell model. In this model, polymer composites with glass
fibers (GFRC), carbon fibers (CFRC), and hybrid fibers (HC) are reinforced with carbon nanotubes
(CNT). The aspect ratio of CNTs was 1000, and they were distributed in the sizing of the fibers. The volume fraction of the CNT reinforcement is 0.46%. As a result of simulations, it was shown that
the CNT enhances the fatigue performance (maximum stress and lifetime) in all considered
composites. For the very low cycle loading, the CNT reinforcement leads to 25–43% increase in the
stress while, for the millions of cycles, the CNT effect increases the stress by 64–120%. Similarly, the
nanoclay particles located in the matrix or in the fiber sizing, ensure drastically enhanced fatigue
lifetimes of composites [62]. h
h
f h
d l
f h
h
h
l f b
h
l
Figure 11 shows a schema of a unit cell model. In this model, polymer composites with glass
fibers (GFRC), carbon fibers (CFRC), and hybrid fibers (HC) are reinforced with carbon nanotubes
(CNT). The aspect ratio of CNTs was 1000, and they were distributed in the sizing of the fibers. The volume fraction of the CNT reinforcement is 0.46%. As a result of simulations, it was shown that
the CNT enhances the fatigue performance (maximum stress and lifetime) in all considered composites. 5.1. Modellling of Hybrid Composites Critical stress (corresponding to D = 50%) plotted versus the fraction of carbon fibers in
hybrid glass/carbon fiber composites (from [57], with kind permission of Elsevier). Figure 10. Critical stress (corresponding to D = 50%) plotted versus the fraction of carbon fibers in
hybrid glass/carbon fiber composites (from [57], with kind permission of Elsevier). Figure 9. Critical stress (corresponding to D = 50%) plotted versus the fraction of carbon fibers in
hybrid glass/carbon fiber composites (from [57], with kind permission of Elsevier). Figure 10. Critical stress (corresponding to D = 50%) plotted versus the fraction of carbon fibers in
hybrid glass/carbon fiber composites (from [57], with kind permission of Elsevier). 5.2. Nanoengineered Composites
5.2. Nanoengineered Composites As a result it was shown that the nanoclay (NC) secondary
reinforcement allows to increase fatigue resistance drastically. Composites with NC achieve the same
fatigue life (taken exemplarily at 5.68 × 107 cycles) as neat composites, while subject to 2–3.5 times
higher loadings. Composites with NC in fiber sizing, with exfoliated NC, and aligned NC ensure
much higher fatigue lifetimes than with NC in the matrix, with clustered or randomly-oriented
NC (respectively). Materials 2017, 10, 1278
11 of 15 Figure 10. Example of a unit cell model of CNT-reinforced hybrid composite. Reprinted from [62]
with kind permission of Elsevier. Figure 11. Example of a unit cell model of CNT-reinforced hybrid composite. Reprinted from [62] with
kind permission of Elsevier. Materials 2017, 10, 1278
11 of 15
Figure 10. Example of a unit cell model of CNT-reinforced hybrid composite. Reprinted from [62]
with kind permission of Elsevier Figure 10. Example of a unit cell model of CNT-reinforced hybrid composite. Reprinted from [62]
with kind permission of Elsevier. Figure 11. Example of a unit cell model of CNT-reinforced hybrid composite. Reprinted from [62] with
kind permission of Elsevier. Figure 10. Example of a unit cell model of CNT-reinforced hybrid composite. Reprinted from [62]
with kind permission of Elsevier (a)
(a) (a)
(b)
(c)
Figure 11. Schema of the model of thehierarchical fiber composite with nanoclay reinforcements (a)
and simulated crack paths in submodels (aligned and randomly-oriented platelets (b,c)). Reprinted
from [66] with kind permission of Elsevier. 6. Conclusions
An overview of computational studies of composites for wind energy applications carried over
(a)
(b)
(c)
Figure 11. Schema of the model of thehierarchical fiber composite with nanoclay reinforcements (a)
and simulated crack paths in submodels (aligned and randomly-oriented platelets (b,c)). Reprinted
from [66] with kind permission of Elsevier. 6. Conclusions
Figure 12. Schema of the model of thehierarchical fiber composite with nanoclay reinforcements
(a) and simulated crack paths in submodels (aligned and randomly-oriented platelets (b,c)). Reprinted from [66] with kind permission of Elsevier. (c)
fiber composite with nanoclay reinforcements (a
and randomly oriented platelets (b c)) Reprinted
(c) (b)
Figure 11. 5.2. Nanoengineered Composites
5.2. Nanoengineered Composites For the very low cycle loading, the CNT reinforcement leads to 25–43% increase in the stress while,
for the millions of cycles, the CNT effect increases the stress by 64–120%. Similarly, the nanoclay
particles located in the matrix or in the fiber sizing, ensure drastically enhanced fatigue lifetimes of
composites [62]. Figure 11 shows a schema of the model of the hierarchical fiber composite with nanoclay
reinforcements (a) and simulated crack paths in in-between platelets observed in the simulations
(aligned and randomly-oriented platelets, b and c) [66]. The composites with nanoclay reinforcement
achieve the same fatigue life (taken exemplarily at 5.68 × 107 cycles) as neat composites, while subject
to 2–3.5 times higher loadings. Composites with the nanoplatelets localized in the fiber/matrix
i te face laye (fibe si i g) e su e
uch highe fatigue lifeti
es tha
those
ith the
a oplatelets
Figure 12a shows a schema of the model of the hierarchical fiber composite with nanoclay
reinforcements (a) and simulated crack paths in in-between platelets observed in the simulations
(aligned and randomly-oriented platelets, b and c) [66]. The composites with nanoclay reinforcement
achieve the same fatigue life (taken exemplarily at 5.68 × 107 cycles) as neat composites, while subject
to 2–3.5 times higher loadings. Composites with the nanoplatelets localized in the fiber/matrix 11 of 15 Materials 2017, 10, 1278 interface layer (fiber sizing) ensure much higher fatigue lifetimes than those with the nanoplatelets in
the matrix: 43–49% higher applied stress corresponding to the selected lifetime of 5.68 × 107 cycles. In further simulations, composites with nanoclay secondary reinforcements were analyzed [62]. Figure 12b,c show the simulated crack path in the interface area of the composite with aligned and
randomly oriented nanoclay particles. As a result it was shown that the nanoclay (NC) secondary
reinforcement allows to increase fatigue resistance drastically. Composites with NC achieve the same
f
i
lif (
k
l
il
5 68
107
l
)
i
hil
bj
2 3 5 i interface layer (fiber sizing) ensure much higher fatigue lifetimes than those with the nanoplatelets in
the matrix: 43–49% higher applied stress corresponding to the selected lifetime of 5.68 × 107 cycles. In further simulations, composites with nanoclay secondary reinforcements were analyzed [62]. Figure 12b,c show the simulated crack path in the interface area of the composite with aligned and
randomly oriented nanoclay particles. 6. Conclusions An overview of computational studies of composites for wind energy applications carried over
from recent years in the Composite and Materials Mechanics Section, DTU Wind, is presented. In this
review, the computational models of the deformation and damage evolution of various composites
are presented. In the numerical experiments, damage mechanisms were analyzed. The competition
between matrix cracking and interface debonding was observed in numerical experiments. In the area,
where the interface is damaged, no matrix cracks form, and vice versa, in the area where the long matrix
cracks are formed, the fiber cracking does not lead to interface damage. One can conclude that local
weak places in composite interfaces can be rather beneficial for the composite strength and toughness:
they can prevent matrix failure (by channeling the fracture energy into interface defects), and even
delay the fiber failure. Practically, this means that a heterogeneous interface (interface with both weak
and strong regions) can prevent matrix failure and, therefore, ensure the integrity of the material. In the statistical model of composite compression, it was observed that the clustered, bundled fiber
arrangement leads to the quicker failure of the composite due to the effect of the load redistribution. While there might be no difference between the clustered and homogeneous fiber arrangement if the
material is not pre-damaged, the clustered arrangement leads to the much quicker failure of fibers at
the second loading (or if the material is pre-damaged). Compression strength of a wrinkled laminate was found to be strongly dependent on the
maximum initial lamina misalignment angle and the plasticity of the matrix material. Finally, it is
found that, in hybrid wrinkles, an increasing amount of carbon increases the compression strength. The undisturbed laminate next to the wrinkle along the length reduces the kinking strength, which
afterwards increases when undisturbed laminate are on top of that added below and/or above
the waviness. Further, the effect of hybrid and nanoengineered structures on the performance of the composite
was studied in computational experiments. It was shown that the strength of the hybrid composite
decreases when the carbon fibers replace glass fibers, reaching the lowest point at a fraction of carbon
fibers of 40–50% (of all fibers) and then begins to grow sharply, to achieve the highest point for pure
carbon composite. Further, it was observed that the nanoscale reinforcement enhances the fatigue
performance (maximum stress and lifetime) in all considered composites. 5.2. Nanoengineered Composites
5.2. Nanoengineered Composites Schema of the model of thehierarchica
d i
l t d
k
th i
b
d l ( li
d
(b) the mo
(b) h nano
(c) a
i
u a e
a
pa
i
u
o e
(a ig e
a
a
o
y o ie
e
p a e e
( , ))
ep i
e
from [66] with kind permission of Elsevier. 6. Conclusions
A
o e
ie
of o
utatio al tudie of o
o ite fo
i d e e
y a
li atio
a ied o e
Figure 11. Schema of the model of thehierarchical fiber composite with nanoclay reinforcements (a)
and simulated crack paths in submodels (aligned and randomly-oriented platelets (b,c)). Reprinted
from [66] with kind permission of Elsevier. 6 Conclusions
Figure 12. Schema of the model of thehierarchical fiber composite with nanoclay reinforcements
(a) and simulated crack paths in submodels (aligned and randomly-oriented platelets (b,c)). Reprinted from [66] with kind permission of Elsevier. Materials 2017, 10, 1278 12 of 15 12 of 15 6. Conclusions For very low cycle loading,
the nanoreinforcement leads to a 25–43% increase in the stress while, for the millions of cycles, the CNT
effect increases the stress by 64–120%. Similarly, the nanoclay particles located in the matrix or in the
fiber sizing ensure drastically enhanced fatigue lifetimes of the composites. Acknowledgments: The authors gratefully acknowledge the financial support of the Danish Council for Strategic
Research (DSF) via the Danish Centre for Composite Structures and Materials for Wind Turbines (DCCSM)
(contract No. 09-067212) and the collaborative project ‘High reliability of large wind turbines via computational
micromechanics based enhancement of materials performances’ (ref. No. 10-094539). Author Contributions: L.P.M. wrote the Sections 2, 3.2 and 4.2. L.M.J. prepared the Sections 3.1, 4.1, 5 and 6 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.
Del Campo, V.; .Ragni, D.; Micallef, D.; Diez, F.J.; Simão Ferreira, C.J. Estimation of loads on a horizon
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3. Del Campo, V.; .Ragni, D.; Micallef, D.; Diez, F.J.; Simão Ferreira, C.J. Estimation of loads on a horizontal axis
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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English
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Functional Characterisation of Germinant Receptors in Clostridium botulinum and Clostridium sporogenes Presents Novel Insights into Spore Germination Systems
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PLOS pathogens
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cc-by
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Functional Characterisation of Germinant Receptors in
Clostridium botulinum and Clostridium sporogenes
Presents Novel Insights into Spore Germination Systems Jason Brunt*, June Plowman, Duncan J. H. Gaskin, Manoa Itchner, Andrew T. Carter, Michael W. Peck
Gut Health and Food Safety, Institute of Food Research (IFR), Norwich Research Park, Colney, Norwich, Norfolk, United Kingdom ason Brunt*, June Plowman, Duncan J. H. Gaskin, Manoa Itchner, Andrew T. Carte
ut Health and Food Safety, Institute of Food Research (IFR), Norwich Research Park, Colney, Norwich, Norfolk, United Kingdom Abstract D t
A
il bilit
Th
th
fi
th t ll d t
d
l i
th fi di
f ll
il bl
ith
t
t i ti
All
l
t d t
ithi
th
d it Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within
ting Information files. Funding: This research was funded by the BBSRC Institute Strategic Programme on Gut Health and Food Safety (BB/J004529/1). 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. * Email: Jason.brunt@ifr.ac.uk * Email: Jason.brunt@ifr.ac.uk C. botulinum is a heterogeneous species that comprises a
complex of four distinct groups of bacteria that share the
common
property
of
forming
the
botulinum
neurotoxin
[3,10,11]. Group I (proteolytic) C. botulinum is associated with
foodborne botulism, infant botulism and wound botulism, and
forms one or more neurotoxins of types A, B, F or H [3,5,10,11]. Strains of Group I C. botulinum that form type A1 neurotoxin
have received the most attention to date because they are often
associated with botulism in humans, the extreme potency of the
type A1 neurotoxin, and due to the use of type A1 neurotoxin as
a pharmaceutical [1,12–15]. Indeed, the first C. botulinum
genome to be sequenced was that of Group I C. botulinum type
A1 strain ATCC3502 [16]. Functional Characterisation of Germinant Receptors in
Clostridium botulinum and Clostridium sporogenes
Presents Novel Insights into Spore Germination Systems Abstract Clostridium botulinum is a dangerous pathogen that forms the highly potent botulinum toxin, which when ingested causes
a deadly neuroparalytic disease. The closely related Clostridium sporogenes is occasionally pathogenic, frequently associated
with food spoilage and regarded as the non-toxigenic equivalent of Group I C. botulinum. Both species form highly resistant
spores that are ubiquitous in the environment and which, under favourable growth conditions germinate to produce
vegetative cells. To improve the control of botulinum neurotoxin-forming clostridia, it is imperative to comprehend the
mechanisms by which spores germinate. Germination is initiated following the recognition of small molecules (germinants)
by a specific germinant receptor (GR) located in the spore inner membrane. The present study precisely defines clostridial
GRs, germinants and co-germinants. Group I C. botulinum ATCC3502 contains two tricistronic and one pentacistronic GR
operons, while C. sporogenes ATCC15579 has three tricistronic and one tetracistronic GR operons. Insertional knockout
mutants, allied with characterisation of recombinant GRs shows for the first time that amino acid stimulated germination in
C. botulinum requires two tri-cistronic encoded GRs which act in synergy and cannot function individually. Spore
germination in C. sporogenes requires one tri-cistronic GR. Two other GRs form part of a complex involved in controlling the
rate of amino-acid stimulated germination. The suitability of using C. sporogenes as a substitute for C. botulinum in
germination studies and food challenge tests is discussed. Citation: Brunt J, Plowman J, Gaskin DJH, Itchner M, Carter AT, et al. (2014) Functional Characterisation of Germinant Receptors in Clostridium botulinum and
Clostridium sporogenes Presents Novel Insights into Spore Germination Systems. PLoS Pathog 10(9): e1004382. doi:10.1371/journal.ppat.1004382 Received May 2, 2014; Accepted August 4, 2014; Published September 11, 2014 Received May 2, 2014; Accepted August 4, 2014; Published September 11, 2014 nt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
tion, and reproduction in any medium, provided the original author and source are credited. Copyright: 2014 Brunt et al. This is an open-access article distributed under the terms of the Creative Commons Attributi
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2014 Brunt 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. Author Summary Clostridium botulinum is a dangerous pathogen that forms
the deadly botulinum neurotoxin. Strains of C. botulinum
are present in the environment as spores. Under suitable
conditions, the dormancy of the bacterial spore is broken,
and germination occurs. Germination is initiated following
the recognition of small molecules by a specific germinant
receptor (GR) located within spores. Currently, the identi-
fication
and
characterisation
of
these
GRs
remains
unknown, but is critical if strategies are to be developed
to either prevent spore germination altogether, or to
germinate all the spores and then inactivate the emergent
sensitive vegetative cells. The present study has charac-
terised two functionally active GRs in C. botulinum which
act in synergy and cannot function individually, and a
related functionally active GR in C. sporogenes. These GRs
respond to amino acids. Other GRs appear to form part of
a complex involved in controlling the speed of germina-
tion, or are not functionally active. This study provides new
insights into the mechanisms involved in germination and
will allow us to develop new strategies to control this
deadly pathogen. Strains of Group I C. botulinum and C. sporogenes are both
present in the environment as spores. This highly resistant
dormant state enables survival in adverse conditions (e.g. absence
of nutrients, UV light, heat treatment, radiation, desiccation, high
pressure, toxic chemicals) that vegetative cells would not survive,
and their formation by Group I C. botulinum and C. sporogenes is
a primary reason why these bacteria present a significant food
safety and food spoilage problem. Significantly, strains of Group I
C. botulinum and C. sporogenes form spores of very high heat
resistance, and the ‘‘botulinum cook’’ has been adopted by the
canning industry as the standard minimum heat treatment (121uC
for 3 min) for low acid canned foods [1]. Under suitable
conditions, the dormancy of bacterial spores is broken, and
germination occurs. This is often initiated by a germinant receptor
(GR) located in the spore inner membrane responding to nutrient
germinants, and is followed by the release of dipicolinic acid and
partial core hydration. Later, cortex-lytic enzymes degrade
peptidoglycan in the spore cortex, enabling further core hydration
and core expansion, and this results in the loss of spore resistance
[24]. Germination involves pre-formed enzymes located in the
dormant spore, and is followed by the initiation of metabolism and
macromolecular synthesis, eventually leading to the emergence of
a cell that is able to multiply. Introduction difficile are able to
germinate effectively in the absence of what is classically
understood as a GR. Such differences between clostridia probably
reflect their wide genetic diversity [1,24]. Spores of Group I C. botulinum and C. sporogenes germinate when specific germinant
nutrients such as a combination of L-alanine and L-lactate (with
less efficient germination in response to other amino acids and L-
lactate or single amino acids [3,44]) interact with a GR located in
the clostridial spore inner membrane. GR operons are well
conserved amongst strains of Group I C. botulinum. Group I C. botulinum type A strains ATCC3502, Hall, ATCC19397, Kyoto
and NCTC2012 possess a pentacistronic GR operon (gerXB-XA2-
XB2-XC2-XB), two tricistronic GR operons (gerXA1-XB1-XC1
and gerXA3-XB3-XC3), and an orphan gerXB homologue [3,45]. Gene clusters resembling the pentacistronic GR operon and
gerXA3-XB3-XC3 have been characterised in C. sporogenes strain
NCIMB701792 and Group I C. botulinum type B strain
NCTC7273, respectively [16,46]. Strains of Group I C. botulinum
type B (Okra) and F (Langeland) possess an additional tricistronic
GR operon (gerXC4-XA4-XB4) [3,45]. It is still not known,
however, to which nutrient germinant(s) the various specific GRs
are responding, or the relative importance of each of the GRs. Author Summary One approach that has interested
microbiologists for many decades is to develop strategies to either
prevent
spore
germination
altogether
and
thereby
prevent
subsequent growth, or to germinate all the spores and then
inactivate the emergent sensitive vegetative cells. Unfortunately
the highly heterogeneous nature of spore germination (as observed
for example with Group II C. botulinum) [25–27] has prevented
the development of suitable processes. However, a greater
understanding of spore germination in Group I C. botulinum
and C. sporogenes may enable the development of novel
intervention strategies to prevent or reduce disease and other
adverse events such as food spoilage. The purpose of the present study was to dissect, at the molecular
level,
spore
germination
in
Group
I
C. botulinum
strain
ATCC3502
and
in
C. sporogenes
strain
ATCC15579. In
particular, the key aims were to establish for each strain which
of the multiple nutrient GRs was active in spore germination, and
for the active nutrient GRs which nutrient germinant(s) they
responded to. This was achieved using a combination of genomic,
genetic and physiological approaches. The spore GRs in the two
clostridia showed a number of interesting similarities, and this
study provided further evidence of differences between spore
germination in Clostridium and Bacillus species. Interestingly,
subtle differences were also noted in spore germination between
that in Group I C. botulinum strain ATCC3502 and that in C. sporogenes strain ATCC15579. This study has provided novel
insights into spore germination in these two important clostridia. Results Involvement of L-lactate in amino acid induced
germination in C. botulinum and C. sporogenes Introduction Clostridium botulinum and Clostridium sporogenes are closely
related anaerobic spore-forming bacteria. C. botulinum is a
dangerous pathogen that forms the deadly botulinum neurotoxin. This is the most potent toxin known, as little as 30–100 ng can be
fatal [1]. Eight distinct types of botulinum neurotoxin (types A to H),
and more than thirty different neurotoxin sub-types (e.g. sub-types
A1 to A5) are recognised [2–5]. The botulinum neurotoxins are
150 kD proteins with zinc-endopeptidase activity that block
acetylcholine transmission in cholinergic nerves, leading to a floppy
paralysis known as botulism, that may prove fatal to both humans
and animals [6,7]. The most frequently reported types of human
botulism are foodborne botulism, infant botulism and wound
botulism [1,8]. Foodborne botulism is an intoxication caused by
consumption of neurotoxin formed by C. botulinum following spore
germination and growth of vegetative cells in food. Infant and
wound botulism are infections involving spore germination, growth
of vegetative cells and neurotoxin formation in the gut of young
infants and in deep wounds (often associated with drug abuse),
respectively. Botulinum neurotoxins are also important pharma-
ceuticals used to treat a range of localised conditions e.g. blepharospasm, hemifacial spasm, and for cosmetic purposes [9]. C. sporogenes is occasionally pathogenic [17], a significant cause
of food spoilage [18], and because of its strong physiological
similarity to Group I C. botulinum is very widely used as a
surrogate for this organism in demonstrating the effectiveness of
food preservation processes [19,20]. Genome sequencing, whole
genome analysis using DNA microarrays, and other typing
methods have confirmed the close genetic relationship of C. sporogenes and Group I C. botulinum [2,21–23]. The formation of
botulinum neurotoxin is used to distinguish strains of Group I C. botulinum from those of C. sporogenes [19]. September 2014 | Volume 10 | Issue 9 | e1004382 1 PLOS Pathogens | www.plospathogens.org Germinant Receptors in Clostridium Bacillus species, although recently significant advancements have
been made with several clostridia including C. perfringens, C. difficile and C. sordellii [24,29–43]. Spore germination in
clostridia often proceeds more slowly than that in Bacillus species
[1], and recent evidence suggests that although there are many
similarities there are also a number of important differences in
spore
germination
between
clostridia
and
Bacillus
species
[24,28,29]. For example, spores of Bacillus species require a
complete GR system to germinate effectively, and while this is also
the case for some clostridia, spores of C. PLOS Pathogens | www.plospathogens.org Involvement of L-lactate in amino acid induced
germination in C. botulinum and C. sporogenes A selection of amino acids at various concentrations (Table S1)
were assessed for their individual effect on germination of spores of
C. botulinum and C. sporogenes. The majority of the amino-acids
tested have previously been reported to contribute as germinants
or co-germinants for spores of Clostridium or Bacillus [1]. Initial
tests showed that spore germination was similar under aerobic and
anaerobic conditions (data not shown). This confirmed previous
reports for C. botulinum and C. sporogenes [47,48]. In the
presence of Tris buffer (pH 7.4), L-lactate (50 mM) and NaHCO3
(50 mM) at 30uC the addition at 100 mM of either L-alanine or L-
cysteine initiated spore germination in C. botulinum and C. sporogenes, although at differing rates (Figure 1a & 1b). L-lactate
was not essential for L-alanine or L-cysteine stimulated germina- Spore germination in Bacillus species generally involves a GR
located in the spore inner membrane. The GR is composed of
three proteins (A, B and C) that are encoded in a tricistronic
operon. The A and B proteins are integral membrane proteins,
while the C proteins are lipoproteins [24,28]. Spore germination
in Clostridium species is not as extensively studied as that in September 2014 | Volume 10 | Issue 9 | e1004382 PLOS Pathogens | www.plospathogens.org 2 Germinant Receptors in Clostridium tion and had no effect on rate or the overall extent of germination
(data not shown). Three amino acids (L-methionine, L-serine, L-
phenylalanine) each required L-lactate for inducement of germi-
nation in both species. The addition of L-lactate on its own failed
to stimulate germination (,10% fall in OD600, equating to ,1%
germination). Spores of C. botulinum, but not those of C. sporogenes, were also germinated by glycine in combination with
L-lactate (Figure 1a). L-cysteine combined with L-lactate pro-
duced the most rapid germination of C. botulinum spores (40% of
initial OD600, approximately 90% germination after 6 h) and C. sporogenes spores (40% of initial OD600, approximately 90%
germination after 4 h). C. sporogenes germination proceeded far
more rapidly to completion with all the tested amino acids
compared to C. botulinum. A number of other amino acids were
tested, but failed to induce spore germination in either C. botulinum or C. sporogenes, both in the presence and absence of
L-lactate (Table S1). For both species, the saturation concentration
of the amino acids was 50–100 mM with L-lactate at 50 mM
(Figure 1c & 1d). Homologues of the C. botulinum nutrient germinant
receptor operons in C. sporogenes p
p
p
g
Homologues of Group I C. botulinum strain ATCC3502 GR
sub-units (gerXA, gerXB, gerXC) were identified by BLASTp
analyses against a draft un-assembled genome of C. sporogenes
strain ATCC15579 (Figure 3). Analysis showed that the C. sporogenes strain contains three tricistronic GR operons and one
tetracistronic
GR
operon. In
comparison
C. botulinum
ATCC3502 has two tricistronic GR operons and one pentacis-
tronic GR operon. Each strain has an additional orphan gerXB
subunit homologue. Alignment using Clustal Omega and using
Jalview to produce a tree showing the average distance based on
amino acid sequence identity (%), revealed homology between the
GR operons (Figure 3). Each operon in C. botulinum had a closely
related operon in C. sporogenes (from 11.6–16.0% difference in
identity for each gerXA), while the CLOSPO_02140 gerXA is
most distant with regards to sequence % identity. Transmembrane
helix (TMH) prediction analysis showed that the gerXA sub-units
of C. botulinum and C. sporogenes have between three and five
TMHs, and the gerXB subunits have ten TMHs. The gerXC
subunits were predicted to be lipoproteins and encode a signal
peptide. Interestingly, more detailed sequence analysis of the
pentacistronic operon of C. botulinum reveals that although the
first gene of the operon, CBO1974, is a full-length member of the
gerXB family, the 59 end of its coding region is overlapped by a
small (162 bp) region of a gerXA gene, annotated as CBO1973A. Comparative analysis between C. botulinum and C. sporogenes ger
homologues also revealed that the gerXA gene, CLOSPO_00838
lacks an uninterrupted region encoding 20 amino acid residues
which map to residues 74–93 of the predicted translation for
CBO0123 (Figure 3b). A database search shows that this deletion
(with respect to C. botulinum strain ATCC3502) is not confined to
C. sporogenes strain ATCC15579, but can be found in 10 of a total
of 19 GerA peptide sequences from proteomes of C. sporogenes (2),
Group I C. botulinum (4) and Group III C. botulinum (4) (data not
shown). Similar deletions were not found in any Group II C. botulinum GerA peptides. The function of this apparently
conserved region remains unknown. Spore production environment affects rate of
germination g
The effect of sporulation medium and sporulation temperature
on the subsequent germination properties of C. sporogenes spores
was determined (Figure 2). Assessment of C. botulinum was
precluded by frequent poor sporulation of this strain, which was
less than 5% and 1% in Robertson’s cooked meat broth (CMB) and
TY respectively, compared to the optimum yield of 30% spores on
RCM plus skimmed milk (RCM+SM) plates at 37uC. C. sporogenes
spores were produced using either RCM+SM, CMB or TY broth at
37uC. Microscopic observations showed sporulation was notably
lower (ca. 40%) using CMB and TY compared to RCM+SM. C. sporogenes spores were also produced at 15, 20, 28, 30, 37 and 42uC
in CMB to evaluate the effect of temperature. Microscopic
observations showed that sporulation was notably lower (ca.50%)
at 15uC compared to 37uC in CMB, with sporulation not observed
at 42uC. Germination of the spores was then evaluated in the
presence of L-alanine + L-lactate (Figure 2). Interestingly, spores
produced in CMB at 37uC germinated at a faster rate compared to
spores produced in the other media at 37uC. Sporulation
temperature also affected germination rates, with spores produced
at 37uC germinating more rapidly than spores produced at other
temperatures (Figure 2). Subsequently, all spore crops used in
germination studies were produced at 37uC in CMB for C. sporogenes and on RCM+SM for C. botulinum. Involvement of L-lactate in amino acid induced
germination in C. botulinum and C. sporogenes For simplicity, L-lactate was included in all
subsequent germination studies. The optimum pH range was
pH 6–8 for germination with most amino acids +
L-lactate. However, spore germination was also observed at pH 10 in the
presence of L-serine, and L-alanine +
L-lactate. The rich
microbiological growth medium, TY medium, was not optimum
for spore germination, with less germination observed than in the
defined system, although the addition of L-lactate did increase the
rate and overall extent of germination in TY medium. Spore
germination measured using the Bioscreen system correlated well
with direct counts of phase-dark spores by phase-contrast
microscopy (data not shown) with an OD600 fall of 40%
correlating to .90% germination of spores. amino acids prevented spore germination (defined as a ,10% fall
in OD600, equating to ,1% germination observed microscopical-
ly) in C. botulinum and C. sporogenes induced by their equivalent
L-amino acid (Table 1). To ascertain if specific D-amino acids
could prevent germination by non-equivalent L-amino acids, D-
serine was tested at a ten-fold excess of each of the five L-amino
acids. Interestingly, D-serine prevented germination in C. spor-
ogenes induced by L-cysteine, L-methionine, L-phenylalanine and
L-serine, and to a lesser extent L-alanine. L-alanine and L-cysteine
induced germination was only slightly affected by a ten-fold excess
of D-serine in C. botulinum. However, when a 100-fold excess of
D-alanine was added, spore germination in the presence of each of
the five L-amino acids was prevented in C. botulinum. Finally, in
order to assess whether the D-amino acid is acting specifically
rather than being simply in excess of the L-amino acid germinant,
we performed further tests using different germinants (L-alanine,
1 mM or L-serine, 20 mM) each combined with an excess
(100 mM and 40 mM respectively) of the non-germinant L-valine. The addition of excess non-germinant in each experiment had no
effect on final germination levels. However, the D-amino acids (in
this case alanine or serine) continued to be inhibitory. C. botulinum requires two tri-cistronic receptors for
germination sporogenes), and triple (C. sporogenes) insertion mutants were
constructed. Furthermore, a C. sporogenes quadruple insertional
knockout GR mutant (gerXA42) was also generated. The current
insertional knockout system does not allow multiple insertion
selection, as following one insertion the single mutant is then
erythromycin resistant. However, the mutant generation system
was shown to be highly efficient, which negated the need for a
different antibiotic selection in the multiple insertion mutants. All
insertion events were tested by PCR which confirmed chromo-
somal integration of the intron (Figure S1). PCR using gene
specific and intron specific primers confirmed insertion of the
intron into the GR gene; this was further confirmed by PCR with
gene specific primers flanking the insertion site producing a ,2 kb
product (Figure S1). Insertion events were verified by Southern
hybridization using an intron specific probe which confirmed the
correct number of insertion events in all the constructed mutants
(Figure S1c). sporogenes), and triple (C. sporogenes) insertion mutants were
constructed. Furthermore, a C. sporogenes quadruple insertional
knockout GR mutant (gerXA42) was also generated. The current
insertional knockout system does not allow multiple insertion
selection, as following one insertion the single mutant is then
erythromycin resistant. However, the mutant generation system
was shown to be highly efficient, which negated the need for a
different antibiotic selection in the multiple insertion mutants. All
insertion events were tested by PCR which confirmed chromo-
somal integration of the intron (Figure S1). PCR using gene
specific and intron specific primers confirmed insertion of the
intron into the GR gene; this was further confirmed by PCR with
gene specific primers flanking the insertion site producing a ,2 kb
product (Figure S1). Insertion events were verified by Southern
hybridization using an intron specific probe which confirmed the
correct number of insertion events in all the constructed mutants
(Figure S1c). g
To characterise the GRs and to identify their cognate
germinants, single insertional knockout mutants (gerXA1-01232,
gerXA2-19752, gerXA3-27972) were created for each of the
identified GR operons in C. botulinum. Spores generated from
these mutants were then analysed for amino acid stimulated
germination using L-alanine, L-cysteine, L-methionine, L-serine,
L-phenylalanine or glycine, all in the presence of L-lactate
(Figure 4a). The OD600 of wild-type spores decreased (,40%)
indicating efficient and complete spore germination in the
presence of each amino acid. There was a ,20% decrease in
OD600 with TY medium + L-lactate. Mutation of putative germinant receptors Unlike their L-isomers, D-alanine, D-cysteine, D-methionine,
D-phenylalanine and D-serine all failed to trigger spore germina-
tion in either C. sporogenes or C. botulinum. Moreover, the D- To characterise the functionality of the putatively identified
germination GRs, a series of single (C. botulinum and C. September 2014 | Volume 10 | Issue 9 | e1004382 PLOS Pathogens | www.plospathogens.org 3 Germinant Receptors in Clostridium Figure 1. Rate of C. botulinum and C. sporogenes spore germination in the presence of selected amino acids. Effect of L-alanine, L-
cysteine, L-methionine, L-serine, L-phenylalanine and glycine (C. botulinum only) at 100 mM on germination of (a) C. botulinum ATCC3502 and (b) C. sporogenes ATCC15579. The effect of increasing L-alanine concentrations (0.5 mM–100 mM) on germination was also tested on (c) C. botulinum
ATCC3502 and (d) C. sporogenes ATCC15579. Tests were conducted in 20 mM Tris buffer, pH 7.4, L-lactate (50 mM) and NaHCO3 (50 mM) at 30uC. Buffer only controls contained 20 mM Tris, pH 7.4, L-lactate (50 mM) and NaHCO3 (50 mM). Data labels (right) refer to percentage germination
observed by phase contrast microscopy at the end of the experiment. Error bars represent the standard deviation of 3 independent experiments. doi:10.1371/journal.ppat.1004382.g001
Germinant Receptors in Clostridium ation in the presence of selected amino acids. Effect of L-alanine, L- Figure 1. Rate of C. botulinum and C. sporogenes spore germination in the presence of selected amino acids. Effect of L-alanine, L-
cysteine, L-methionine, L-serine, L-phenylalanine and glycine (C. botulinum only) at 100 mM on germination of (a) C. botulinum ATCC3502 and (b) C. sporogenes ATCC15579. The effect of increasing L-alanine concentrations (0.5 mM–100 mM) on germination was also tested on (c) C. botulinum
ATCC3502 and (d) C. sporogenes ATCC15579. Tests were conducted in 20 mM Tris buffer, pH 7.4, L-lactate (50 mM) and NaHCO3 (50 mM) at 30uC. Buffer only controls contained 20 mM Tris, pH 7.4, L-lactate (50 mM) and NaHCO3 (50 mM). Data labels (right) refer to percentage germination
observed by phase contrast microscopy at the end of the experiment. Error bars represent the standard deviation of 3 independent experiments. doi:10.1371/journal.ppat.1004382.g001 PLOS Pathogens | www.plospathogens.org September 2014 | Volume 10 | Issue 9 | e1004382 A single tri-cistronic receptor in C. sporogenes is essential
for germination A single tri-cistronic receptor in C. sporogenes is essential
for germination Figure 2. The effect of spore production environment on
subsequent germination of C. sporogenes spores. For germination
tests, spores were incubated in 20 mM Tris buffer, pH 7.4, L-alanine
(100 mM), L-lactate (50 mM) and NaHCO3 (50 mM) at 30uC. Spores were
produced in CMB at different temperatures; 15uC, 20uC, 28uC, 30uC,
37uC. Spores were also produced in TY broth at 37uC and on RCM+SM
plates at 37uC. Spores produced at 37uC in CMB and incubated in
20 mM Tris, pH 7.4, L-lactate (50 mM) and NaHCO3 (50 mM) only, were
included as a negative control (WT Buffer). Spore germination was
confirmed by phase contrast microscopy. Data labels (right) refer to
percentage germination observed by phase contrast microscopy at the
end of the experiment. Error bars represent the standard deviation of 3
independent experiments. doi:10.1371/journal.ppat.1004382.g002 To characterise the C. sporogenes GRs and to identify their
cognate germinants, single insertional knockout mutants (gerXA1-
008382, gerXA2-030062, gerXA3-022172, gerXA4-021402) were
created for each of the identified GR operons. The OD600 of wild-
type spores decreased (,40%) indicating efficient and complete
spore germination (Figure 4b). Mutant gerXA3-022172 failed to
germinate (,10% fall in OD600; spore germination not observed
microscopically) with any of the amino acids tested after 20 h of
exposure (Figure 4b & 4c). Spore germination was not observed
with a nutrient rich broth (TY + L-lactate) demonstrating that
CLOSPO_02217 is required for amino acid stimulated germina-
tion. Germination of mutant gerXA2-030062 showed an initial
delay of one hour compared to the wild type with all the amino
acids tested (e.g. Figure 4c). However, following this interval,
germination proceeded to the same extent, albeit at a slower rate
compared to the wild type (Figure 4b & 4c). Similarly, gerXA4-
021402 had an initial germination postponement of four hours
and then proceeded to germinate fully, but also at a slower rate. No discernible phenotype was observed following insertional
inactivation of the gerXA1 (mutant gerXA1-008382) GR com-
pared to the WT (Figure 4b & 4c). p
p
doi:10.1371/journal.ppat.1004382.g002 nutrient rich broth (TY + L-lactate) suggesting that CBO2797 is
essential for amino acid stimulated germination. The mutant
gerXA1-01232 also failed to germinate to the same extent as the
wild type, with a small decrease (,10%) in OD600 observed with
L-cysteine and with nutrient rich broth (TY + L-lactate). C. botulinum requires two tri-cistronic receptors for
germination In contrast, the gerXA3-
27972 mutant failed to germinate with any of the amino acids
tested even after 20 h of exposure (,1% germination observed
microscopically). Moreover, germination was not observed with a September 2014 | Volume 10 | Issue 9 | e1004382 September 2014 | Volume 10 | Issue 9 | e1004382 PLOS Pathogens | www.plospathogens.org 4 Germinant Receptors in Clostridium Figure 2. The effect of spore production environment on
subsequent germination of C. sporogenes spores. For germination
tests, spores were incubated in 20 mM Tris buffer, pH 7.4, L-alanine
(100 mM), L-lactate (50 mM) and NaHCO3 (50 mM) at 30uC. Spores were
produced in CMB at different temperatures; 15uC, 20uC, 28uC, 30uC,
37uC. Spores were also produced in TY broth at 37uC and on RCM+SM
plates at 37uC. Spores produced at 37uC in CMB and incubated in
20 mM Tris, pH 7.4, L-lactate (50 mM) and NaHCO3 (50 mM) only, were
included as a negative control (WT Buffer). Spore germination was
confirmed by phase contrast microscopy. Data labels (right) refer to
percentage germination observed by phase contrast microscopy at the
end of the experiment. Error bars represent the standard deviation of 3
independent experiments. doi:10.1371/journal.ppat.1004382.g002 (fdx) from C. sporogenes. Complementation was successful for one
of the two GerXAs observed to be important for nutrient-induced
germination. Introduction of the receptor CBO0123-CBO0124-
CBO0125
complementation
vector
(pMTL83151esp
or
pMTL83151fdx) successfully restored germination to the mutant
gerXA1-01232, albeit at a different rate compared to that of the
wild type (Figure 5a). Introduction of the GR CBO2797-
CBO2796-CBO2795 complementation vector (pMTL83151esp
or pMTL83151fdx) drastically reduced sporulation efficiency,
giving insufficient spores to allow assessment of the germination
phenotype. Finally, these results were further supported by examining the
number of colonies formed on TY plates after incubation for 2
days
at
37uC. All spore
crops
were
adjusted
to
a
final
concentration of ,16108 spores/ml, serially diluted, and plated
on to TY agar. Single mutant gerXA2-19752 showed comparable
numbers of colonies to the wild-type. In contrast, mutants gerXA1-
01232 and gerXA3-27972 exhibited a greatly reduced colony
forming efficiency (Figure 6). Figure 2. The effect of spore production environment on
subsequent germination of C. sporogenes spores. For germination A single tri-cistronic receptor in C. sporogenes is essential
for germination In neither
case could spore germination be observed microscopically. These
results suggest that CBO0123 is also essential for amino acid
induced germination. Interestingly, the gerXA2-19752 mutant
showed similar germination patterns to those of WT spores
(Figure 4a). Complementation was performed by two different
approaches; using plasmid pMTL83151esp, which relies on the
putative native promoter of the gene, or using pMTL83151fdx
which includes the powerful promoter Pfdx of the ferredoxin gene To further understand the function of the individual GRs in
germination,
triple
insertional
knockout
GerXA
mutants
(gerXA3200838+,
gerXA3202140+,
gerXA3202217+,
gerXA32
03006+) and a quadruple insertional knockout GerXA mutant Table 1. Minimum D-amino acid concentration required to prevent* germination by its equivalent L-amino acid. D-amino acid concentration
L-amino acid/concentration
C. sporogenes
C. botulinum
L-alanine/1 mM
10 mM
100 mM
L-cysteine/10 mM
100 mM
100 mM
L-serine/10 mM
20 mM
100 mM
L-phenylalanine/10 mM
20 mM
NT
L-methionine/10 mM
100 mM
100 mM
Tests were conducted in 20 mM Tris buffer, pH 7.4, L-amino acid (1–10 mM), D-amino acid (10–100 mM) + L-lactate (50 mM) and NaHCO3 (50 mM) at 30uC. NT = D-
phenylalanine was not inhibitory at 20 mM and could not be tested at a higher concentration due to low solubility. *Defined as a ,10% fall in OD600, equating to ,1% germination observed microscopically. doi:10.1371/journal.ppat.1004382.t001 Tests were conducted in 20 mM Tris buffer, pH 7.4, L-amino acid (1–10 mM), D-amino acid (10–100 mM) + L-lactate (50 mM) and NaHCO3 (50 mM) at 30uC. NT = D-
phenylalanine was not inhibitory at 20 mM and could not be tested at a higher concentration due to low solubility. *Defined as a ,10% fall in OD600, equating to ,1% germination observed microscopically. doi:10.1371/journal.ppat.1004382.t001 September 2014 | Volume 10 | Issue 9 | e1004382 September 2014 | Volume 10 | Issue 9 | e1004382 PLOS Pathogens | www.plospathogens.org 5 Germinant Receptors in Clostridium Figure 3. Alignment of C. botulinum and C. sporogenes germinant receptor proteins. Homologues of C. botulinum (strain ATCC3502) GRs
were identified by BLASTp analyses using the draft un-assembled genome of C. sporogenes (strain ATCC15579). (a) Tree calculated (using Jalview [76])
from the pairwise sequence distances between GerXA only (determined from % sequence identities) of C. sporogenes (CLOSPO_number.) and C. botulinum (CBOnumber.) GRs, using the UPGMA algorithm [76]; average distances between GerXA (green) are shown on the branches. A single tri-cistronic receptor in C. sporogenes is essential
for germination Mutation of three GerXAs resulted in one
remaining potentially functional GerXA so any possible interac-
tion between GerXAs is excluded and so enables dissection of the
specific germinant recognised. As anticipated, spores from the
quadruple insertional knockout mutant gerXA42 failed to germi-
nate (,10% fall in OD600, germination not observed microscop-
ically) with any of the amino acids or in nutrient rich broth (TY +
L-lactate) (Figure 4b). Furthermore, the three triple mutants
gerXA3200838+, gerXA3202140+, and gerXA3203006+ also failed
to germinate with the amino acid systems or in nutrient rich broth
(TY + L-lactate). Mutant gerXA3202217+ which has only a single
active GerXA present (CLOSPO_02217), displayed comparable
germination rates to the wild type with all the amino acids tested
and the nutrient rich broth (TY + L-lactate) (Figure 4b). Complementation of all the GerXAs further confirmed these
findings. Particularly important was the introduction of the GR
CLOSPO_02217- CLOSPO_02218- CLOSPO_02219 comple-
mentation vectors (pMTL83151esp or pMTL83151fdx), as these
fully restored germination to the mutant gerXA3-022172 to WT
levels (Figure 5b). gerXA42 were created. Mutation of three GerXAs resulted in one
remaining potentially functional GerXA so any possible interac-
tion between GerXAs is excluded and so enables dissection of the
specific germinant recognised. As anticipated, spores from the
quadruple insertional knockout mutant gerXA42 failed to germi-
nate (,10% fall in OD600, germination not observed microscop-
ically) with any of the amino acids or in nutrient rich broth (TY +
L-lactate) (Figure 4b). Furthermore, the three triple mutants
gerXA3200838+, gerXA3202140+, and gerXA3203006+ also failed
to germinate with the amino acid systems or in nutrient rich broth
(TY + L-lactate). Mutant gerXA3202217+ which has only a single
active GerXA present (CLOSPO_02217), displayed comparable
germination rates to the wild type with all the amino acids tested
and the nutrient rich broth (TY + L-lactate) (Figure 4b). Complementation of all the GerXAs further confirmed these
findings. Particularly important was the introduction of the GR
CLOSPO_02217- CLOSPO_02218- CLOSPO_02219 comple-
mentation vectors (pMTL83151esp or pMTL83151fdx), as these
fully restored germination to the mutant gerXA3-022172 to WT
levels (Figure 5b). A single tri-cistronic receptor in C. sporogenes is essential
for germination GerXB (red)
and GerXC (yellow) are shown on the same tree (UPGMA produced identical-topology trees for each of the GerXB, GerXC proteins; distances not
shown). (black triangle) Position of insertion sites of retargeted introns for mutations (in equivalent DNA sequence). Small green coloured region of
CBO1974 represents a small protein fragment (CBO1973A), the coding sequence of which overlaps that of CBO1974, with homology to the C
terminus of a GerXA protein. Blue square; 20 amino acid section that is deleted in its C. sporogenes homologue, CLOSPO_00838. (b) More detailed
version of part of the above tree, showing the amino acid sequence encoded by a region in CBO0123 that is deleted from its C. sporogenes
homologue, CLOSPO_00838. doi:10.1371/journal.ppat.1004382.g003 Figure 3. Alignment of C. botulinum and C. sporogenes germinant receptor proteins. Homologues of C. botulinum (strain ATCC3502) GRs
were identified by BLASTp analyses using the draft un-assembled genome of C. sporogenes (strain ATCC15579). (a) Tree calculated (using Jalview [76])
from the pairwise sequence distances between GerXA only (determined from % sequence identities) of C. sporogenes (CLOSPO_number.) and C. botulinum (CBOnumber.) GRs, using the UPGMA algorithm [76]; average distances between GerXA (green) are shown on the branches. GerXB (red)
and GerXC (yellow) are shown on the same tree (UPGMA produced identical-topology trees for each of the GerXB, GerXC proteins; distances not
shown). (black triangle) Position of insertion sites of retargeted introns for mutations (in equivalent DNA sequence). Small green coloured region of
CBO1974 represents a small protein fragment (CBO1973A), the coding sequence of which overlaps that of CBO1974, with homology to the C
terminus of a GerXA protein. Blue square; 20 amino acid section that is deleted in its C. sporogenes homologue, CLOSPO_00838. (b) More detailed
version of part of the above tree, showing the amino acid sequence encoded by a region in CBO0123 that is deleted from its C. sporogenes
homologue, CLOSPO_00838. doi:10.1371/journal.ppat.1004382.g003 serially diluted, and plated on to TY agar. The wild-type and
single insertion mutants gerXA1-008382, gerXA4-021402 and
gerXA2-030062 formed comparable numbers of colonies. In
contrast, mutant gerXA3-022172 showed a greatly reduced colony
forming efficiency (Figure 6). Triple mutants gerXA3200838+,
gerXA3202140+, gerXA3203006+ and the quadruple insertional
knockout mutant gerXA42 exhibited a significantly reduced colony
forming efficiency compared to the WT. Importantly, the triple
mutant gerXA3202217+, revealed comparable numbers of colonies
to the wild type (Figure 6). gerXA42 were created. PLOS Pathogens | www.plospathogens.org Discussion One important feature that has contributed to the success of
botulinum neurotoxin-forming clostridia, and all other clostridia,
is their ability to form highly resistant endospores. Under suitable
conditions the spores germinate with associated loss in resistance
properties, and cell multiplication recommences. Spore germina-
tion occurs through a number of steps that are poorly understood
in clostridia. The present study has identified which nutrient
germinants are able to stimulate spore germination in Group I C. botulinum ATCC3502 and in C. sporogenes ATCC15579, has for
the first time identified which of the individual GRs are responding Finally, the number of colonies formed on TY plates after
incubation for 2 days at 37uC was determined. All spore crops
were adjusted to a final concentration of ,16108 spores/ml, PLOS Pathogens | www.plospathogens.org September 2014 | Volume 10 | Issue 9 | e1004382 6 Germinant Receptors in Clostridium Germinant Receptors in Clostridium to these nutrient germinants. A survey of the available genomes of
G
I C b t li
d it
l
l ti
C
p
l
presence of multiple gerXB genes in the pentacistronic operon of
G
I C b t li
ATCC3502 (CBO1974 1978)
d i
it
Figure 4. Mutation of specific receptors precludes amino acid stimulated germination. Spores were incubated in 20 mM Tris buffer,
pH 7.4, amino acid (100 mM) + L-lactate (50 mM) and NaHCO3 (50 mM) at 30uC for 20 hours with L-alanine, L-cysteine, L-methionine, L-serine, L-
phenylalanine, glycine (C. botulinum only) or in TY + L-lactate (50 mM). (a) C. botulinum single insertional knockout mutants and wild type spore
germination. (b) C. sporogenes single insertional knockout mutants, triple insertional knockout mutants, quadruple insertional knockout GR mutant
and wild type spore germination. * L-cysteine is a relatively insoluble amino acid and precipitates out of solution after 2 hours. Due to the 4 hour
delay in germination of the mutant gerXA4-021402 cysteine precipitation caused OD600 readings to be unrepresentative and therefore germination
was confirmed by microscopy. (c) Alanine induced germination rates of single insertional knockout GR mutants for C. sporogenes were determined
using spores generated from the wild type (C. sporogenes ATCC15579) and mutants gerXA1-008382, gerXA4-021402, gerXA3-022172, gerXA2-030062. WT spores incubated in buffer only (see above), were included as a negative control (WT Buffer). Data labels (right) refer to percentage germination
observed by phase contrast microscopy at the end of the experiment. Discussion Error bars in (a–c) represent the standard deviation of 3 independent
experiments. Spore germination was confirmed by phase contrast microscopy. doi:10.1371/journal.ppat.1004382.g004 Figure 4. Mutation of specific receptors precludes amino acid stimulated germination. Spores were incubated in 20 mM Tris buffer,
pH 7.4, amino acid (100 mM) + L-lactate (50 mM) and NaHCO3 (50 mM) at 30uC for 20 hours with L-alanine, L-cysteine, L-methionine, L-serine, L-
phenylalanine, glycine (C. botulinum only) or in TY + L-lactate (50 mM). (a) C. botulinum single insertional knockout mutants and wild type spore
germination. (b) C. sporogenes single insertional knockout mutants, triple insertional knockout mutants, quadruple insertional knockout GR mutant
and wild type spore germination. * L-cysteine is a relatively insoluble amino acid and precipitates out of solution after 2 hours. Due to the 4 hour
delay in germination of the mutant gerXA4-021402 cysteine precipitation caused OD600 readings to be unrepresentative and therefore germination
was confirmed by microscopy. (c) Alanine induced germination rates of single insertional knockout GR mutants for C. sporogenes were determined
using spores generated from the wild type (C. sporogenes ATCC15579) and mutants gerXA1-008382, gerXA4-021402, gerXA3-022172, gerXA2-030062. WT spores incubated in buffer only (see above), were included as a negative control (WT Buffer). Data labels (right) refer to percentage germination
observed by phase contrast microscopy at the end of the experiment. Error bars in (a–c) represent the standard deviation of 3 independent
experiments. Spore germination was confirmed by phase contrast microscopy. doi:10.1371/journal.ppat.1004382.g004 presence of multiple gerXB genes in the pentacistronic operon of
Group I C. botulinum ATCC3502 (CBO1974-1978), and in its
tetracistronic
equivalent
in
C. sporogenes
ATCC15579
(CLOSPO_03006-03003). Less obvious evidence includes the
small fragment of a gerXA gene apparently inserted into the
beginning of the first ‘extra’ gerXB gene of the Group I C. botulinum ATCC3502 pentacistronic operon, and the extra (or
deleted) 20 codons discovered by comparison of the coding
sequences of CBO0123 and CLOSPO_00838. Site-directed
mutagenesis studies will be required to determine the functional
status of these genetic differences. presence of multiple gerXB genes in the pentacistronic operon of
Group I C. botulinum ATCC3502 (CBO1974-1978), and in its
tetracistronic
equivalent
in
C. sporogenes
ATCC15579
(CLOSPO_03006-03003). Less obvious evidence includes the
small fragment of a gerXA gene apparently inserted into the
beginning of the first ‘extra’ gerXB gene of the Group I C. Discussion botulinum ATCC3502 pentacistronic operon, and the extra (or
deleted) 20 codons discovered by comparison of the coding
sequences of CBO0123 and CLOSPO_00838. Site-directed
mutagenesis studies will be required to determine the functional
status of these genetic differences. to these nutrient germinants. A survey of the available genomes of
Group I C. botulinum and its close relative, C. sporogenes reveals
that although the general trend is to possess three or four operons
encoding spore GR proteins, the fine detail of this organisation
varies. The gerXC subunits were predicted to be lipoproteins and
encode a signal peptide. In Bacillus, a lipobox consensus sequence
(GCX) has been recognised within the first 30 residues of the GRs
C subunits, where the cysteine in this motif is diacylglycerylated to
facilitate cleavage of the signal peptide. In C. botulinum this
lipobox was observed in gerXC gene CBO0125 and in two C. sporogenes gerXC genes CLOSPO_00836 and CLOSPO_02139. However, diacylglycerol addition does not appear to be essential
for function [29]. In addition to the existence of orphan ger genes
(such as CBO2300 in Group I C. botulinum ATCC3502), there is
evidence of genetic recombination at these loci, most obviously the to these nutrient germinants. A survey of the available genomes of
Group I C. botulinum and its close relative, C. sporogenes reveals
that although the general trend is to possess three or four operons
encoding spore GR proteins, the fine detail of this organisation
varies. The gerXC subunits were predicted to be lipoproteins and
encode a signal peptide. In Bacillus, a lipobox consensus sequence
(GCX) has been recognised within the first 30 residues of the GRs
C subunits, where the cysteine in this motif is diacylglycerylated to
facilitate cleavage of the signal peptide. In C. botulinum this
lipobox was observed in gerXC gene CBO0125 and in two C. sporogenes gerXC genes CLOSPO_00836 and CLOSPO_02139. However, diacylglycerol addition does not appear to be essential
for function [29]. In addition to the existence of orphan ger genes
(such as CBO2300 in Group I C. botulinum ATCC3502), there is
evidence of genetic recombination at these loci, most obviously the Spore germination in Group I C. botulinum ATCC3502 and C. sporogenes ATCC15579 was triggered by a variety of amino acids,
often in combination with L-lactate. Discussion Germination rates of complemented GR mutants for
C. botulinum and C. sporogenes. Spores were incubated in 20 mM
Tris buffer, pH 7.4, amino acid (100 mM) + L-lactate (50 mM) + NaHCO3
(50 mM) at 30uC with L-cysteine (C. botulinum), L-alanine (C. spor-
ogenes). (a) C. botulinum mutant gerXA1-01232 complemented with
plasmid pMTL8315esp (gerXA1-01232esp) or plasmid pMTL8315fdx
(gerXA1-01232fdx). (b) C. sporogenes mutant gerXA3-022172 comple-
mented with plasmid pMTL8315esp (gerXA3-022172esp) or plasmid
pMTL8315fdx (gerXA3-022172fdx). There were two negative controls. Firstly, the uncomplemented mutant (gerXA1-01232 or gerXA3-022172),
secondly WT spores incubated in 20 mM Tris buffer (pH 7.4) + L-lactate
(50 mM) + NaHCO3 (50 mM) only (WT Buffer). Spore germination was
confirmed by phase contrast microscopy. Error bars represent the
standard deviation of 3 independent experiments. Data labels (right)
refer to percentage germination observed by phase contrast micros-
copy at the end of the experiment. doi:10.1371/journal.ppat.1004382.g005
Figure 6. Capacity of WT or mutant spores to germinate a
form colonies. C. botulinum and C. sporogenes wild type and mut
spore suspensions were enumerated using a haemocytometer a
adjusted to a final concentration of ,16108 spores/ml. Spores w
heat activated (80uC, 15 min), serially diluted in 0.85% saline, and pla
in triplicate on to TY agar before anaerobic incubation (37uC, 48 h
after which colonies were enumerated. Data presented represent
mean log10 colony-forming units/ml from triplicate plates, with er
bars representing the standard deviation of the mean. doi:10.1371/journal.ppat.1004382.g006 Figure 5. Germination rates of complemented GR mutants for
C. botulinum and C. sporogenes. Spores were incubated in 20 mM
Tris buffer, pH 7.4, amino acid (100 mM) + L-lactate (50 mM) + NaHCO3
(50 mM) at 30uC with L-cysteine (C. botulinum), L-alanine (C. spor-
) ( ) C b
li
XA
2
l
d
i h Figure 6. Capacity of WT or mutant spores to germinate and
form colonies. C. botulinum and C. sporogenes wild type and mutant
spore suspensions were enumerated using a haemocytometer and
adjusted to a final concentration of ,16108 spores/ml. Spores were
heat activated (80uC, 15 min), serially diluted in 0.85% saline, and plated
in triplicate on to TY agar before anaerobic incubation (37uC, 48 hrs),
after which colonies were enumerated. Data presented represent the
mean log10 colony-forming units/ml from triplicate plates, with error
bars representing the standard deviation of the mean. doi:10.1371/journal.ppat.1004382.g006 Figure 6. Capacity of WT or mutant spores to germinate and
form colonies. C. botulinum and C. Discussion Any effect is unlikely to be due to the hydrophobic
nature of L-phenylalanine, as L-alanine and L-cysteine also have
polar side chains and induce germination efficiently. In the present
study, germination was more rapid when induced by single amino
acids with L-lactate than in the nutrient rich medium TY; a similar
observation has been made for Group II C. botulinum [54]. However,
the
addition
of
L-lactate
to
TY
increased
the
germination rate significantly. Figure 5. Germination rates of complemented GR mutants for
C. botulinum and C. sporogenes. Spores were incubated in 20 mM
Tris buffer, pH 7.4, amino acid (100 mM) + L-lactate (50 mM) + NaHCO3
(50 mM) at 30uC with L-cysteine (C. botulinum), L-alanine (C. spor-
ogenes). (a) C. botulinum mutant gerXA1-01232 complemented with
plasmid pMTL8315esp (gerXA1-01232esp) or plasmid pMTL8315fdx
(gerXA1-01232fdx). (b) C. sporogenes mutant gerXA3-022172 comple-
mented with plasmid pMTL8315esp (gerXA3-022172esp) or plasmid
pMTL8315fdx (gerXA3-022172fdx). There were two negative controls. Firstly, the uncomplemented mutant (gerXA1-01232 or gerXA3-022172),
secondly WT spores incubated in 20 mM Tris buffer (pH 7.4) + L-lactate
(50 mM) + NaHCO3 (50 mM) only (WT Buffer). Spore germination was
confirmed by phase contrast microscopy. Error bars represent the
standard deviation of 3 independent experiments. Data labels (right)
refer to percentage germination observed by phase contrast micros-
copy at the end of the experiment. doi:10.1371/journal.ppat.1004382.g005 The production of spore crops is usually performed under
conditions that maximise the spore yield [55,56]. However,
mounting evidence now suggests that sporulation conditions may
have a direct effect on germination efficiency in Bacillus [55–57]. In the present study, a greater yield of C. sporogenes spores was
achieved on RCM+SM plates compared with CMB and TY
broths. However, the germination rate was initially more rapid
with spores produced in CMB, albeit all spore crops achieved a
similar extent of germination after 16 h. B. subtilis spores
produced in a liquid medium germinated more readily than
spores produced on plates [58]. Similarly, germination of B. subtilis spores with dodecylamine was also highly dependent on py
p
doi:10.1371/journal.ppat.1004382.g005 C. botulinum, while L-cysteine + L-lactate were the most effective
germinant for C. sporogenes. L-lactate had no discernible effect on
spore germination in the presence of L-alanine or L-cysteine, but
was essential for germination induced by L-methionine, L-serine
or L-phenylalanine. Previous studies have reported a variable
effect of L-lactate on amino acid induced spore germination in
Group I C. botulinum and C. sporogenes [1,44,46]. Discussion sporogenes wild type and mutant
spore suspensions were enumerated using a haemocytometer and
adjusted to a final concentration of ,16108 spores/ml. Spores were
heat activated (80uC, 15 min), serially diluted in 0.85% saline, and plated
in triplicate on to TY agar before anaerobic incubation (37uC, 48 hrs),
after which colonies were enumerated. Data presented represent the
mean log10 colony-forming units/ml from triplicate plates, with error
bars representing the standard deviation of the mean. doi:10.1371/journal.ppat.1004382.g006 induced by L-methionine + L-lactate in Group I C. botulinum
appears to be a novel finding. L-methionine triggered germination
has been previously reported in C. sporogenes [49], B. anthracis
[50] and C. tetani [51]. Germination in C. botulinum and C. sporogenes was also induced by L-phenylalanine + L-lactate, as
reported previously in C. bifermentans [52] and C. sordellii [30],
whereas the GR GerI in B. cereus interacts with L-phenylalanine
in combination with inosine [53]. Moreover, L-phenylalanine
stimulation of C. botulinum germination was more effective than
that obtained with L-alanine. It may be that L-phenylalanine and
L-lactate interact before interacting with the GR or that L-lactate
and L-phenylalanine may directly affect the GR together or
sequentially. Any effect is unlikely to be due to the hydrophobic
nature of L-phenylalanine, as L-alanine and L-cysteine also have
polar side chains and induce germination efficiently. In the present
study, germination was more rapid when induced by single amino
acids with L-lactate than in the nutrient rich medium TY; a similar
observation has been made for Group II C. botulinum [54]. However,
the
addition
of
L-lactate
to
TY
increased
the
germination rate significantly. induced by L-methionine + L-lactate in Group I C. botulinum
appears to be a novel finding. L-methionine triggered germination
has been previously reported in C. sporogenes [49], B. anthracis
[50] and C. tetani [51]. Germination in C. botulinum and C. sporogenes was also induced by L-phenylalanine + L-lactate, as
reported previously in C. bifermentans [52] and C. sordellii [30],
whereas the GR GerI in B. cereus interacts with L-phenylalanine
in combination with inosine [53]. Moreover, L-phenylalanine
stimulation of C. botulinum germination was more effective than
that obtained with L-alanine. It may be that L-phenylalanine and
L-lactate interact before interacting with the GR or that L-lactate
and L-phenylalanine may directly affect the GR together or
sequentially. Discussion L-phenylalanine + L-lactate
and L-cysteine + L-lactate were the most effective germinants for September 2014 | Volume 10 | Issue 9 | e1004382 September 2014 | Volume 10 | Issue 9 | e1004382 PLOS Pathogens | www.plospathogens.org 7 Germinant Receptors in Clostridium Figure 5. Germination rates of complemented GR mutants for
C. botulinum and C. sporogenes. Spores were incubated in 20 mM
Tris buffer, pH 7.4, amino acid (100 mM) + L-lactate (50 mM) + NaHCO3
(50 mM) at 30uC with L-cysteine (C. botulinum), L-alanine (C. spor-
ogenes). (a) C. botulinum mutant gerXA1-01232 complemented with
plasmid pMTL8315esp (gerXA1-01232esp) or plasmid pMTL8315fdx
(gerXA1-01232fdx). (b) C. sporogenes mutant gerXA3-022172 comple-
mented with plasmid pMTL8315esp (gerXA3-022172esp) or plasmid
pMTL8315fdx (gerXA3-022172fdx). There were two negative controls. Firstly, the uncomplemented mutant (gerXA1-01232 or gerXA3-022172),
secondly WT spores incubated in 20 mM Tris buffer (pH 7.4) + L-lactate
(50 mM) + NaHCO3 (50 mM) only (WT Buffer). Spore germination was
confirmed by phase contrast microscopy. Error bars represent the
standard deviation of 3 independent experiments. Data labels (right)
refer to percentage germination observed by phase contrast micros-
copy at the end of the experiment. doi:10.1371/journal.ppat.1004382.g005 induced by L-methionine + L-lactate in Group I C. botulin
appears to be a novel finding. L-methionine triggered germinat
has been previously reported in C. sporogenes [49], B. anthra
[50] and C. tetani [51]. Germination in C. botulinum and
sporogenes was also induced by L-phenylalanine + L-lactate,
reported previously in C. bifermentans [52] and C. sordellii [3
whereas the GR GerI in B. cereus interacts with L-phenylalan
in combination with inosine [53]. Moreover, L-phenylalan
stimulation of C. botulinum germination was more effective th
that obtained with L-alanine. It may be that L-phenylalanine a
L-lactate interact before interacting with the GR or that L-lact
and L-phenylalanine may directly affect the GR together
sequentially. Any effect is unlikely to be due to the hydropho
nature of L-phenylalanine, as L-alanine and L-cysteine also ha
polar side chains and induce germination efficiently. In the pres
study, germination was more rapid when induced by single am
acids with L-lactate than in the nutrient rich medium TY; a simi
observation has been made for Group II C. botulinum [5
However,
the
addition
of
L-lactate
to
TY
increased
germination rate significantly. The production of spore crops is usually performed und
conditions that maximise the spore yield [55 56]
Howev
Figure 5. PLOS Pathogens | www.plospathogens.org September 2014 | Volume 10 | Issue 9 | e1004382 Discussion Insertion mutant (gerXA2-19752) showed no
attributable phenotype, with a similar germination pattern to the
wild type strain. The pseudogene CBO1973A may hint at the
possibility of recombinational events that have occurred at this locus
that may have disrupted the normal control regions for correct GR
expression. It cannot be ruled out that germination may be
stimulated by suitable environmental stimuli that are not found in
the nutrient rich medium, TY broth or any of the specific
germinants tested in this work. In the present study, D-amino acids failed to trigger spore
germination and also prevented germination induced by their
respective L-amino acid, as reported previously for other strains of
Group I C. botulinum and C. sporogenes [44,47,48,62–64]. It is
noted that D-alanine was previously reported to be a competitive
inhibitor of L-alanine induced germination in C. sporogenes [48]. However, D-alanine did not prevent germination of spores of
Group II C. botulinum types B, E and F in L-alanine + L-lactate +
NaHCO3 (pH 7?0) when added at ten-times the concentration of
L-alanine [54]. Interestingly, in the present study, D-serine
prevented germination induced by L-amino acids in C. sporogenes,
and D-alanine prevented germination induced by L-amino acids
in C. botulinum. These observations are consistent with those
made by Montville et al. (1985), who reported that L-cysteine
triggered germination was inhibited by D-alanine as well as by D-
cysteine, and that L-alanine-triggered germination was inhibited
by D-cysteine as well as by D-alanine in Group I C. botulinum
strains B-aphis and Ba410 [64]. Montville et al. suggested that
alanine and cysteine shared a common germinant binding site in
spores of these two strains [64]. However, kinetic studies (e.g. [43])
are required to establish if the position is the same for Group I C. botulinum ATCC3502 and C. sporogenes ATCC15579. Studies
with B. megaterium and B. subtilis suggest that the B-protein
subunit of the GR presents the site for the receptor-ligand binding
[65,66], and although no evidence is presently available, the
position may be similar in C. botulinum and C. sporogenes. Furthermore, C. botulinum (ATCC3502) and C. sporogenes
(ATCC15579) both contain five putative alanine racemase genes. Alanine racemase is able to convert the germinant L-alanine into
inhibitory D-alanine in B. cereus [67]. However, despite these
clostridia containing five putative racemase genes, germination of
Group I botulinum spores appeared not to be influenced by L-
alanine racemase activity [44]. C. Discussion Germination
of Group I C. botulinum and C. sporogenes with L-serine and
glycine has been reported previously [44,49], while germination September 2014 | Volume 10 | Issue 9 | e1004382 PLOS Pathogens | www.plospathogens.org 8 Germinant Receptors in Clostridium this operon are functional. The requirement for two GRs for
germination has been previously reported in B. subtilis [68]. In this
bacterium the GRs GerB and GerK interact and responded to a
cocktail of L-asparagine, D-glucose, D-fructose, and K+ (AGFK)
[68]. The B. anthracis GRs GerK and GerL also act cooperatively
with alanine to stimulate the germination pathway [69]. Impor-
tantly, they can also act individually and initiate germination with
proline and methionine (GerK) or serine and valine (GerL) as
cogerminants in conjunction with inosine [69]. It is presently
unclear how pairs of GRs come together to induce germination;
potential hypotheses include: (i) one GR of the pair is involved in the
binding of the germinant and the second GR is involved in a
signalling capacity; (ii) both GRs together may be required to form
the germinant binding site; (iii) one GR may physically stabilise the
receptor that receives the germinant. What is clear is that more
evidence is required to characterise the individual role of each GR
in germinant recognition. In the present study, single C. botulinum
GRs failed to induce germination, either with single or with
combinations of amino acids, or with components of a rich growth
medium. Complementation restored wild type levels of germination
to the mutant gerXA1-01232, albeit at a slower rate compared to
that of the wild type. However restoration of germination efficiency
could not be assessed for the complementation mutant gerXA3-
27972 due to its poor sporulation efficiency. The failure of plasmid
complemented mutants to regain wild type sporulation levels has
been reported previously in C. perfringens [70]. Moreover, the use
of multicopy plasmids can fail to restore the phenotype to wild type
levels in clostridia [71]. It may be that in this instance inappropri-
ately elevated levels of the GR proteins in the complementation
mutant gerXA3-27972 diminished sporulation efficiency. Un-
doubtedly, until techniques become available for stable integration
of a single chromosomal copy of the complementing DNA,
complementation studies in clostridia will remain challenging. Group I C. botulinum ATCC3502 also has a pentacistronic putative
GR. PLOS Pathogens | www.plospathogens.org September 2014 | Volume 10 | Issue 9 | e1004382 Discussion This third GR, CBO1975-1977 (gerXA2-XB2-XC2), is
unusual as it is flanked by two additional gerXB genes (CBO1974
and CBO1978) and is closely related to a GR gene cluster
characterized in C. sporogenes ATCC15579 (this work). Further-
more, upstream of CBO1974 there is a pseudogene, CBO1973A
with a disrupted ORF which would encode the C-terminal region of
a GerA protein. This pseudogene overlaps the putative start of
CBO1974 (gerXB). Insertion mutant (gerXA2-19752) showed no
attributable phenotype, with a similar germination pattern to the
wild type strain. The pseudogene CBO1973A may hint at the
possibility of recombinational events that have occurred at this locus
that may have disrupted the normal control regions for correct GR
expression. It cannot be ruled out that germination may be
stimulated by suitable environmental stimuli that are not found in
the nutrient rich medium, TY broth or any of the specific
germinants tested in this work. C. sporogenes is often regarded as the non-toxigenic equivalent the method used for spore preparation [59]. Although the present
study shows that media composition for sporulation does have an
impact on germination, the reasons for these findings remain
unclear. The effect of sporulation temperature in CMB on the
yield of spores and their subsequent germination was also assessed. A greater number of spores were formed at 37uC than at lower
temperatures, and they also germinated more readily. Thus, on
this occasion spore yield and spore germination were positively
correlated. For Group II C. botulinum, sporulation temperature
affected spore yield and fatty acid content, but not heat resistance
or germination [60]. For B. subtilis, sporulation temperature
affected resistance to wet heat and spore coat protein levels [61]. Certainly, for C. sporogenes it is apparent that sporulation
conditions have a direct effect on subsequent germination with
the selected amino acids. It remains to be established whether this
effect is due to the number and/or state of GRs, or to as yet
unknown proteins that are involved in the germination pathway. However, it is clear from these results that sporulation conditions
should be considered, especially when Clostridium studies in the
food industry are to be performed to evaluate processing strategies,
as these typically use spores produced under conditions where the
yield has been maximised. p
q
germination has been previously reported in B. subtilis [68]. Discussion In this
bacterium the GRs GerB and GerK interact and responded to a
cocktail of L-asparagine, D-glucose, D-fructose, and K+ (AGFK)
[68]. The B. anthracis GRs GerK and GerL also act cooperatively
with alanine to stimulate the germination pathway [69]. Impor-
tantly, they can also act individually and initiate germination with
proline and methionine (GerK) or serine and valine (GerL) as
cogerminants in conjunction with inosine [69]. It is presently
unclear how pairs of GRs come together to induce germination;
potential hypotheses include: (i) one GR of the pair is involved in the
binding of the germinant and the second GR is involved in a
signalling capacity; (ii) both GRs together may be required to form
the germinant binding site; (iii) one GR may physically stabilise the
receptor that receives the germinant. What is clear is that more
evidence is required to characterise the individual role of each GR
in germinant recognition. In the present study, single C. botulinum
GRs failed to induce germination, either with single or with
combinations of amino acids, or with components of a rich growth
medium. Complementation restored wild type levels of germination
to the mutant gerXA1-01232, albeit at a slower rate compared to
that of the wild type. However restoration of germination efficiency
could not be assessed for the complementation mutant gerXA3-
27972 due to its poor sporulation efficiency. The failure of plasmid
complemented mutants to regain wild type sporulation levels has
been reported previously in C. perfringens [70]. Moreover, the use
of multicopy plasmids can fail to restore the phenotype to wild type
levels in clostridia [71]. It may be that in this instance inappropri-
ately elevated levels of the GR proteins in the complementation
mutant gerXA3-27972 diminished sporulation efficiency. Un-
doubtedly, until techniques become available for stable integration
of a single chromosomal copy of the complementing DNA,
complementation studies in clostridia will remain challenging. Group I C. botulinum ATCC3502 also has a pentacistronic putative
GR. This third GR, CBO1975-1977 (gerXA2-XB2-XC2), is
unusual as it is flanked by two additional gerXB genes (CBO1974
and CBO1978) and is closely related to a GR gene cluster
characterized in C. sporogenes ATCC15579 (this work). Further-
more, upstream of CBO1974 there is a pseudogene, CBO1973A
with a disrupted ORF which would encode the C-terminal region of
a GerA protein. This pseudogene overlaps the putative start of
CBO1974 (gerXB). Discussion sporogenes may have brought about an evolutionary pressure
which has allowed adaption of this organism to survive with only a
single functional GR operon (or at least a single functional
GerXA). Certainly the germination mechanism of C. sporogenes
ATCC15579 is different to that of C. botulinum ATCC3502. Alignment of putative GRs with known functioning GRs appears
to be problematic, as at present this is done over the whole protein
in the absence of detailed knowledge of the functionality of the
putative GR. However, based on comparison of the whole
proteins, the GerXA of homologous GR proteins CBO2795-
2797/CLOSPO_02217-02219 are functionally active, while no
discernible function could be identified for the GerXA of GR
proteins CBO1974-1978/CLOSPO_03003-03006. The GerXAs
of other GR proteins were either essential (in C. botulinum
ATCC3502)
or
contribute
to
the
rate
(in
C. sporogenes
ATCC15579). The results also call into the question the use of
C. sporogenes as a suitable substitute for C. botulinum with regards
to germination rates and germination substrates. More work is
required to fully understand the role of each GR in clostridia and
indeed why some species contain multiple GR operons and others
can function with just one. The testing of additional strains would
seem to be appropriate. The long term aim is that as more is understood of the complex
germination systems in clostridia, it may be possible to devise
specific strategies to disrupt this process. This would be of great
b
fi
h l
l
h
i
l
idi
f
l
i
h Bacterial strains and growth conditions Proteolytic C. botulinum strain ATCC3502 (neurotoxin subtype
A1) and C. sporogenes ATCC15579 were grown anaerobically at
37uC in tryptone-yeast medium (TY). The Escherichia coli strain
Top10 (Invitrogen) was used for plasmid maintenance and the E. coli strain CA434 [79] was used for conjugal transfer. Both strains
of E. coli were grown aerobically in Luria-Bertani medium (LB) at
37uC. Where appropriate, growth medium was supplemented with
antibiotics
at
the
following
final
concentrations;
ampicillin
100 mg/ml, chloramphenicol 25 mg/ml, cycloserine 250 mg/ml,
thiamphenicol 15 mg/ml, erythromycin 500 mg/ml (E. coli),
20 mg/ml (C. botulinum) 2.5 mg/ml (C. sporogenes), and the
chromogenic substrate 5-bromo-4-chloro-3-indolyl-b-D-galacto-
pyranoside (X-Gal) 80 mg/ml. All bacterial media supplements
were purchased from Sigma. Spores of C. botulinum and C. sporogenes strains were prepared in TY, Reinforced Clostridial
Medium plus skimmed milk (RCM+SM) or Robertson’s cooked
meat broth (CMB) (Southern Group Laboratories) and incubated
at 15, 20, 28, 30, 37 or 42uC for a period of 10 days. Spores were
cleaned and stored as described previously [54]. PCR, cloning and Southern hybridisation Constructed mutants and plasmids utilised in this study are
presented in Table 2. Primers used for verification of successful
insertion events and the Southern blot probes are listed in Table
S2 (supplementary material). PCR experiments were carried out
using Phusion High-Fidelity PCR Master Mix with GC Buffer kit
(Thermo Fisher). Plasmid isolation and PCR purification was
performed using the Wizard Plus SV Minipreps DNA Purification
System and the Wizard SV Gel and PCR Clean-Up System
(Promega) respectively, as described in the provided Technical
Manual. Chromosomal DNA isolation from suspected mutants
were prepared as previously described [16]. Restriction endonu-
cleases and T4 DNA ligase were purchased from New England
BioLabs and used according to the manufacturer’s instructions. Southern hybridisation was performed to confirm the correct
number of insertion events had occurred. The hybridisation probe
was constructed by PCR to target the inserted intron using the
primers Erm-F and Erm-R (Table S2). Genomic DNA (1 mg) was
digested overnight with HindIII restriction enzyme and the
fragments separated on a 1% agarose gel. Southern blot analysis
was performed with ECL detection using a commercial kit
(Amersham ECL Direct Nucleic Acid Labelling and Detection
System) according to the manufacturer’s instructions. The long term aim is that as more is understood of the complex
germination systems in clostridia, it may be possible to devise
specific strategies to disrupt this process. This would be of great
benefit to help control pathogenic clostridia, for example in the Receptor identification and alignment Homologues of C. botulinum ATCC3502 GR sub-units (gerXA,
gerXB, gerXC) were identified by BLASTp analyses against a draft
un-assembled genome of C. sporogenes strain ATCC15579. Alignment of C. sporogenes receptors with C. botulinum was
performed using Clustal Omega [75] and Jalview [76] was utilised
to produce a tree showing the average distance using % identity. Protein
domain
analysis
was
performed
using
Pfam
[77]. Transmembrane helix prediction analysis of the GR sub-units
was implemented using TMHMM [78]. Discussion The long term aim is that as more is understood of the complex food industry, and might also help to control spore-disseminated
nosocomial infections such as those caused by C. difficile. CLOSPO_02217-02219 GR. Indeed, preliminary proteomics
data
from
purified
spores
reveals
that
the
gerXC
gene
CLOSPO_00836 is translated into a protein (data not shown). Although in Bacillus all subunits of GRs are required for a
response to amino acids, the function of each individual subunit
still remains to be elucidated [72]. The finding that, in C. sporogenes ATCC15579, some or all of the proteins from two GRs
(CLOSPO_02139-02141 and CLOSPO_03003-03006) contribute
to increase the rate of spore germination induced by a third GR
(CLOSPO_02217-02219; or at least CLOSPO_02217) implies a
close interaction between GR proteins. Different GRs may
interact directly (and/or compete) with each other [73] or possibly
one GR may facilitate access of the germinant to another GR. Although these two GRs were not stimulated by individual or a
combination of amino acids, or by components of a rich
microbiological growth medium, it is possible that they may
individually respond to some other as yet unknown germinant. Interestingly, when the wild-type and various mutants containing
an active CLOSPO_02217 were plated out on a rich growth
medium recovery was complete (,107 CFU/ml), while in mutants
where CLOSPO_02217 was insertionally inactivated (including in
the quadruple gerXA42 mutant) the number of colonies recovered
was significantly lower (,103–104 CFU/ml). The recovery, at a
very
low
frequency,
of
any
spores
in
the
absence
of
CLOSPO_02217 (including in the quadruple gerXA42 mutant)
may be due to an alternative low efficiency receptor system distinct
from the ger family, or perhaps to stochastic effects (e.g. cortex-
lytic enzymes or ion/water channels). Similar observations have
been made in Bacillus subtilis [74]. It is perhaps not surprising that
this GR (CLOSPO_02217-02219) was involved in germination
with the selected amino acids as it shares .85% homology with
the
functional
and
now
characterised
C. botulinum
GR
(CBO2795-2797). It is interesting that this GerXA can operate
independently of any other GerXA, unlike in C. botulinum. One
hypothesis is that the short deletion in gerXA of CLOSPO_00838
(when compared to its active C. botulinum homologue, CBO0123)
is associated with loss of function. This mutational event in C. PLOS Pathogens | www.plospathogens.org Discussion sporogenes is often regarded as the non-toxigenic equivalent
of Group I C. botulinum [2,19]. Comparisons of C. botulinum and
C. sporogenes are important if C. sporogenes is to be used as a valid
surrogate model in spore germination and other studies. There are
four genes encoding GerXAs in C. sporogenes ATCC15579, and
only one of these (CLOSPO_02217) was essential for amino acid
induced germination. Two other GerXA proteins (products of
CLOSPO_02140 and CLOSPO_03006) increased the rate of
germination, providing that the product of CLOSPO_02217 was
also present. Therefore, one GR (CLOSPO_02217-02219; and at
least CLOSPO_02217) is required for amino acid stimulated spore
germination. The remaining three GerXAs were not essential, but
it cannot be ruled out that other products (GerXB and GerXC) of
these operons may be functional and act synergistically with the Molecular dissection of spore germination in Group I C. botulinum strain ATCC3502 demonstrated that two GerXAs were
required for amino acid stimulated germination. The interruption
of either gene CBO0123 or CBO2797 (mutants gerXA1-01232 and
gerXA3-27972) resulted in no observable germination. Thus, it has
been shown that for amino acid stimulated germination there is a
minimum requirement for the GerXAs produced by these two GRs,
and while the product of gene CBO1975 appears to be inactive, it
cannot be ruled out that the other products (GerXB and GerXC) of September 2014 | Volume 10 | Issue 9 | e1004382 9 Germinant Receptors in Clostridium CLOSPO_02217-02219 GR. Indeed, preliminary proteomics
data
from
purified
spores
reveals
that
the
gerXC
gene
CLOSPO_00836 is translated into a protein (data not shown). Although in Bacillus all subunits of GRs are required for a
response to amino acids, the function of each individual subunit
still remains to be elucidated [72]. The finding that, in C. sporogenes ATCC15579, some or all of the proteins from two GRs
(CLOSPO_02139-02141 and CLOSPO_03003-03006) contribute
to increase the rate of spore germination induced by a third GR
(CLOSPO_02217-02219; or at least CLOSPO_02217) implies a
close interaction between GR proteins. Different GRs may
interact directly (and/or compete) with each other [73] or possibly
one GR may facilitate access of the germinant to another GR. Although these two GRs were not stimulated by individual or a
combination of amino acids, or by components of a rich
microbiological growth medium, it is possible that they may
individually respond to some other as yet unknown germinant. Discussion Interestingly, when the wild-type and various mutants containing
an active CLOSPO_02217 were plated out on a rich growth
medium recovery was complete (,107 CFU/ml), while in mutants
where CLOSPO_02217 was insertionally inactivated (including in
the quadruple gerXA42 mutant) the number of colonies recovered
was significantly lower (,103–104 CFU/ml). The recovery, at a
very
low
frequency,
of
any
spores
in
the
absence
of
CLOSPO_02217 (including in the quadruple gerXA42 mutant)
may be due to an alternative low efficiency receptor system distinct
from the ger family, or perhaps to stochastic effects (e.g. cortex-
lytic enzymes or ion/water channels). Similar observations have
been made in Bacillus subtilis [74]. It is perhaps not surprising that
this GR (CLOSPO_02217-02219) was involved in germination
with the selected amino acids as it shares .85% homology with
the
functional
and
now
characterised
C. botulinum
GR
(CBO2795-2797). It is interesting that this GerXA can operate
independently of any other GerXA, unlike in C. botulinum. One
hypothesis is that the short deletion in gerXA of CLOSPO_00838
(when compared to its active C. botulinum homologue, CBO0123)
is associated with loss of function. This mutational event in C. sporogenes may have brought about an evolutionary pressure
which has allowed adaption of this organism to survive with only a
single functional GR operon (or at least a single functional
GerXA). Certainly the germination mechanism of C. sporogenes
ATCC15579 is different to that of C. botulinum ATCC3502. Alignment of putative GRs with known functioning GRs appears
to be problematic, as at present this is done over the whole protein
in the absence of detailed knowledge of the functionality of the
putative GR. However, based on comparison of the whole
proteins, the GerXA of homologous GR proteins CBO2795-
2797/CLOSPO_02217-02219 are functionally active, while no
discernible function could be identified for the GerXA of GR
proteins CBO1974-1978/CLOSPO_03003-03006. The GerXAs
of other GR proteins were either essential (in C. botulinum
ATCC3502)
or
contribute
to
the
rate
(in
C. sporogenes
ATCC15579). The results also call into the question the use of
C. sporogenes as a suitable substitute for C. botulinum with regards
to germination rates and germination substrates. More work is
required to fully understand the role of each GR in clostridia and
indeed why some species contain multiple GR operons and others
can function with just one. The testing of additional strains would
seem to be appropriate. Germinants and spore germination The potential germinants, including; L-alanine, L-serine, L-
cysteine,
L-methionine,
L-phenylalanine,
glycine
(0.5 mM– September 2014 | Volume 10 | Issue 9 | e1004382 10 Germinant Receptors in Clostridium Table 2. Constructed mutants and plasmids utilised in this study. 100 mM) and antagonists D-alanine, D-cysteine, D-methionine, D-
phenylalanine, D-serine (10 mM–200 mM depending on solubility)
(Sigma) were all prepared in Tris-HCl buffer (20 mM, pH 7.4) with
extent of germination by approximately 10% [54]. Germinant
solutions were prepared and filter sterilised (0.45-mm syringe filter,
Millipore, Bedford, MA). The pH of the germinant solutions was
Strain
CDS annotation
Locus
Insertion site
Mutant
Description
C. botulinum
(ATCC3502)
gerXA1
CBO0123
42s
gerXA1-01232
Single insertional
knockout
gerXA2
CBO1975
56s
gerXA2-19752
Single insertional
knockout
gerXA3
CBO2797
1344a
gerXA3-27972
Single insertional
knockout
C. sporogenes
(ATCC15579)
gerXA1
CLOSPO_00838
175s
gerXA1-008382
Single insertional
knockout
gerXA2
CLOSPO_03006
795s
gerXA2-030062
Single insertional
knockout
gerXA3
CLOSPO_02217
61s
gerXA3-022172
Single insertional
knockout
gerXA4
CLOSPO_02140
601s
gerXA4-021402
Single insertional
knockout
gerXA1,
CLOSPO_00838,
175s,
gerXA3202140+
Triple insertional
knockout leaving one
functional GerXA gerXA4
gerXA2,
CLOSPO_03006,
795s,
gerXA3
CLOSPO_02217
61s
gerXA1,
CLOSPO_00838,
175s,
gerXA3202217+
Triple insertional
knockout leaving one
functional GerXA gerXA3
gerXA2,
CLOSPO_03006,
795s,
gerXA4
CLOSPO_02140
601s
gerXA1,
CLOSPO_00838,
175s,
gerXA3203006+
Triple insertional
knockout leaving one
functional GerXA gerXA2
gerXA3,
CLOSPO_02217,
61s,
gerXA4
CLOSPO_02140
601s
gerXA2,
CLOSPO_03006,
795s,
gerXA3200838+
Triple insertional
knockout leaving one
functional GerXA gerXA1
gerXA3,
CLOSPO_02217,
61s,
gerXA4
CLOSPO_02140
601s
gerXA1,
CLOSPO_00838,
175s,
gerXA42
Quadruple insertional
knockout. Insertion in all
known gerXA sub-units
gerXA2,
CLOSPO_03006,
795s,
gerXA3,
CLOSPO_02217,
61s,
gerXA4
CLOSPO_02140
601s
Plasmids
pMTL007C-E2
Clostridial mutagenesis
plasmid
pMTL8315esp
GR expression plasmid
with no fdx promoter. Relies on native
promoter of inserted
genes
pMTL8315fdx
GR expression plasmid
with fdx promoter
2 indicates the gene is absent; + indicates that only that gene is functional; no2 describes the number of genes knocked out. a = antisense orientation insertion site;
s = sense orientation insertion site. The designation of ‘‘X’’ before the letter A is used in clostridia as unlike Bacillus, GRs in these species have not yet been attributed to
specific germinants. doi:10.1371/journal.ppat.1004382.t002 2 indicates the gene is absent; + indicates that only that gene is functional; no2 describes the number of genes knocked out. a = antisense orientation insertion site;
s = sense orientation insertion site. The designation of ‘‘X’’ before the letter A is used in clostridia as unlike Bacillus, GRs in these species have not yet been attributed to
specific germinants. September 2014 | Volume 10 | Issue 9 | e1004382 Mutant generation Clostridium mutants were generated using the Clostron system,
which inserts an erythromycin resistance cassette into the targeted
gene of interest. Target sites were identified using the Pertuka
method [80] and mutants were generated (Table 2) as described
by Heap et al. [81]. gerXA GR subunits were targeted in all
germination operons. Re-targeted introns were ligated into the
pMTL007C-E2 vector following restriction digest with HindIII/
BsrGI. All retargeted introns were sequence verified before
transformation into E. coli CA434. Confirmed sequenced plasmids
were then conjugated into their respective hosts. Finally, primers
were designed and used to confirm that the intron was present and
in the correct orientation in the target gene/genes of interest
(Table S2). The current insertional knockout system does not allow
selective isolation of clones containing a second intron insertion as
following one insertion the mutant strain is then erythromycin
resistant. To create the double, triple and quadruple insertional
knockout mutants an alternative approach was taken in which
clones containing intron insertions were identified by screening
large numbers of colonies rather than by antibiotic selection. Plasmid re-targeting was carried out as above and transferred in to
C. botulinum or C. sporogenes using E. coli CA434. Confirmation
of successful trans-conjugation events were screened on TY agar
plates containing cycloserine (250 mg/ml) and thiamphenicol
(15 mg/ml). To create the multiple insertional knockout mutants
the process was repeated using successive rounds of plasmid
targeting and gene insertion. Successful integration of introns into
the target genes was confirmed by PCR using primers flanking the
target sites (Table S2). Furthermore, to confirm that the required
number of insertion events had occurred, genomic DNA from the
mutants was digested with HindIII and subjected to Southern
analysis using an intron specific probe. Figure S2
Constructed plasmids used for expression of
Clostridium receptors in complementation experiments. Complementation plasmid components; catP (chloramphenicol
acetyltransferase) conferring thiamphenicol resistance in E.coli and
chloramphenicol resistance in Clostridium. repH (pCB102); Gram-
positive replicon. ColE1; Gram-negative replicon. traJ; plasmid
conjugative transfer protein. Esp3I; restriction enzyme site used
for insertion of GR operons. Plasmid pMTL8315fdx contains the
strong promoter Pfdx of the ferredoxin gene (fdx) from C. sporogenes NCIMB 10696. Plasmid pMTL8315esp contains no
additional promoter and relies on a wild type copy of the inserted
gene under the control of its native promoter (assumed to be
located in the intergenic region upstream of the first CDS of the
operon). Complementation For
complementation,
two
plasmids,
pMTL8315esp
and
pMTL8315fdx were constructed (Figure S2). Primers, with Esp3I
restriction sites were designed to amplify two GR fragments; one
included the 59 noncoding region encompassing the putative
promoter and was ligated into pMTL8315esp; the second fragment
contained the coding operon only and was ligated into the plasmid
pMTL8315-fdx which contains the strong promoter Pfdx of the
ferredoxin gene (fdx) from C. sporogenes NCIMB 10696. Following
confirmation by sequencing, GR plasmids were then transconjugated
into their respective mutants using E. coli CA434 as described earlier. Table S2
Primers used in this study. + denotes resulting
product contains .500 bp upstream of the named gene to be
certain that a native promoter is included. (PDF) Germinants and spore germination d i
/j
l doi:10.1371/journal.ppat.1004382.t002 100 mM) and antagonists D-alanine, D-cysteine, D-methionine, D-
phenylalanine, D-serine (10 mM–200 mM depending on solubility)
(Sigma) were all prepared in Tris-HCl buffer (20 mM, pH 7.4) with
NaHCO3 (50 mM), with or without L-lactate (50 mM). NaHCO3
was a non-essential component that increases the rate and overall extent of germination by approximately 10% [54]. Germinant
solutions were prepared and filter sterilised (0.45-mm syringe filter,
Millipore, Bedford, MA). The pH of the germinant solutions was
adjusted to evaluate the effect of pH on spore germination at pH 3
to pH 10. Prior to the addition of germinants all spore suspensions September 2014 | Volume 10 | Issue 9 | e1004382 11 PLOS Pathogens | www.plospathogens.org Germinant Receptors in Clostridium Spore crops were produced as above, except with the addition of
thiamphenicol (15 mg/ml) to the media to maintain the plasmid. were heat activated at 80uC for 10 min. Germination of spores at
30uC was measured by a decrease in optical density (OD) at 600 nm
every 5 min using a Bioscreen C analyser system (Labsystems,
Basingstoke, UK) under aerobic conditions. Germination was
expressed in terms of measured OD600 as a percentage of the initial
OD600. To validate the OD600 measurements, at the completion of
each test the proportion of germinated spores was visualised by the
assessment of 200 spores in at least ten fields using phase-contrast
microscopy. Typically, full germination was indicated when the
OD600 fell to ,40% of its initial value. In some tests a small fall in
OD600 was observed (,10% of initial value). This was attributed to
settling of spores in the Bioscreen wells, and was not accompanied
by microscopic observation of spore germination. Finally, the
capacity of spores to germinate and form colonies was assessed. Spore suspensions were enumerated using a haemocytometer and
adjusted to a final concentration of ,16108 spores/ml. Spores were
then heat activated (80uC, 15 min), serially diluted in 0.85% saline,
and plated in triplicate on to TY agar before incubation
anaerobically (37uC, 48 hrs). Mutant generation Both plasmids are adaptations of the pMTL80000
modular plasmid system of Heap et al., 2009 [82]. (PDF) Table
S1
Effect
of
amino
acid
concentrations
on
clostridial spore germination. * C. botulinum only, no
germination detected in C. sporogenes at 100 mM. A selection of
amino
acids,
were
assessed
for
their
individual
effect
on
germination of spores of C. botulinum and C. sporogenes in the
presence of Tris-HCl buffer (pH 7.4), L-lactate (50 mM) and
NaHCO3 (50 mM). Germination was determined by a decrease in
optical density (OD) at 600 nm using a Bioscreen C analyser and
validated by phase-contrast microscopy. (PDF) Supporting Information Figure S1
Confirmation of successful insertional muta-
genesis. A, PCR screens of C. botulinum ger gene mutants
confirms that the intron has successfully inserted into the target
gene. Primer set 1 anneals to the ErmRAM and confirms that the
RAM is spliced and therefore integrated (expected band size of
mutants 900 bp). Primer set 2, intron binding EBS universal primer
plus gene specific forward primer amplified sequence across the
intron-exon junction to confirm intron insertion into the desired
gene (expected band size of mutants 250 bp). Primer set 3 anneals to
each end of the target gene and confirms the intron is present in the
target gene (expected band size of mutants ,2 kb). Lanes labelled
WT show the PCR products (primer set 3) generated for each ger A
gene when wild type DNA is used as template. B, Diagrammatic
representation of results shown in A revealing primer binding sites
and length of pcr products. C, Southern hybridisation was
performed to confirm the correct number of insertion events had
occurred. The hybridisation probe was designed to target the
chromosomally inserted ErmRAM sequence. Genomic DNA of all
strains was digested overnight with HindIII. Expected band sizes
were as follows; gerXA1-01232 2.9 kb, gerXA2-19752 6 kb,
gerXA3-27972 4 kb. The negative control, WT genomic DNA,
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Clostridium botulinum in nonthermally processed canned butter. J Dairy Sci 96:
2754–2764. 46. Broussolle V, Alberto F, Shearman CA, Mason DR, Botella L, et al. Acknowledgments The authors would like to thank Dr. Fran Mulholland for his advice on
proteomics data. September 2014 | Volume 10 | Issue 9 | e1004382 PLOS Pathogens | www.plospathogens.org 12 Germinant Receptors in Clostridium reagents/materials/analysis tools: DJHG. Contributed to the writing of the
manuscript: JB MWP ATC. Author Contributions Conceived and designed the experiments: JB MWP ATC. Performed the
experiments: JB JP MI ATC. Analyzed the data: JB MWP. Contributed Conceived and designed the experiments: JB MWP ATC. Performed the
experiments: JB JP MI ATC. Analyzed the data: JB MWP. Contributed Germinant Receptors in Clostridium Rose R, Setlow B, Monroe A, Mallozzi M, Driks A, et al. (2007) Comparison of
the properties of Bacillus subtilis spores made in liquid or on agar plates. J Appl
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Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25: 1189–1191. 61. Melly E, Genest PC, Gilmore ME, Little S, Popham DL, et al. (2002) Analysis of
the properties of spores of Bacillus subtilis prepared at different temperatures. J Appl Microbiol 92: 1105–1115. 77. Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, et al. (2012) The Pfam
protein families database. Nucleic Acids Res 40: D290–301. J
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designed group II introns to disrupt Escherichia coli DExH/D-box protein and
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ium botulinum and C-sporogenes. Anaerobe 8: 89–100. 21. Collins MD, Lawson PA, Willems A, Cordoba JJ, Fernandez-Garayzabal J, et al. (1994) The phylogeny of the genus Clostridium: proposal of five new genera and
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surrogate for proteolytic Clostridium botulinum. J Bacteriol 194: 1631–1632. 49. Ishimori T, Takahashi K, Goto M, Nakagawa S, Kasai Y, et al. (2012)
Synergistic effects of high hydrostatic pressure, mild heating, and amino acids on
germination and inactivation of Clostridium sporogenes spores. Appl Environ
Microbiol 78: 8202–8207. 24. Setlow P (2014) Germination of Spores of Bacillus Species: What We Know and
Do Not Know. J Bacteriol 196: 1297–1305. 50. Ross C, Abel-Santos E (2010) The Ger receptor family from sporulating
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and incubation temperature on germination and outgrowth of individual spores
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2712–2719. 51. Shoesmith JG, Holland KT (1972) The germination of spores of Clostridium
tetani. J Gen Microbiol 70: 253–261. PLOS Pathogens | www.plospathogens.org PLOS Pathogens | www.plospathogens.org September 2014 | Volume 10 | Issue 9 | e1004382 September 2014 | Volume 10 | Issue 9 | e1004382 13 Germinant Receptors in Clostridium Christie G, Lowe CR (2007) Role of chromosomal and plasmid-borne receptor
homologues in the response of Bacillus megaterium QM B1551 spores to
germinants. J Bacteriol 189: 4375–4383. 81. Heap JT, Kuehne SA, Ehsaan M, Cartman ST, Cooksley CM, et al. (2010) The
ClosTron: Mutagenesis in Clostridium refined and streamlined. J Microbiol
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66. Moir A, Corfe BM, Behravan J (2002) Spore germination. Cell Mol Life Sci 59:
403–409. 67. Dodatko T, Akoachere M, Muehlbauer SM, Helfrich F, Howerton A, et al. (2009) Bacillus cereus spores release alanine that synergizes with inosine to
promote germination. PLoS ONE 4: e6398. 82. Heap JT, Pennington OJ, Cartman ST, Minton NP (2009) A modular system for
Clostridium shuttle plasmids. J Microbiol Methods 78: 79–85. September 2014 | Volume 10 | Issue 9 | e1004382 PLOS Pathogens | www.plospathogens.org 14
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Search for <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:mi>C</mml:mi><mml:mi>P</mml:mi></mml:math> violation in <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:msubsup><mml:mi mathvariant="normal">Ξ</mml:mi><mml:mi>b</mml:mi><mml:mo>−</mml:mo></mml:msubsup><mml:mo stretchy="false">→</mml:mo><mml:mi>p</mml:mi><mml:msup><mml:mi>K</mml:mi><mml:mo>−</mml:mo></mml:msup><mml:msup><mml:mi>K</mml:mi><mml:mo>−</mml:mo></mml:msup></mml:math…
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Physical review. D/Physical review. D.
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I. INTRODUCTION Breaking of CP symmetry has not yet been observed in
the properties of any baryon. Tests of this symmetry have
been performed through studies of Λ0
b baryon decays to
pπ−, pK−[7,17], K0
Spπ−[18], ΛKþK−, ΛKþπ−[19],
pπ−πþπ−, pπ−KþK−, pK−πþπ−and pK−KþK−[20–22]
final states, as well as Ξ0
b decays to pK−πþπ−and
pK−πþK−[21,22]. No significant evidence of CP viola-
tion has been found in any of these studies, nor in
measurements of the properties of charm baryon decays
[23]. In light of the large CP -violation effects observed in
three-body charmless decays of B mesons, it is of great
interest to extend the range of searches in b -baryon decays. In particular, the recently observed Ξ−
b →pK−K−decay
[24] provides an interesting new opportunity to search for
CP -violation effects. In the Standard Model (SM), CP violation, defined as the
breaking of symmetry under the combined charge conjuga-
tion and parity operations, owes its origin to a single
irreducible complex phase in the Cabibbo-Kobayashi-
Maskawa (CKM) matrix [1,2]. All effects of CP violation
in particle decays observed so far are consistent with this
paradigm. However, the degree of CP violation permitted in
the SM is inconsistent with the observed matter-antimatter
asymmetry in the Universe [3,4]. This motivates further
searches for sources of CP violation beyond the SM. Interference between two amplitudes with different weak
and strong phases leads to CP violation in decay, where
weak phases are those that change sign under CP con-
jugation while strong phases do not. In the SM, weak phases
are associated with the complex elements of the CKM matrix
and strong phases are associated with hadronic final-state
effects. Two such amplitudes are potentially present in
decays of b hadrons to final states that do not contain
charm quarks, which therefore provide fertile ground for
studies of CP violation. Significant asymmetries have been
observed between B and ¯B partial widths in ¯B0 →K−πþ
[5–9] ¯B0 →πþπ−[5,6,10] and ¯B0s →Kþπ−[7,8] decays. Even larger CP -violation effects have been observed in
regions of the phase space of B−decays to πþπ−π−,
K−πþπ−, KþK−K−and KþK−π−final states [11–16]. In this paper, the first amplitude analysis of Ξ−
b →
pK−K−decays is reported. This is also the first amplitude
analysis of any b -baryon decay mode allowing for
CP -violation effects. A search for the previously unob-
served Ω−
b →pK−K−decay is also presented. Published by the American Physical Society under the terms of
the Creative Commons Attribution 4.0 International license.
Further distribution of this work must maintain attribution to
the author(s) and the published article’s title, journal citation,
and DOI. Funded by SCOAP3. (Received 3 May 2021; accepted 11 August 2021; published 20 September 2021) A search for CP violation in charmless three-body Ξ−
b →pK−K−decays is performed using pp
collision data recorded with the LHCb detector, corresponding to integrated luminosities of 1 fb−1 at a
center-of-mass energy
ffiffiffis
p ¼ 7 TeV, 2 fb−1 at
ffiffiffis
p ¼ 8 TeV and 2 fb−1 at
ffiffiffis
p ¼ 13 TeV. A good
description of the phase-space distribution is obtained with an amplitude model containing contributions
from Σð1385Þ, Λð1405Þ, Λð1520Þ, Λð1670Þ, Σð1775Þ and Σð1915Þ resonances. The model allows for CP -
violation effects, which are found to be consistent with zero. The branching fractions of Ξ−
b →Σð1385ÞK−,
Ξ−
b →Λð1405ÞK−, Ξ−
b →Λð1520ÞK−, Ξ−
b →Λð1670ÞK−, Ξ−
b →Σð1775ÞK−and Ξ−
b →Σð1915ÞK−
decays are also reported. In addition, an upper limit is placed on the product of ratios of Ω−
b and Ξ−
b
fragmentation fractions and the Ω−
b →pK−K−and Ξ−
b →pK−K−branching fractions. DOI: 10.1103/PhysRevD.104.052010 *Full author list given at the end of the article. PHYSICAL REVIEW D 104, 052010 (2021) PHYSICAL REVIEW D 104, 052010 (2021) © 2021 CERN, for the LHCb Collaboration II. DETECTOR, TRIGGER AND SIMULATION The LHCb detector [25,26] 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 surrounding the
pp interaction region, a large-area silicon-strip detector
located upstream of a dipole magnet with a bending power
of about 4 Tm, and three stations of silicon-strip detectors
and straw drift tubes placed downstream of the magnet. The
tracking system provides a measurement of the momentum,
p, of charged particles with a relative uncertainty that varies
from 0.5% at low momentum to 1.0% at 200 GeV.1 The
minimum distance of a track to a primary pp collision
vertex (PV), the impact parameter (IP), is measured with a
resolution of ð15 þ 29=pTÞ μm, where pT is the compo-
nent of the momentum transverse to the beam direction, in
GeV. Different types of charged hadrons are distinguished
using information from two ring-imaging Cherenkov
detectors. Photons, electrons and hadrons are identified
by a calorimeter system consisting of scintillating-pad and
preshower detectors, an electromagnetic and a hadronic
calorimeter. Muons are identified by a system composed
of alternating layers of iron and multiwire proportional
chambers. The magnetic field deflects oppositely charged
particles in opposite directions and this can lead to
detection asymmetries. Periodically reversing the magnetic
field polarity throughout the data-taking reduces this effect
to a negligible level. Approximately 60% of 2011 data,
50% of 2012 data, 61% of 2015 data and 53% of 2016 data
were collected in the “down” polarity configuration and the
rest in the “up” configuration. Simulation samples are used to investigate background
from other b -hadron decays and to study the detection and
reconstruction efficiency of the signal. In the simulation,
pp collisions are generated using PYTHIA [30] with a
specific LHCb configuration [31]. Decays of unstable
particles are described by EVTGEN [32], in which final-
state radiation is generated using PHOTOS [33]. The inter-
action of the generated particles with the detector, and its
response, are implemented using the GEANT4 toolkit [34] as
described in Ref. [35]. 1Natural units with ℏ¼ c ¼ 1 are used throughout this paper. I. INTRODUCTION The analysis
reported here is performed using proton-proton (pp)
collision data recorded with the LHCb detector, corre-
sponding to integrated luminosities of 1 fb−1 at a center-of-
mass energy of
ffiffiffis
p ¼ 7 TeV collected in 2011, 2 fb−1 at
ffiffiffis
p ¼ 8 TeV in 2012 and 2 fb−1 at
ffiffiffis
p ¼ 13 TeV in 2015
and 2016. The data-taking period of 2011 and 2012 is
referred to hereafter as Run 1 and that of 2015 and 2016 as
Run 2. The inclusion of charge-conjugate processes is
implied throughout the paper, except where asymmetries
are discussed. *Full author list given at the end of the article. Published by the American Physical Society under the terms of
the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to
the author(s) and the published article’s title, journal citation,
and DOI. Funded by SCOAP3. This paper is organized as follows. Section II gives a
brief description of the LHCb detector, trigger requirements
and simulation software. The signal candidate selection 2470-0010=2021=104(5)=052010(29) © 2021 CERN, for the LHCb Collaboration 052010-1 PHYS. REV. D 104, 052010 (2021) R. AAIJ et al. candidate, fulfilling any hardware trigger requirement. At
the software stage, it is required that at least one charged
particle associated to the b -hadron candidate has high pT
and high χ2
IP, where χ2
IP is defined as the difference in PV fit
χ2 with and without the inclusion of a specific particle. A
multivariate algorithm [27] is used to identify secondary
vertices consistent with being a two- or three-track
b -hadron decay. The PVs are fitted with and without
the tracks that comprise the b -baryon candidate, and the
PV that gives the smallest χ2
IP is associated with the
candidate. Finally, the momentum scale for charged
particles is calibrated using samples of J=ψ →μþμ−,
Bþ →J=ψKþ and Λ →pπ−decays collected concurrently
with the data sample used for this analysis [28,29]. procedure is set out in Sec. III. In Sec. IV, the procedure for
estimating the signal and background yields that enter the
amplitude fit is explained. Section V covers the modeling
of the distribution of decays across the phase space. Sections VI and VII contain a description of the systematic
uncertainties associated with the analysis procedure and a
presentation of the results, respectively. A brief summary of
the analysis is given in Sec. VIII. III. OFF-LINE SELECTION A threshold on the output of the MVA is chosen to
maximize N S=
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
N S þ N B
p
, where N S and N B represent
the estimated numbers of Ξ−
b →pK−K−signal and com-
binatorial background candidates, respectively, within a
signal region of 40 MeV around the Ξ−
b mass from
Ref. [39]. This range corresponds to approximately 2.5
times the Ξ−
b →pK−K−invariant mass resolution. The
value of N S is estimated using the signal efficiency
evaluated from simulation, multiplied by the Ξ−
b →
pK−K−branching fraction, the Ξ−
b fragmentation fraction,
the b¯b production cross-section [40] and the integrated
luminosity for the relevant data-taking period. The product
of the Ξ−
b →pK−K−branching fraction and the Ξ−
b
fragmentation fraction is obtained from the results of
Ref. [24], where the B−→KþK−K−channel is used
for normalization, by multiplying the B−fragmentation
fraction in the relevant kinematic range [41] and the
B−→KþK−K−branching fraction [23]. The value of
N B is estimated from data by fitting the region 6125 <
mðpK−K−Þ < 6470 MeV with a linear function and
extrapolating the result into the signal region. The MVA
output requirements have efficiencies of about 52% and
61% for Run 1 and Run 2, respectively, with combinatorial
background rejection of about 98% for both data-taking
periods. The choice of N S=
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
N S þ N B
p
as the figure of
merit is intended to obtain a sufficiently large data sample
to make an amplitude analysis viable. After all off-line
selection requirements are applied, each selected event
contains a single X−
b candidate. Variables
that
exhibit
good
discriminating
power
between the signal and background samples are chosen
as inputs to the MVA. III. OFF-LINE SELECTION These are as follows: the angle
between the X−
b candidate’s momentum vector and the line
connecting its decay vertex to its associated PV; the scalar
sum of the pT of all final-state tracks; the χ2
IP of the highest
pT final-state track and of the X−
b candidate; the square of
the significance of the distance between the X−
b decay
vertex and its associated PV; the vertex fit χ2 per degree
of freedom of the X−
b candidate; the minimum change
in the X−
b candidate vertex fit χ2 when including an
additional track; variables that characterize the PID infor-
mation of the proton and kaon candidates; and a variable
that quantifies the isolation of the X−
b candidate. The last of
these is defined as the pT asymmetry between the X−
b
candidate and the tracks within a circle, centered on the X−
b
candidate (but excluding its decay products), with a
radius
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
δη2 þ δϕ2
p
< 1.7 in the space of pseudorapidity
η and azimuthal angle ϕ (in radians) around the beam
direction [36]. The variables describing the phase space of the decay,
which are used in the amplitude analysis, are calculated
following a kinematic fit in which the X−
b candidate mass
is fixed to the Ξ−
b mass from Ref. [39]. This procedure
improves resolution of these variables and ensures that all
decays remain within the phase-space boundary. The
difference between the Ξ−
b mass value used in this fit
and recent more precise results [42–44] has negligible
impact on the analysis. The experimental resolution of the
mðpK−Þ invariant mass, in the region with the narrowest
resonance considered in this analysis, the Λð1520Þ state, is
expected to be around 1.5 MeV. This is smaller than
the Λð1520Þ width, and therefore effects related to To describe accurately the proton and kaon PID varia-
bles, the quantities in simulation are resampled according
to values obtained from data calibration samples of
Λ0
b →Λþc π−, Dþs →ϕπþ and Dþ →D0πþ decays [37]. The procedure accounts for correlations between the
variables associated to a particular track, as well as the
dependence
of
the
PID
response
on
pT,
η,
and
the multiplicity of tracks in the event. All other MVA
input variables show good agreement between simulation
and data, as validated with a control sample of B−→p ¯pK−
decays. III. OFF-LINE SELECTION It is trained with a signal sample comprised of simulated
Ξ−
b →pK−K−decays generated uniformly across the
phase space and a background sample obtained from
candidates in data in the sideband regions 5545.0 <
mðpK−K−Þ < 5634.4 MeV and 6209.0 < mðpK−K−Þ <
6470.0 MeV. The latter of these regions is dominated by
combinatorial background, as its lower threshold excludes
possible Ω−
b →pK−K−decays from the sample. The
former region includes also contributions from sources
of partially reconstructed background such as Ξ−
b →
Nðpπ0ÞK−K−or Ξ−
b →K−ðK−π0ÞK−p decays. Potential
cross-feed background from B−→KþK−K−decays is
removed by assigning the proton candidate the kaon mass
and vetoing the mðKþK−K−Þ region within 45 MeV
around the known B−mass [23]. This veto corresponds to
approximately 3 times the invariant mass resolution for
B−→KþK−K−decays. of one or more final-state particles. The MVA classifier is
designed primarily to reduce combinatorial background,
while retaining high signal efficiency, but also has some
discriminating power against the other background sources. It is trained with a signal sample comprised of simulated
Ξ−
b →pK−K−decays generated uniformly across the
phase space and a background sample obtained from
candidates in data in the sideband regions 5545.0 <
mðpK−K−Þ < 5634.4 MeV and 6209.0 < mðpK−K−Þ <
6470.0 MeV. The latter of these regions is dominated by
combinatorial background, as its lower threshold excludes
possible Ω−
b →pK−K−decays from the sample. The
former region includes also contributions from sources
of partially reconstructed background such as Ξ−
b →
Nðpπ0ÞK−K−or Ξ−
b →K−ðK−π0ÞK−p decays. Potential
cross-feed background from B−→KþK−K−decays is
removed by assigning the proton candidate the kaon mass
and vetoing the mðKþK−K−Þ region within 45 MeV
around the known B−mass [23]. This veto corresponds to
approximately 3 times the invariant mass resolution for
B−→KþK−K−decays. Several types of MVA classifiers are investigated, with a
gradient boosted decision tree algorithm giving the best
performance [38]. Four classifiers are trained separately
with samples separated by the data-taking period (Run 1 or
Run 2) and by even or odd event numbers. The event
number identifies the proton-proton bunch crossing, from
which the X−
b candidate was recorded, in a certain opera-
tional period of the experiment. To avoid possible MVA
overtraining, for each data-taking period the classifier
trained on the sample with even event numbers is validated
and employed on the sample with odd event numbers, and
vice versa. III. OFF-LINE SELECTION The off-line selection consists of an initial filtering stage
followed by a requirement on the output of a multivariate
algorithm (MVA). Compared to the procedure applied to
select the Ξ−
b →pK−K−channel in Ref. [24], improve-
ments in both stages lead to a significant increase in
efficiency. In particular, the inclusion in the multivariate
algorithm of particle identification (PID) variables that
distinguish the final-state charged hadrons from misidenti-
fied particles is found to separate signal from background
effectively. y
In the filtering stage, tracks are required to be of good
quality, to satisfy p > 1500 MeV and pT > 250 MeV, and
to be displaced from all PVs. Tracks associated to proton
candidates must, at this stage, satisfy a loose PID require-
ment and all tracks are required to not be associated to hits
in the muon system. Each b -hadron (henceforth denoted as
X−
b) candidate must form a good-quality decay vertex that is
separated significantly from any PVand must be consistent
with originating from its associated PV. Only X−
b candi-
dates with pT > 3500 MeV and invariant mass 5545 <
mðpK−K−Þ < 6470 MeV are retained for further analysis. In the selected mðpK−K−Þ range there are three
main categories of background that contribute: combina-
torial background that results from random association of
unrelated tracks; partially reconstructed background due to
b -hadron decays into final states similar to the signal, but
with additional soft particles that are not reconstructed; and
cross-feed background that results from misidentification The online event selection is performed by a trigger,
which consists of a hardware stage, based on information
from the calorimeter and muon systems, followed by a
software stage, which applies a full event reconstruction. During off-line analysis, reconstructed candidates are
associated with trigger decisions. Events considered in
the analysis are required to have been triggered at the
hardware level in one of two ways: either through one of the
final-state tracks of the signal decay depositing sufficient
energy in the calorimeter system, or by one of the other
tracks in the event, not reconstructed as part of the signal 052010-2 SEARCH FOR CP VIOLATION IN Ξ−
b →pK−K−… PHYS. REV. D 104, 052010 (2021) of one or more final-state particles. The MVA classifier is
designed primarily to reduce combinatorial background,
while retaining high signal efficiency, but also has some
discriminating power against the other background sources. IV. X −
b CANDIDATE MASS FIT Distributions of mðpK−K−Þ for selected X−
b candidates
are shown in Fig. 1 for Run 1 and Run 2 separately. The
signal yields are obtained from unbinned extended maxi-
mum-likelihood fits to these distributions. The fit model is
composed of signal and background components, whose
shape parameters are mostly obtained from fits to the
corresponding simulation samples, after imposing the same
selection requirements as on the data. One exception is the
combinatorial background component, which is modeled
by an exponential function with slope parameter allowed to
vary freely in the fit to data. Partially reconstructed and combinatorial background
contributions are also included in the fit model. It is found
that the mðpK−K−Þ distributions of various potential
sources of partially reconstructed background, such as
Ξ−
b →Nðpπ0ÞK−K−or Ξ−
b →K−ðK−π0ÞK−p decays,
are very similar [46]. Therefore, the baseline fit model
includes a single partially reconstructed background com-
ponent,
which
is
modeled
from
simulated
Ξ−
b →
K−ðK−π0ÞK−p decays with an ARGUS function [48]
convolved with a Gaussian function. The threshold of the
ARGUS function is fixed to the known value of mΞ−
b −mπ0
[23,42], and the width parameter of the Gaussian function
is taken from the fit to simulation and scaled by the same
factor as the signal components. Negligible contributions
are expected from partially reconstructed Ω−
b decays, such
as Ω−
b →K−ðK−π0ÞK−p. Signal Ξ−
b →pK−K−and Ω−
b →pK−K−components
are each modeled with the sum of two Crystal Ball (CB)
functions [45], where the core width and peak position are
shared and with independent power-law tails on both sides. The tail parameters and the relative normalization of the CB
functions are determined from simulation. The peak posi-
tions are fixed to the Ξ−
b mass from Ref. [42] and the known
Ω−
b mass [23], and a scale factor relating the width in data to
that in simulation is introduced. The results of the fits to Run 1 and Run 2 data are shown
in Table I and Fig. 1. The free parameters of each fit are the
two signal yields, the partially reconstructed and combi-
natorial background yields, the width scale factor and the
exponential shape parameter of the combinatorial back-
ground, while the cross feed background yield is con-
strained to its expectation relative to the Ξ−
b →pK−K−
signal yield. III. OFF-LINE SELECTION The MVA input variables are also found to not be
correlated strongly either with X−
b candidate mass or with
position in the phase space of the decay. 052010-3 PHYS. REV. D 104, 052010 (2021) R. AAIJ et al. finite resolution in the phase-space variables are not
considered further. scaled by the same factor as the signal components. The
phase-space distribution of these decays is not known, and
the simulation sample is weighted according to a model,
inspired by the mðpK−Þ and mðpπ−Þ mass spectra
observed in Λ0
b →J=ψpK−[46] and Λ0
b →J=ψpπ−[47]
decays, which consists of the Λð1405Þ, Λð1520Þ, Λð1690Þ,
Nð1440Þ, Nð1520Þ, Nð1535Þ and Nð1650Þ resonances. The yield of the Ξ−
b →pK−π−cross feed component is
expressed relative to the Ξ−
b →pK−K−signal yield
and constrained within uncertainty according to the pre-
vious branching fraction ratio measurement [24] and
relative selection efficiency. The expected relative yields
are 0.15 0.06 and 0.14 0.03 for Run 1 and Run 2,
respectively. The expected Ξ−
b →pK−K−signal efficiency, assuming
uniform distribution of decays across the phase space
and taking into account the LHCb detector acceptance,
reconstruction and both online and off-line selection
criteria, is ð1.159 0.005Þ% for Run 1 and ð1.748
0.006Þ% for Run 2. The corresponding Ω−
b →pK−K−
signal efficiencies are ð1.257 0.005Þ% and ð1.921
0.006Þ%. The quoted uncertainties are due to the limited
size of the simulation samples only. IV. X −
b CANDIDATE MASS FIT A possible cross feed background contribution from
Ξ−
b →pK−π−decays [24], where the pion is misidentified
as a kaon, is modeled with the sum of two CB functions. All shape parameters of this function are fixed according to
the values obtained from a fit to simulation but the width is 5600
5800
6000
6200
6400
) [MeV]
−
K
−
K
p
(
m
0
20
40
60
80
100
120
Entries / (23.125 MeV)
LHCb
-1
3 fb
Data
Total fit
signal
−
b
signal
−
b
cross-feed
−
−
K
p
−
b
Part. rec. bkgd. Comb. bkgd. 5600
5800
6000
6200
6400
) [MeV]
−
K
−
K
p
(
m
0
20
40
60
80
100
120
140
160
180
Entries / (23.125 MeV)
LHCb
-1
2 fb
Data
Total fit
signal
−
b
signal
−
b
cross-feed
−
−
K
p
−
b
Part. rec. bkgd. Comb. bkgd. FIG. 1. Distributions of pK−K−invariant mass for X−
b candidates in (left) Run 1 and (right) Run 2 data with results of the unbinned
extended maximum-likelihood fits superimposed. The total fit result is shown as the blue solid curve, with individual components shown
as indicated in the legend. 5600
5800
6000
6200
6400
) [MeV]
−
K
−
K
p
(
m
0
20
40
60
80
100
120
140
160
180
Entries / (23.125 MeV)
LHCb
-1
2 fb
Data
Total fit
signal
−
b
signal
−
b
cross-feed
−
−
K
p
−
b
Part. rec. bkgd. Comb. bkgd. 5600
5800
6000
6200
6400
) [MeV]
−
K
−
K
p
(
m
0
20
40
60
80
100
120
Entries / (23.125 MeV)
LHCb
-1
3 fb
Data
Total fit
signal
−
b
signal
−
b
cross-feed
−
−
K
p
−
b
Part. rec. bkgd. Comb. bkgd. FIG. 1. Distributions of pK−K−invariant mass for X−
b candidates in (left) Run 1 and (right) Run 2 data with results of the unbinned
extended maximum-likelihood fits superimposed. The total fit result is shown as the blue solid curve, with individual components shown
as indicated in the legend. 052010-4 052010-4 PHYS. REV. D 104, 052010 (2021) SEARCH FOR CP VIOLATION IN Ξ−
b →pK−K−… TABLE I. Yields obtained from fits to the mðpK−K−Þ distri-
butions, in the full invariant mass range. The quoted uncertainties
are statistical only. A. Modeling of the signal component The probability density function (PDF) for the signal
component is expressed as IV. X −
b CANDIDATE MASS FIT distribution of m2ðpK−
1 Þ and m2ðpK−
2 Þ has a symmetry
under interchange of the variables. This motivates the use
of the variables m2
low and m2
high, which denote the lower and
higher of m2ðpK−
1 Þ and m2ðpK−
2 Þ, respectively, effectively
removing a duplicated half of the ðm2ðpK−
1 Þ; m2ðpK−
2 ÞÞ
plane. References hereafter to the Dalitz plot (DP) of Ξ−
b →
pK−K−decays refer to the two-dimensional ðm2
low; m2
highÞ
distribution. The DP distributions of selected candidates in
Run 1 and Run 2 are shown in the top row of Fig. 2. Parameter
Run 1
Run 2
Ξ−
b →pK−K−yield
193 21
297 23
Ω−
b →pK−K−yield
−4 6
15 9
Partially reconstructed background yield
231 34
442 36
Combinatorial background yield
721 50
775 51 It is common practice in amplitude analysis to use the so-
called “square” Dalitz plot (SDP) variables [15,51], which
in this case are defined as Only candidates in the mðpK−K−Þ signal region of
40 MeV around the Ξ−
b mass from Ref. [39] are retained
for the amplitude analysis. In this region, the yields of
the signal, cross feed, and combinatorial components are
Nsig ¼ 181 20, Ncf ¼ 16 7 and Ncomb ¼ 90 6 for
Run 1, and Nsig ¼ 278 21, Ncf ¼ 25 6 and Ncomb ¼
95 6 for Run 2, where the quoted uncertainties are
statistical only. These correspond to signal purities of
ð63 3Þ% and ð70 2Þ% for Run 1 and Run 2, respec-
tively. The contribution from the partially reconstructed
background in the signal region is negligible. m0 ¼ 1
π arccos
2 mðK−K−Þ −mminðK−K−Þ
mmaxðK−K−Þ −mminðK−K−Þ
and
θ0 ¼ 1
π θðK−K−Þ:
ð2Þ ð2Þ Here mminðK−K−Þ ¼ 2mK and mmaxðK−K−Þ ¼ mΞb −mp
represent the kinematic limits of mðK−K−Þ for Ξ−
b →
pK−K−decay, and θðK−K−Þ is the angle between one
K−direction in the K−K−center-of-mass frame and the
direction of the K−K−system in the Ξ−
b center-of-mass
frame. The symmetry of the final state requires that
distributions are symmetric with respect to θ0 ¼ 0.5, so
only the region θ0 ∈½0; 0.5 is considered. IV. X −
b CANDIDATE MASS FIT These SDP
variables provide improved granularity, when using uni-
form binning, in the regions close to the DP boundaries
that tend to be populated most densely. This is beneficial,
for example, in the modeling of the signal efficiency. Furthermore, the mapping to a square space aligns the
bin boundaries to the kinematic boundaries of the phase
space. As such, all efficiencies and background distribu-
tions in the analysis are obtained as functions of the SDP
variables. The SDP distributions of selected candidates in
Run 1 and Run 2 are shown in the bottom row of Fig. 2. No significant signal from the Ω−
b →pK−K−decay is
observed. The results of the fits are used to set limits on the
product of its branching fraction with the fragmentation
fraction for Ω−
b production, normalized to the correspond-
ing quantities for Ξ−
b →pK−K−decay, i.e., R ¼
fΩ−
b
fΞ−
b
× BðΩ−
b →pK−K−Þ
BðΞ−
b →pK−K−Þ
¼ ϵðΞ−
b →pK−K−Þ
ϵðΩ−
b →pK−K−Þ × NðΩ−
b →pK−K−Þ
NðΞ−
b →pK−K−Þ ;
ð1Þ ð1Þ where N and ϵ denote yield and efficiency, respectively, for
the indicated mode, while fΞ−
b and fΩ−
b are the Ξ−
b and Ω−
b
fragmentation fractions. Results for the ratio R are
reported, both for Run 1 and Run 2 separately and
combined, in Sec. VII. V. AMPLITUDE ANALYSIS The phase space of the three-body decay of a, potentially
polarized, b baryon has 5 degrees of freedom. A baseline
assumption is made that Ξ−
b baryons produced in pp
collisions within the LHCb acceptance have negligible
polarization, as observed for Λ0
b baryons [49,50]. As a
result, the phase space of the Ξ−
b →pK−
1 K−
2 decay is
characterized by two independent kinematic variables
(subscripts here distinguish the two kaons in the final
state). Since no resonances are expected to decay to K−
1 K−
2 ,
these variables chosen are the squared invariant masses
m2ðpK−
1 Þ and m2ðpK−
2 Þ. The presence of two identical
kaons in the final state imposes a Bose symmetry such that
the decay amplitudes must be invariant under the exchange
of these two particles. As a result, the two-dimensional PQ
sigðΩÞ ¼ ϵQðΩÞ
Γ
dΓQ
dΩ ;
ð3Þ ð3Þ ð3Þ where Q ¼ þ1 for Ξ−
b decays and Q ¼ −1 for ¯Ξþ
b decays
and Ω denotes the phase space in terms of the DP variables. where Q ¼ þ1 for Ξ−
b decays and Q ¼ −1 for ¯Ξþ
b decays
and Ω denotes the phase space in terms of the DP variables. The efficiency is denoted by ϵQðΩÞ and can differ for
Q ¼ þ1 and −1 to accommodate efficiency asymmetries;
as described in Sec. V B, the efficiency maps are deter-
mined using SDP coordinates, denoted Ω0, but at any point
in the phase space ϵQðΩÞ ¼ ϵQðΩ0Þ. The term dΓQ=dΩ
describes the differential decay densities for Ξ−
b and ¯Ξþ
b
decays, including both local and overall rate asymmetries,
and the normalization factor Γ is 052010-5 PHYS. REV. D 104, 052010 (2021) R. AAIJ et al. V. AMPLITUDE ANALYSIS 5
10
15
]
2
) [GeV
−
K
p
(
low
2
m
5
10
15
20
25
]
2
) [GeV
−
K
p
(
high
2
m
LHCb
-1
3 fb
5
10
15
]
2
) [GeV
−
K
p
(
low
2
m
5
10
15
20
25
]
2
) [GeV
−
K
p
(
high
2
m
LHCb
-1
2 fb
0
0.2
0.4
0.6
0.8
1
m'
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
LHCb
-1
3 fb
0
0.2
0.4
0.6
0.8
1
m'
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
LHCb
-1
2 fb
FIG. 2. Distributions of selected candidates from (left) Run 1 and (right) Run 2 data in the (top) Dalitz-plot and (bottom) square Dalitz-
plot representations of the phase space. 5
10
15
]
2
) [GeV
−
K
p
(
low
2
m
5
10
15
20
25
]
2
) [GeV
−
K
p
(
high
2
m
LHCb
-1
3 fb 5
10
15
]
2
) [GeV
−
K
p
(
low
2
m
5
10
15
20
25
]
2
) [GeV
−
K
p
(
high
2
m
LHCb
-1
2 fb 5
10
15
]
2
) [GeV
−
K
p
(
low
2
m
5
10
15
20
25
]
2
) [GeV
−
K
p
(
high
2
m
LHCb
-1
2 fb
0
0.2
0.4
0.6
0.8
1
m'
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
LHCb
-1
2 fb 0
0.2
0.4
0.6
0.8
1
m'
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
LHCb
-1
3 fb 0
0.2
0.4
0.6
0.8
1
m'
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
LHCb
-1
2 fb FIG. 2. Distributions of selected candidates from (left) Run 1 and (right) Run 2 data in the (top) Dalitz-plot and (bottom) square Dalitz-
plot representations of the phase space. Γ¼
Z
Ω
ϵQ¼þ1ðΩÞdΓQ¼þ1
dΩ
þϵQ¼−1ðΩÞdΓQ¼−1
dΩ
dΩ:
ð4Þ incoherently over the spins of the initial and final states
(corresponding to an average over initial states) and
coherently over all contributing intermediate states. V. AMPLITUDE ANALYSIS The angle ζ gives the Wigner rotation that is
required to relate the proton helicity state jλ0pi, defined in the
pK−
low rest frame to the proton helicity state jλpi, defined in
the K−K−rest frame. This angle, computed in the proton rest
frame, is formed between the momenta of K−
low and of the
K−K−system. Mathematical definitions of these three
angles, each defined in the range ½0; π, are systems, respectively. The Ξ−
b, R and proton spins are
denoted by JΞb, JR and Jp, respectively. The three functions of the form dJ
λ;λ0 in Eq. (7) are the
small Wigner d-matrix elements [55] that impose angular
momentum conservation giving rise to the condition
jλRj ≤1=2. As a result, for intermediate states with any
half-integer spin, only helicities corresponding to λR ¼
1=2 contribute to the amplitude. The three angles θR,
θp and ζ are functions of the DP variables. The angle θR,
defined in the Ξ−
b rest frame, is formed between the direction
opposite to the proton momentum and the combined cos θR ¼
ðm2
Ξb þ m2
K −m2
lowÞðm2
Ξb þ m2p −m2
KKÞ −2m2
Ξbðm2
high −m2
K −m2pÞ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Kðm2
Ξb; m2p; m2
KKÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Kðm2
Ξb; m2
low; m2
KÞ
q
;
ð8Þ
cos θp ¼
2m2
lowðm2
high −m2
K −m2pÞ −ðm2
low þ m2p −m2
KÞðm2
Ξb −m2
low −m2
KÞ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Kðm2
Ξb; m2
K; m2
lowÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Kðm2
low; m2p; m2
KÞ
q
;
ð9Þ
cos ζ ¼
2m2pðm2
high −m2
Ξb −m2
KÞ þ ðm2
Ξb þ m2p −m2
KKÞðm2
low −m2p −m2
KÞ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Kðm2
Ξb; m2p; m2
KKÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Kðm2
low; m2p; m2
KÞ
q
;
ð10Þ ð8Þ 2m2
lowðm2
high −m2
K −m2pÞ −ðm2
low þ m2p −m2
KÞðm2
Ξb −m2
low −m2
KÞ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Kðm2
Ξb; m2
K; m2
lowÞ
q
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Kðm2
low; m2p; m2
KÞ
q
;
ð9Þ ð9Þ ð10Þ where m2
KK ¼m2
Ξb þ2m2
K þm2p −m2
low −m2
high, the Käll´en
function
is
given
by
Kða; b; cÞ ¼ a2 þ b2 þ c2 −
2ðab þ ac þ bcÞ, and the Ξ−
b, K and p masses are denoted
by mΞb, mK and mp, respectively. The term Rðm2
lowÞ, in Eq. (7), describes the line shape of
each resonant or nonresonant contribution. V. AMPLITUDE ANALYSIS Equations (3) and (4) assume no asymmetry in the
production rates of Ξ−
b and ¯Ξþ
b baryons produced within
the LHCb acceptance from high-energy pp collisions,
consistent with measurement [44]. The effect of such a
production asymmetry would, in this analysis, mimic a
global (i.e., phase-space independent) difference between
ϵQ¼þ1 and ϵQ¼−1 and, therefore, the systematic uncertainty
due to this assumption can be evaluated straightforwardly. The helicity formalism is used to parametrize the decay
dynamics. A detailed description of this formalism can be
found in Refs. [46,47,52–54]. In particular, the Dalitz-plot
decomposition procedure [54] is followed to express the
symmetrized decay amplitude as AQ
R;MΞb;λpðm2
low;m2
highÞ¼TQ
R;MΞb;λpðm2
low;m2
highÞ
þð−1ÞMΞbþλpTQ
R;MΞb;λpðm2
high;m2
lowÞ:
ð6Þ The differential decay density is expressed as ð6Þ dΓQ
dΩ ¼
1
ð8πmΞbÞ3
X
MΞb;λp
X
RAQ
R;MΞb;λpðΩÞ
2
;
ð5Þ ð5Þ The first term corresponds to the amplitude for the weak
decay Ξ−
b →RK−
high, where R decays to pK−
low via the
strong interaction. This decay amplitude is expressed as where AQ
R;MΞb;λp denotes the symmetrized decay amplitude
for a given intermediate state R, Ξ−
b spin component along a
chosen quantization axis MΞb, and proton helicity λp. The
quantization axis is chosen to be the direction opposite to
the proton momentum in the Ξ−
b rest frame, and the proton
helicity is defined in the rest frame of the K−K−system to
ensure explicit symmetry between the pK−
low and pK−
high
decay chains. Here K−
low is the kaon whose four-momentum
is used in the definition of m2
low and K−
high denotes the
other kaon. The amplitude in Eq. (5) has been summed TQ
R;MΞb;λpðm2
low;m2
highÞ¼
X
λR;λ0p
ðd
JΞb
MΞb;λRðθRÞdJR
λR;λ0pðθpÞd
Jp
λ0p;λpðζÞ
×ηλ0pð−1Þλ0p−λphQ
R;λRRðm2
lowÞÞ;
ð7Þ where the amplitude is summed coherently over the
allowed helicities of the intermediate state λR, and of the
proton λ0p defined in the rest frames of the Ξ−
b and the pK−
low 052010-6 PHYS. REV. D 104, 052010 (2021) SEARCH FOR CP VIOLATION IN Ξ−
b →pK−K−… momentum of the pK−
low system. The angle θp is between
the direction opposite to the K−
high momentum in the Ξ−
b rest
frame and the proton momentum in the rest frame of the
pK−
low system. V. AMPLITUDE ANALYSIS Resonances are
parametrized with relativistic Breit–Wigner (RBW) func-
tions, FRBW, that are modified by Blatt–Weisskopf barrier
factors, BLΞb and BLR, and are given by The second term in Eq. (6) corresponds to the weak
decay of Ξ−
b →RK−
low, where R now decays to R →pK−
high. The expression for this amplitude can be obtained by
interchanging m2
low ↔m2
high in Eqs. (7)–(10) Rðm2xÞ ¼ BLΞbðpjp0; dÞ
p
mΞb
LΞbFRBWðm2xjm0; Γ0Þ
× BLRðqjq0; dÞ
q
m0
LR
:
ð12Þ g
The term ηλ0p, in Eq. (7), arises as a consequence of parity
conservation in the strong decay of the intermediate state R. It is defined as ð12Þ ηλ0p ¼
1 when λ0p ¼ 1=2;
ð−1Þ
3
2−JRηR when λ0p ¼ −1=2; Here m2x is either m2
low or m2
high, while p is the magnitude of
the resonance momentum in the Ξ−
b center-of-mass frame,
and q is the magnitude of the proton momentum in the
resonance center-of-mass frame. The symbols p0 and q0
denote the values of these quantities at the resonance
peak, i.e., when mx ¼ m0. The orbital angular momentum
released in the Ξ−
b decay is denoted LΞb, while that in the
resonance decay is denoted LR. Angular momentum con-
servation
in
the
Ξ−
b
decay
imposes
the
condition
JR −1=2 ≤LΞb ≤JR þ 1=2. The minimal value LΞb ¼
JR −1=2 is assumed when calculating Rðm2xÞ. Angular
momentum conservation in the resonance decay limits LR
to JR 1
2, which is then uniquely defined by parity
conservation in the decay, ηR ¼ ð−1ÞLRþ1. Here m2x is either m2
low or m2
high, while p is the magnitude of
the resonance momentum in the Ξ−
b center-of-mass frame,
and q is the magnitude of the proton momentum in the
resonance center-of-mass frame. The symbols p0 and q0
denote the values of these quantities at the resonance
peak, i.e., when mx ¼ m0. The orbital angular momentum
released in the Ξ−
b decay is denoted LΞb, while that in the
resonance decay is denoted LR. Angular momentum con-
servation
in
the
Ξ−
b
decay
imposes
the
condition
JR −1=2 ≤LΞb ≤JR þ 1=2. The minimal value LΞb ¼
JR −1=2 is assumed when calculating Rðm2xÞ. V. AMPLITUDE ANALYSIS Angular
momentum conservation in the resonance decay limits LR
to JR 1
2, which is then uniquely defined by parity
conservation in the decay, ηR ¼ ð−1ÞLRþ1. ηλ0p ¼
ð−1Þ
3
2−JRηR when λ0p ¼ −1=2; where ηR is the intrinsic parity of R. Q where ηR is the intrinsic parity of R.
Q where ηR is the intrinsic parity of R. Q The complex coefficient hQ
R;λR, in Eq. (7), encapsulates
the combined couplings of the weak decay of the initial
state and the strong decay of the intermediate state. This
coefficient, subsequently referred to as the helicity cou-
pling, can be expressed as hQ
R;λR ¼ ðxR;λR þ QδxR;λRÞ þ iðyR;λR þ QδyR;λRÞ;
ð11Þ ð11Þ where xR;λR and yR;λR denote the real and imaginary
components of the CP -conserving part of the coupling,
while δxR;λR and δyR;λR are CP -violating parameters. 052010-7 PHYS. REV. D 104, 052010 (2021) R. AAIJ et al. The Blatt–Weisskopf barrier functions are The Blatt–Weisskopf barrier functions are where α is a slope parameter that is determined from the fit,
and m0 is fixed to be the midpoint of the mlow range, i.e.,
2.83 GeV. B0ðkjk0; dÞ ¼ 1;
ð13Þ
B1ðkjk0; dÞ ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
1 þ ðk0dÞ2
1 þ ðkdÞ2
s
;
ð14Þ
ðkjk0; dÞ ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
9 þ 3ðk0dÞ2 þ ðk0dÞ4
9 þ 3ðkdÞ2 þ ðkdÞ4
s
;
ð15Þ B0ðkjk0; dÞ ¼ 1;
ð13Þ
B1ðkjk0; dÞ ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
1 þ ðk0dÞ2
1 þ ðkdÞ2
s
;
ð14Þ
B2ðkjk0; dÞ ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
9 þ 3ðk0dÞ2 þ ðk0dÞ4
9 þ 3ðkdÞ2 þ ðkdÞ4
s
;
ð15Þ
B3ðkjk0; dÞ ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
225 þ 45ðk0dÞ2 þ 6ðk0dÞ4 þ ðk0dÞ6
225 þ 45ðkdÞ2 þ 6ðkdÞ4 þ ðkdÞ6
s
;
ð16Þ ð13Þ The primary outputs of the amplitude analysis are the
CP -conserving and CP -violating components of the
helicity couplings introduced in Eq. (11). However, since
these depend on the choice of phase convention, amplitude
formalism and normalization, they can be difficult to
compare between analyses. where ηR is the intrinsic parity of R.
Q It is therefore more useful to
report the fit fractions F i for each intermediate component i
of the fit model, defined by ð15Þ F i ¼
R
ΩðdΓþ
i =dΩ þ dΓ−
i =dΩÞdΩ
R
ΩðdΓþ=dΩ þ dΓ−=dΩÞdΩ ;
ð21Þ B3ðkjk0; dÞ ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
225 þ 45ðk0dÞ2 þ 6ðk0dÞ4 þ ðk0dÞ6
225 þ 45ðkdÞ2 þ 6ðkdÞ4 þ ðkdÞ6
s
; B3ðkjk0; dÞ ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
225 þ 45ðk0dÞ2 þ 6ðk0dÞ4 þ ðk0dÞ6
225 þ 45ðkdÞ2 þ 6ðkdÞ4 þ ðkdÞ6
s
;
ð16 ð21Þ ð16Þ where where and account for suppression creating high values of the
orbital angular momentum L, which depends on the
momentum of one of the decay products, k, in the center-
of-mass frame of the decaying particle and on the size of
the decaying particle given by the constant d. The value
d ¼ 5.0 GeV−1 is used for Ξ−
b decays while 1.5 GeV−1 is
used for resonances [53]. dΓQ
i
dΩ ¼
1
ð8πmΞbÞ3
X
MΞb;λp
jAQ
i;MΞb;λpðΩÞj2:
ð22Þ ð22Þ It is also useful to report the interference fit fractions Iij
between the two intermediate components i and j,
defined by The relativistic Breit–Wigner amplitude is given by Iij ¼
R
ΩðdΓþ
ij=dΩ þ dΓ−
ij=dΩÞdΩ
R
ΩðdΓþ=dΩ þ dΓ−=dΩÞdΩ ;
ð23Þ ð23Þ FRBWðm2xjm0; Γ0Þ ¼
1
m2
0 −m2x −im0ΓðmxÞ ;
ð17Þ where where where dΓQ
ij
dΩ ¼
1
ð8πmΞbÞ3
X
MΞb;λp;i;j
2ReðAQ
i;MΞb;λpðAQ
j;MΞb;λpÞÞ:
ð24Þ ð24Þ ΓðmxÞ ¼ Γ0
q
q0
2LRþ1 m0
mx
B0
LRðq; q0; dÞ2:
ð18Þ The parameters of CP violation ACP
i , associated with each
component i of the model, are also reported. These are
defined as The parameters of CP violation ACP
i , associated with each
component i of the model, are also reported. These are
defined as Here m0 and Γ0 denote the pole mass and width of the
resonance, respectively. In the case of the Λð1405Þ reso-
nance, which peaks below the pK−threshold, m0 is
replaced by an effective mass in the kinematically allowed
region [56], ACP
i
¼
R
ΩðdΓþ
i =dΩ −dΓ−
i =dΩÞdΩ
R
ΩðdΓþ
i =dΩ þ dΓ−
i =dΩÞdΩ :
ð25Þ ð25Þ meff
0 ¼mmin þðmmax −mminÞ
1þtanh
m0 −mmaxþmmin
2
mmax −mmin
;
ð19Þ B. Modeling of signal efficiency
and background distributions 0
0.2
0.4
0.6
0.8
1
m'
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0
5
10
15
20
25
30
3
−
10
×
decay
+
b
Ξ
Efficiency of
LHCb Simulation 0
0.2
0.4
0.6
0.8
1
m'
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0
5
10
15
20
25
30
3
−
10
×
decay
−
b
Efficiency of
LHCb Simulation 0
0.2
0.4
0.6
0.8
1
m'
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0
5
10
15
20
25
30
3
−
10
×
decay
+
b
Ξ
Efficiency of
LHCb Simulation 0
0.2
0.4
0.6
0.8
1
m'
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0
5
10
15
20
25
30
3
−
10
×
decay
−
b
Ξ
Efficiency of
LHCb Simulation FIG. 3. Efficiency as a function of square Dalitz-plot position for (left) Ξ−
b and (right) ¯Ξþ
b decays, for (top) Run 1 and (bottom) Run 2. FIG. 3. Efficiency as a function of square Dalitz-plot position for (left) Ξ−
b and (right) ¯Ξþ
b decays, for (top) Run 1 and (bottom) Run 2. FIG. 3. Efficiency as a function of square Dalitz-plot position for (left) Ξ−
b and (right) ¯Ξþ
b decays, fo effects of discontinuity at the bin edges. No significant
detection asymmetry is observed. After imposing the selection criteria and splitting the
data sample by the initial state charge, too few candidates
are available in the sideband region to train the neural
networks. As a result, the neural networks are trained
using the combined sample of Ξ−
b and ¯Ξþ
b candidates,
and no asymmetry in the shape of the combinatorial
background SDP distribution is assumed in the base-
line model. Candidates selected in data in the sideband 5890 <
mðpK−K−Þ < 6470 MeV are used to model the SDP
distribution of the combinatorial background, which domi-
nates this region as discussed in Sec. IV. The effect of
the Ξ−
b mass constraint used when calculating the SDP
variables causes a distortion of the distribution from that
of combinatorial background in the signal region. B. Modeling of signal efficiency
and background distributions ð19Þ The detector geometry and the on-line and off-line
selection procedure can induce variation in the signal
efficiency across the phase space of the decay. This is
accounted for, as shown in Eq. (3), by determining the
efficiency as a function of the SDP variables. The efficiency
maps are obtained from simulation, but with effects related
to PID calibrated using data as outlined in Sec. III. The
efficiency maps for Ξ−
b and ¯Ξþ
b decays can be seen in Fig. 3
separately for Run 1 and Run 2. These maps are obtained
by employing a uniform 10 × 10 binning scheme and
smoothing with a two-dimensional cubic spline to mitigate where mmax and mmin are the upper and lower limits of the
kinematically allowed range, respectively. In this case, the
q0 value in Eq. (18) is the value of q at m ¼ meff
0 . This
parametrization ensures that only the tail of the RBW
function enters the fit model as a virtual contribution. Nonresonant components are modeled using an exponen-
tial line shape, RNRðm2xÞ ¼
p
mΞb
LR
Ξb
q
m0
LR
expð−αm2xÞ;
ð20Þ ð20Þ 052010-8 SEARCH FOR CP VIOLATION IN Ξ−
b →pK−K−… PHYS. REV. D 104, 052010 (2021) 0
0.2
0.4
0.6
0.8
1
m'
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0
5
10
15
20
25
30
3
−
10
×
decay
−
b
Efficiency of
LHCb Simulation
0
0.2
0.4
0.6
0.8
1
m'
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0
5
10
15
20
25
30
3
−
10
×
decay
+
b
Ξ
Efficiency of
LHCb Simulation
0
0.2
0.4
0.6
0.8
1
m'
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0
5
10
15
20
25
30
3
−
10
×
decay
−
b
Ξ
Efficiency of
LHCb Simulation
0
0.2
0.4
0.6
0.8
1
m'
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0
5
10
15
20
25
30
3
−
10
×
decay
+
b
Ξ
Efficiency of
LHCb Simulation
FIG. 3. Efficiency as a function of square Dalitz-plot position for (left) Ξ−
b and (right) ¯Ξþ
b decays, for (top) Run 1 and (bottom) Run 2. B. Modeling of signal efficiency
and background distributions This is
accounted for using a method [57] in which the unnor-
malized
function
describing
the
mðpK−K−Þ
and
Ω0 ¼ ðm0; θ0Þ space is expressed as The SDP distribution of cross feed background from
misidentified Ξ−
b →pK−π−decays that enter the signal
region is modeled using simulation and is shown in the
bottom row of Fig. 4 separately for Run 1 and Run 2. These distributions are described in terms of a uniform
10 × 10 binned SDP histogram, smoothed with a two-
dimensional cubic spline. As described in Sec. IV, the
simulation is weighted to reproduce resonance structures
expected in the phase space of Ξ−
b →pK−π−decays. Differences in selection requirements, together with the
limited statistics of the Ξ−
b →pK−π−simulation samples,
cause the PDFs to differ between Run 1 and Run 2. In the
baseline fit it is assumed that there is no asymmetry
between Ξ−
b and ¯Ξþ
b candidates in the cross feed yields or
SDP distributions. FðmðpK−K−Þ; Ω0Þ ¼ jf0ðΩ0Þ þ exp ð−βmðpK−K−ÞÞ
× f1ðΩ0Þj2;
ð26Þ ð26Þ where β is a free parameter determined from a fit to the
sideband candidates. The functions f0 and f1 are modeled
using neural networks that are trained using candidates
from the data sideband region. This model is then extrapo-
lated to predict the PDF of the combinatorial background at
the Ξ−
b mass, i.e., PcombðΩ0Þ ¼ FðmΞb; Ω0Þ=N, where the
normalisation factor N ¼ R
Ω0 FðmΞb; Ω0ÞdΩ0. The PDF in
terms of DP variables is obtained using PcombðΩÞ ¼
jJjPcombðΩ0Þ, where jJj is the Jacobian determinant of
the transformation between variables, dΩ ¼ jJjdΩ0. The
SDP distributions of the combinatorial background for Run
1 and Run 2 are shown in the top row of Fig. 4. 2All Σ resonances considered are neutral; the conventional
charge superscripts are omitted for brevity of notation. C. Fitting procedure The function that is minimized, twice the negative log-
likelihood, is ð27Þ ð27Þ −2 ln L ¼ −2
X
r
X
Nr
i
ln ðPQ
totðΩiÞÞ
;
ð28Þ ð28Þ The yields of signal, combinatorial background and cross
feed background components are denoted by Nsig, Ncomb
and Ncf, respectively, and are obtained as described in
Sec. IV, separately for Run 1 and Run 2. The quantity
Ntot ¼ Nsig þ Ncomb þ Ncf is the total yield in the signal
region. The PDFs for the signal, combinatorial background
and cross feed background components are denoted by
PQ
sigðΩÞ, PcombðΩÞ and PcfðΩÞ, respectively, where the
former is given in Eq. (3) and the latter two are displayed in
Fig. 4 in terms of the SDP variables. In the baseline model,
only the signal PDF can differ for Ξ−
b and ¯Ξþ
b candidates,
although a possible global combinatorial background
asymmetry, Acomb, is a free parameter of the model. where the index i runs over the Nr candidates in the data
sample from run period r (Run 1 or Run 2), and Ωi denotes
the DP coordinates of candidate i. C. Fitting procedure The total PDF that is used to model the phase-
space distributions of Ξ−
b →pK−K−and its conjugate
decay is 052010-9 PHYS. REV. D 104, 052010 (2021) R. AAIJ et al. 0
0.2
0.4
0.6
0.8
1
m'
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
3
−
10
×
Combinatorial density
LHCb
-1
3 fb
0
0.2
0.4
0.6
0.8
1
m'
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0
0.05
0.1
0.15
0.2
0.25
0.3
3
−
10
×
Combinatorial density
LHCb
-1
2 fb
0
0.2
0.4
0.6
0.8
1
m'
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0
0.5
1
1.5
2
2.5
3
−
10
×
Cross-feed density
LHCb Simulation
0
0.2
0.4
0.6
0.8
1
m'
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
3
−
10
×
Cross-feed density
LHCb Simulation
FIG. 4. SDP distributions of (top) combinatorial and (bottom) cross feed background components for (left) Run 1 and (right) Run 2. 0
0.2
0.4
0.6
0.8
1
m'
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
3
−
10
×
Combinatorial density
LHCb
-1
3 fb 0
0.2
0.4
0.6
0.8
1
m'
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0
0.05
0.1
0.15
0.2
0.25
0.3
3
−
10
×
Combinatorial density
LHCb
-1
2 fb Combinatorial density 0
0.2
0.4
0.6
0.8
1
m'
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
3
−
10
×
Cross-feed density
LHCb Simulation 0
0.2
0.4
0.6
0.8
1
m'
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0
0.5
1
1.5
2
2.5
3
−
10
×
Cross-feed density
LHCb Simulation Cross-feed density FIG. 4. SDP distributions of (top) combinatorial and (bottom) cross feed background components for (left) Run 1 and (right) Run 2. PQ
totðΩÞ ¼ 1
Ntot
NsigPQ
sigðΩÞ
þ Ncomb
ð1 −QAcombÞ
2
PcombðΩÞ þ Ncf
2 PcfðΩÞ
:
ð27Þ
a
wi
Th
lik a fitting package based on TENSORFLOW [58], interfaced
with the MINUIT function minimization algorithm [59,60]. E. Fit to data In an attempt to find the global minimum, a large number
of fits to data are performed, where the initial values of the
helicity couplings are randomized. The baseline results
are obtained from the fit that returns the smallest −2 ln L
value out of this ensemble. This procedure is found to
converge successfully to the global minimum without any
secondary minima. The Dalitz-plot distribution of the combined Run 1 and
Run 2 data sample is compared to the model obtained from
the fit to data, separately for Ξ−
b and ¯Ξþ
b candidates, in
Fig. 5. Projections of the fit results onto mlow and mhigh are
compared to the data in Fig. 6. Further comparisons of the
fit result and the data in regions of the phase space are
presented in Appendix. There is no indication of CP
violation, i.e., no significant difference between Ξ−
b and
¯Ξþ
b decays, in the distributions. Resonances with spin J ≥7=2 are excluded from consid-
eration as these would require LΞb ≥3 and are therefore
expected to be significantly suppressed. In this analysis, as the normalization of the decay density
is arbitrary, the Λð1520Þ resonance is chosen as the
reference component. This implies that the coupling with
the positive helicity of the Λð1520Þ resonance aQ
R;λR¼þ1=2 is
real. Explicitly, for the reference Λð1520Þ resonance,
yR;λR¼þ1=2 ¼ δyR;λR¼þ1=2 ¼ 0 and xR;λR¼þ1=2 ¼ 1, while
δxR;λR¼þ1=2 is free to vary in the fit to allow for CP
violation in the Λð1520Þ amplitude. The analysis is found
to be insensitive to the coupling of the Λð1520Þ component
with
negative
helicity,
and,
therefore,
xR;λR¼−1=2 ¼
δxR;λR¼−1=2 ¼ yR;λR¼−1=2 ¼ δyR;λR¼−1=2 ¼ 0 for the refer-
ence resonance. The helicity couplings of all other resonant
and nonresonant components are left free to vary in the fit. b
The overall agreement between the data and the model is
good, with unbinned goodness-of-fit tests using the mixed-
sample and point-to-point dissimilarity approaches [61]
giving p-values of 0.20 and 0.25, respectively. In Fig. 6,
there is an apparent discrepancy between the model and the
data at mhigh between 3.4 GeV to 3.7 GeV, predominantly
in the ¯Ξþ
b sample. However, due to the symmetry of the
final state, any structure that appears in mhigh should also
appear in mlow, where no such structure is observed. D. Model selection The Ξ−
b →pK−K−decay can proceed via intermediate
pK−resonances. Various Λ and Σ resonances that are
known to decay to pK−are considered as potential
components of the signal model.2 The Particle Data
Group (PDG) [23] reports a large number of such states;
those that are sufficiently well established are considered in
this study and are shown in Table II. Masses and widths of
all resonance components are fixed to either the central
value or the midpoint in the range of values quoted
in Table II. Nonresonant components, labelled NRðJPÞ,
with spin-parity JP ¼ 1
2
þ; 1
2
−; 3
2
þ; 3
2
−are also considered. An unbinned maximum-likelihood fit is performed to the
combined sample of candidates for Ξ−
b →pK−K−and its
conjugate decay to determine the parameters of the model,
which are the CP -conserving and CP -violating coef-
ficients of the helicity couplings of Eq. (11). The fit is
performed simultaneously to the Run 1 and Run 2 data
samples, which have separate efficiency and background
models as described above. The fit model is implemented in 052010-10 SEARCH FOR CP VIOLATION IN Ξ−
b →pK−K−… PHYS. REV. D 104, 052010 (2021) To establish a baseline fit model, the Λð1520Þ compo-
nent alone is initially included in the model, with additional
components added iteratively in the order that maximizes
the change in −2 ln L obtained from fits to the data with
prospective models. Components with different spin and
parity should have zero interference fit fractions due to
the orthogonality relation satisfied by the small Wigner
d-matrix elements. However, the symmetrization of the
Dalitz plot can lead to nonzero values for such interference
fit fractions in this analysis. As a result, in establishing the
baseline model, it is possible to encounter “unphysical”
interference fit fractions (> 40%) between two compo-
nents. When such a case occurs, the component that gives
the minimal change in −2 ln L, when removed from the fit
model, is discarded. The procedure is terminated when the
change in −2 ln L from including any further contribution
is less than 9 units, limiting the potential for the model to be
influenced by statistical fluctuations. This approach leads
to a model that contains Σð1385Þ, Λð1405Þ, Λð1520Þ,
Λð1670Þ, Σð1775Þ and Σð1915Þ components. The potential
for additional components to be present in the true under-
lying model is considered as a source of systematic
uncertainty. D. Model selection TABLE II. Summary of the considered Λ and Σ resonances,
ranked either **** or *** by the PDG [23]. Note that the pK−
threshold is at 1432 MeV. Resonances marked † are included in
the baseline model, as described in the text. The spin-parity of the
Σð2250Þ is not known and is assumed to be 3
2
þ. For many of these
states, the PDG does not report masses and widths with central
values and uncertainties, but rather gives real and imaginary parts
of the pole position. This reflects the fact that a simple Breit–
Wigner parametrization of these resonances may not fully
describe their line shapes; however, more sophisticated para-
metrizations are beyond the scope of the current analysis. Name
JP Mass ðMeVÞ Width ðMeVÞ
Main decay
channels
****
† Λð1405Þ 1
2
−
1405.1þ1.3
−1.0
50.5 2.0
Σπ
† Λð1520Þ 3
2
−1518 to 1520
15 to 17
N ¯K, Σπ
† Λð1670Þ 1
2
−1660 to 1680
25 to 50
N ¯K, Σπ, Λη
Λð1690Þ 3
2
−1685 to 1695
50 to 70
N ¯K, Σπ, Λππ, Σππ
Λð1820Þ 5
2
þ 1815 to 1825
70 to 90
N ¯K
Λð1830Þ 5
2
−1810 to 1830
60 to 110
Σπ
Λð1890Þ 3
2
þ 1850 to 1910
60 to 200
N ¯K
† Σð1385Þ 3
2
þ
1383.7 1
36 5
Λπ; Σπ
Σð1670Þ 3
2
−1665 to 1685
40 to 80
Σπ
† Σð1775Þ 5
2
−1770 to 1780
105 to 135
N ¯K, ΛðÞπ
† Σð1915Þ 5
2
þ 1900 to 1935
80 to 160
not clear
***
Λð1600Þ 1
2
þ 1560 to 1700
50 to 250
N ¯K, Σπ
Λð1800Þ 1
2
−1720 to 1850
200 to 400
N ¯KðÞ, Σπ, Λη
Λð1810Þ 1
2
þ 1750 to 1850
50 to 250
N ¯KðÞ, Σπ, Λη, ΞK
Λð2110Þ 5
2
þ 2090 to 2140
150 to 250
N ¯KðÞ, Σπ, ΛΩ
Σð1660Þ 1
2
−1630 to 1690
40 to 200
N ¯K, Σπ, Λπ
Σð1750Þ 1
2
−1730 to 1800
60 to 160
N ¯K, Σπ, Λπ, Ση
Σð1940Þ 3
2
−1900 to 1950
150 to 300
N ¯K, Σπ, Λπ
Σð2250Þ ?? 2210 to 2280
60 to 150
N ¯K, Σπ, Λπ Name
JP Mass ðMeVÞ Width ðMeVÞ
Main decay
channels E. Fit to data 0
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b and (bottom) ¯Ξþ
b candidates. E. Fit to data 2
3
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(
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(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
FIG. 6. Distributions of (top) mlow and (bottom) mhigh for (left) Ξ−
b and (right) ¯Ξþ
b candidates, with results of the fits superimposed. The
total fit result is shown as the blue solid curve, with contributions from individual signal components and from combinatorial (Comb)
and cross feed (Crsfd) background shown as indicated in the legend. E. Fit to data Addition of extra components to the fit model does not
significantly improve the data description. Moreover, the
apparent discrepancy in Fig. 6 does not take into account
the systematic uncertainty in the mismodeling of the 052010-11 PHYS. REV. D 104, 052010 (2021) R. AAIJ et al. 0
2
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+
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LHCb
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FIG. 5. Dalitz-plot distributions from (left) data and (right) the fit model for (top) Ξ−
b and (bottom) ¯Ξþ
b candidates. E. Fit to data 2
3
4
) [GeV]
+
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p
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+
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p
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5
) [GeV]
+
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(
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(
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(
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−
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Fit
)
1405
(
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1520
(
)
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(
)
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(
)
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(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb ) [GeV]
+
K
p
(
high
m FIG. 6. Distributions of (top) mlow and (bottom) mhigh for (left) Ξ−
b and (right) ¯Ξþ
b candidates, with results of the fits superimposed. The
total fit result is shown as the blue solid curve, with contributions from individual signal components and from combinatorial (Comb)
and cross feed (Crsfd) background shown as indicated in the legend. 052010-12 SEARCH FOR CP VIOLATION IN Ξ−
b →pK−K−… PHYS. REV. D 104, 052010 (2021) Finally, potential fit bias is investigated by generating
multiple pseudoexperiments with yield and fit parameters
obtained from the nominal mðpK−K−Þ fit. The difference
between the mean fit result of the ensemble and the nominal
value is assigned as the associated systematic uncertainty. combinatorial background, which is the largest component
at mhigh ∼3.7 GeV. Therefore, this feature is not consid-
ered to be significant and is not investigated further. B. Selection efficiency maps The efficiency maps are altered to evaluate systematic
uncertainties from six sources. In each case the difference
between the results obtained using the alternative efficiency
maps and that with the baseline efficiency maps is assigned
as the systematic uncertainty. The first source reflects uncertainties in the pT distri-
bution of Ξ−
b baryons produced in the LHCb acceptance. Alternative efficiency maps are obtained where the simu-
lation samples are weighted so that the pT distribution
matches that of the background-subtracted data. Since there
is no significant signal of Ω−
b baryons, they are assumed
to have the same pT distribution as Ξ−
b baryons, and the
Ω−
b →pK−K−efficiency map is altered in the same way. The second source is a possible mismatch of the
hardware trigger efficiency between simulation and data,
which could arise due to miscalibration of transverse
energy measurements from the calorimeter. Alternative
efficiency maps are obtained by applying corrections that
are calculated, as a function of track pT, using control
samples of kaons from Dþ →D0ðK−πþÞπþ decays and
protons from Λ0
b →Λþc ðpK−πþÞπ−decays. The first source reflects uncertainties in the pT distri-
bution of Ξ−
b baryons produced in the LHCb acceptance. Alternative efficiency maps are obtained where the simu-
lation samples are weighted so that the pT distribution
matches that of the background-subtracted data. Since there
is no significant signal of Ω−
b baryons, they are assumed
to have the same pT distribution as Ξ−
b baryons, and the
Ω−
b →pK−K−efficiency map is altered in the same way. VI. SYSTEMATIC UNCERTAINTIES The outcomes of the analysis are the ratio R of Ω−
b and
Ξ−
b branching fractions and fragmentation fractions [see
Eq. (1)], as well as the fit fractions, interference fit fractions
and CP-asymmetry parameters obtained from the ampli-
tude analysis. Various sources of systematic uncertainty can
affect these measurements. These are discussed one by one
in this section, concluding with a summary. Systematic
uncertainties are evaluated separately for Run 1 and Run 2,
where appropriate. A. Invariant mass fits The fits to the mðpK−K−Þ invariant mass distributions
determine the signal and background yields, which are used
in both the calculation of R and the amplitude analysis. Four sources of systematic uncertainty arising from these
fits are considered. The first is due to the limited size of the
data sample. This enters the calculation of R as statistical
uncertainty, but is a source of systematic uncertainty in the
amplitude analysis where the signal and background yields
are fixed parameters. To evaluate the associated systematic
uncertainty, these yields are varied according to the
covariance matrix obtained from the mðpK−K−Þ fit, and
for each variation the fit to the phase-space distribution is
repeated. The root-mean-square (rms) of the distribution of
the change in each result of the amplitude analysis is
assigned as the corresponding systematic uncertainty. The second source is a possible mismatch of the
hardware trigger efficiency between simulation and data,
which could arise due to miscalibration of transverse
energy measurements from the calorimeter. Alternative
efficiency maps are obtained by applying corrections that
are calculated, as a function of track pT, using control
samples of kaons from Dþ →D0ðK−πþÞπþ decays and
protons from Λ0
b →Λþc ðpK−πþÞπ−decays. The third source is due to uncertainty arising from
binning the phase space when evaluating the efficiency
maps. Alternative efficiency maps are obtained employing
a different SDP binning scheme. An additional systematic
uncertainty is associated to the efficiency of Ω−
b →pK−K−
decays and arises from their unknown phase-space distri-
bution. The standard deviation of the variation of the
efficiency across the binned SDP histogram is assigned
as the corresponding uncertainty. g
p
g y
y
The second source relates to the mðpK−K−Þ fit model. Models for each of the components are varied to evaluate
associated systematic uncertainties: the signal model is
replaced with a Hypatia function [62]; the combinatorial
background
model
is
replaced
with
a
second-order
Chebyshev polynomial; the cross feed and partially recon-
structed background models are replaced with kernel density
estimates; additional partially reconstructed background
components are included; the model used to describe the
phase-space distribution of the Ξ−
b →pK−π−cross feed
background is varied. In each case, the change in each result
from its baseline value is taken as the systematic uncertainty. The third source concerns fixed parameters in the
mðpK−K−Þ fit that are taken from simulation. D. Background asymmetry Each resonant contribution has fixed parameters in the
amplitude fit. These include masses, widths and Blatt–
Weisskopf radius parameters. An ensemble of fits is
obtained varying the masses and widths of all resonances
within the range of values quoted by the PDG and given in
Table II. The Blatt–Weisskopf radius parameter associated
with the Ξ−
b baryon is varied in the range 3–7 GeV−1 and
that associated with the resonances is varied in the range
0–3.5 GeV−1. The rms of the distribution of the change in
each fitted parameter is taken as the systematic uncertainty. Each resonant contribution has fixed parameters in the
amplitude fit. These include masses, widths and Blatt–
Weisskopf radius parameters. An ensemble of fits is
obtained varying the masses and widths of all resonances
within the range of values quoted by the PDG and given in
Table II. The Blatt–Weisskopf radius parameter associated
with the Ξ−
b baryon is varied in the range 3–7 GeV−1 and
that associated with the resonances is varied in the range
0–3.5 GeV−1. The rms of the distribution of the change in
each fitted parameter is taken as the systematic uncertainty. For the Λð1405Þ and Σð1385Þ resonances that peak
below the mlow threshold, the effective RBW used in the
baseline fit is replaced with a line shape equivalent to
the Flatt´e parametrization as done in Ref. [46]. The total
width is modified to account for the Σþπ−channel, i.e.,
ΓðmÞ ¼ ΓpK−ðmÞ þ ΓΣþπ−ðmÞ, assuming equal couplings
to both channels. For each of these line shape variations, the
differences in the results between fits with the alternative
and baseline models are assigned as the associated sys-
tematic uncertainties. In the baseline model, it is assumed that there is no local
asymmetry in the combinatorial background as described in
Sec. V B. The associated systematic uncertainty is evalu-
ated by considering separate background distributions for
Ξ−
b and ¯Ξþ
b candidates. In order to obtain sufficiently large
background samples to determine these separate distribu-
tions, the MVA output requirement for candidates in the
sideband region is relaxed. A possible global combinatorial background asymmetry
is accounted for in the baseline fit, while cross feed
background is assumed to have no asymmetry. A. Invariant mass fits An ensemble
of pseudoexperiments is generated using the nominal
values of these parameters, and each is then fitted many
times with parameters fixed to alternative values obtained
using the covariance matrices of the fits to the simulation
samples. The standard deviation of the change in each
result is evaluated for every pseudoexperiment, and its
average value over the ensemble is assigned as the
systematic uncertainty. The second source relates to the mðpK−K−Þ fit model. Models for each of the components are varied to evaluate
associated systematic uncertainties: the signal model is
replaced with a Hypatia function [62]; the combinatorial
background
model
is
replaced
with
a
second-order
Chebyshev polynomial; the cross feed and partially recon-
structed background models are replaced with kernel density
estimates; additional partially reconstructed background
components are included; the model used to describe the
phase-space distribution of the Ξ−
b →pK−π−cross feed
background is varied. In each case, the change in each result
from its baseline value is taken as the systematic uncertainty. The remaining sources relate to particle identification. The PID variables used in the MVA are drawn from
data calibration samples accounting for dependence on
the signal kinematics. Systematic uncertainties in this
procedure arise from the limited statistics of both the
simulation and calibration samples and the modeling of
the PID variables in the calibration samples. The limitations
due to both simulation and calibration sample size are
evaluated by bootstrapping to create multiple samples and
repeating the procedure for each sample. The impact of
potential mismodeling of the PID variables in the calibra-
tion samples is evaluated by describing the corresponding
distributions using density estimates with different kernel
widths. For each of these cases, alternative efficiency maps
are produced to determine the associated uncertainties on
the results of the analysis. The third source concerns fixed parameters in the
mðpK−K−Þ fit that are taken from simulation. An ensemble
of pseudoexperiments is generated using the nominal
values of these parameters, and each is then fitted many
times with parameters fixed to alternative values obtained
using the covariance matrices of the fits to the simulation
samples. The standard deviation of the change in each
result is evaluated for every pseudoexperiment, and its
average value over the ensemble is assigned as the
systematic uncertainty. In principle, mismodeling of the proton and kaon
reconstruction efficiencies, and associated asymmetries, 052010-13 PHYS. REV. D 104, 052010 (2021) R. AAIJ et al. E. Production asymmetry The baseline fit model assumes no asymmetry in the Ξ−
b
production rates in the LHCb acceptance, consistent with
measurements [44]. To evaluate the associated systematic
uncertainty, the model is adjusted to include production
asymmetries within the experimentally allowed range by
introducing a global asymmetry in the efficiency maps. An
ensemble of fits with varied Ξ−
b production asymmetries is
performed, and the rms of the distribution of the change in A. Invariant mass fits each result, with respect to its baseline value, is assigned as
the systematic uncertainty. could be a source of systematic uncertainty. However, such
effects are known to be negligible at the level of precision
achieved in this analysis [63,64] and therefore are not
accounted for explicitly. F. Polarization The transverse polarization of Ξ−
b baryons produced in pp
collisions is assumed to be consistent with zero, as observed
for Λ0
b baryons [49,50]. The distributions of m2
low and m2
high
are independent of the Ξ−
b polarization. However, if the Ξ−
b
baryons produced in LHC collisions are polarized, efficiency
variation across additional phase-space variables should be
considered in the analysis. To evaluate the systematic
uncertainty due to potential Ξ−
b polarization, two sets of
pseudoexperiments are generated, with the Ξ−
b polarization
set to 20% in one case and −8% in the other. This
corresponds to the 2σ range measured for the Λ0
b baryon
in Refs. [49,50], where σ indicates the Gaussian standard
deviation. A conservatively broad range is taken to allow for
differences between Ξ−
b and Λ0
b polarization. The pseudoex-
periments are generated using a signal model whose helicity
couplings are set to the values from the nominal fit and
where the measured efficiency variation over the additional
phase-space observables is introduced. A fit to the Dalitz-
plot variables is then performed using the baseline model. The largest deviation of the parameters from the nominal
case is assigned as the systematic uncertainty. C. Background shapes The combinatorial background SDP distribution is
obtained by extrapolating from an mðpK−K−Þ sideband
region and has uncertainties related to the available yield in
the sideband and the extrapolation procedure itself. The
former is evaluated by bootstrapping to create multiple
combinatorial background samples and repeating the
amplitude fit with each. The rms of the distribution of
the change in each result is taken as the systematic
uncertainty. The latter is evaluated by changing the archi-
tecture of the neural network, with the change in each result
with respect to its baseline value assigned as the associated
systematic uncertainty. In the baseline fit, the Ξ−
b →pK−π−cross feed back-
ground is described with a model consisting of Λð1405Þ,
Λð1520Þ, Λð1690Þ, Nð1440Þ, Nð1520Þ, Nð1535Þ and
Nð1650Þ resonances. To evaluate the systematic uncer-
tainty arising from this assumption, the model is modified
by adding Λð1600Þ, Λð1670Þ, Λð1800Þ and Nð1720Þ
components, and removing the Nð1520Þ component. The
change in each result, with respect to its baseline value, is
assigned as the associated systematic uncertainty. D. Background asymmetry A fit
allowing for a global cross feed background asymmetry
is performed, and the differences between the results in this
fit and their nominal values is assigned as the systematic
uncertainty arising from this assumption. For the Λð1405Þ and Σð1385Þ resonances that peak
below the mlow threshold, the effective RBW used in the
baseline fit is replaced with a line shape equivalent to
the Flatt´e parametrization as done in Ref. [46]. The total
width is modified to account for the Σþπ−channel, i.e.,
ΓðmÞ ¼ ΓpK−ðmÞ þ ΓΣþπ−ðmÞ, assuming equal couplings
to both channels. For each of these line shape variations, the
differences in the results between fits with the alternative
and baseline models are assigned as the associated sys-
tematic uncertainties. 1. Ratio of fragmentation and branching fractions Since separate fits are performed to the mðpK−K−Þ
distributions from the Run 1 and Run 2 samples, and the
signal efficiencies are also determined separately, results
for R in each of the two samples are obtained. Systematic
uncertainties on R are considered as being either com-
pletely uncorrelated or 100% correlated between the two
results. The systematic uncertainties that are uncorrelated
between Run 1 and Run 2 are folded into their respective
likelihood functions, by convolution with a Gaussian of
appropriate width. The correlated systematic uncertainties
are later folded into the combined likelihood that is
obtained by multiplying the likelihood functions of the
two samples. uncertainty on ACP is due to variation of the Λð1405Þ line
shape which has the same spin and parity as this compo-
nent, whereas for F, it is due to use of an alternate fit
model. For Σð1385Þ, the dominant systematic uncertainty
on ACP is due to use of an alternate fit model, whereas for
F, it is due to variation of the Blatt–Weisskopf radius
parameters. For Λð1405Þ, the largest systematic uncertainty
on ACP is due to use of an alternate fit model, whereas for
F, it is due to variation in its line shape. For Σð1775Þ,
the dominant systematic uncertainty on ACP is due to the
limited size of the sample used for modeling of the
combinatorial background, whereas for F, it is due to
use of an alternate fit model. For Σð1915Þ, the dominant
systematic uncertainty on both ACP and F is due to use of
an alternate fit model. The uncorrelated systematic uncertainties are those that
are related to the fixed parameters in the fit model, the fit
bias and the impact on the efficiency of the Ξ−
b production
kinematics, and the descriptions of the hardware trigger and
particle identification response. The correlated systematic
uncertainties are those related to knowledge of the phase-
space distributions of the decays and the fit model choice. Slightly different procedures are used to obtain the total
uncertainty for the two sources of correlated systematic
uncertainty. That which is related to knowledge of the
phase-space distribution affects the efficiency, and hence is
a constant relative uncertainty. The method to evaluate the
uncertainty due to fit model choice gives different relative
uncertainties between Run 1 and Run 2. H. Alternative fit model The effect of including additional signal components in
the fit model is examined to assign systematic uncertainty
due to the composition of the baseline model. The Λð1690Þ,
Λð1820Þ, Σð1670Þ and NRð3
2
þÞ components are added to the 052010-14 PHYS. REV. D 104, 052010 (2021) SEARCH FOR CP VIOLATION IN Ξ−
b →pK−K−… TABLE III. Absolute systematic uncertainties on R, in units of
10−3, from (top) uncorrelated and (bottom) correlated sources. The total is the sum in quadrature of all contributions. nominal model individually. These modifications of the
model are chosen since they improve −2 ln L, although
not by a significant amount. The largest deviation among the
four cases, from the nominal value of each measured
quantity, is taken as the associated systematic uncertainty. Uncorrelated sources
Run 1
Run 2
Ξ−
b pT distribution
<0.1
0.7
Hardware trigger efficiency
0.1
1.6
PID efficiency
0.1
0.6
Fixed parameters
0.8
0.5
Fit bias
0.5
<0.1
Total
1.0
1.9
Correlated sources
Run 1
Run 2
Combined
Phase-space distribution
8.9
22.5
10.6
Fit model choice
9.1
13.1
8.6
Total
13.6 1. Ratio of fragmentation and branching fractions Since the two
samples have approximately equal statistical weight in the
combination, the average of the relative uncertainties is
taken and assigned to the combined result. Table III
summarizes the systematic uncertainties on R. For interference fit fractions, the largest systematic
uncertainties are mainly due to the use of an alternate fit
model, the limited size of the sample used for modeling
of the combinatorial background, variation of the resonance
line shapes and variation of Blatt–Weisskopf radius
parameters. VII. RESULTS The results of the amplitude analysis are the CP-asym-
metry parameters ACP, defined in Eq. (25), the fit fractions F
defined in Eq. (21) and the interference fit fractions I
defined in Eq. (23). A summary of the systematic uncer-
tainties on these quantities is shown in Table IV. A. Ratio of fragmentation and branching fractions A. Ratio of fragmentation and branching fractions The results for the ratio R of the relative fragmentation
and branching fractions for Ω−
b →pK−K−and Ξ−
b →
pK−K−decays are The most precise results are those related to the Λð1520Þ
and Λð1670Þ resonances. For Λð1520Þ, the dominant
systematic uncertainty on ACP is due to ignoring the
efficiency variation over angular variables if Ξ−
b baryons
are produced polarized, whereas for F, it is due to the
limited size of the sample used for combinatorial back-
ground modeling and the variation of the Blatt–Weisskopf
radius parameters. For Λð1670Þ, the largest systematic R¼ð−2030ðstatÞ1ðuncorr systÞÞ×10−3 for Run1and
R¼ð 5128ðstatÞ2ðuncorrsystÞÞ×10−3 for Run2; R¼ð−2030ðstatÞ1ðuncorr systÞÞ×10−3 for Run1and
R¼ð 5128ðstatÞ2ðuncorrsystÞÞ×10−3 for Run2; where the first uncertainty is statistical and the second
includes only the uncorrelated systematic effects presented
in Table III. The negative log-likelihood functions for these
two results are added to obtain a combined result, 052010-15 PHYS. REV. D 104, 052010 (2021) R. AAIJ et al. TABLE IV. Summary of absolute systematic uncertainties, in units of 10−2, on the results of the amplitude analysis: (top) CP -
asymmetry parameters ACP and fit fractions F, (bottom) interference fit fractions I. A. Ratio of fragmentation and branching fractions The total is the sum in quadrature of all individual
sources Component & Parameter
Mass
fits
Bkg
shapes
Bkg
asym
Eff
Prod
asym
Polarisation
RBW
params
Lineshapes
Alt fit
model
Total
Σð1385Þ
ACP
3.3
20.6
4.4
8.2
6.9
15.0
7.0
12.6
65.5
72.7
F
1.4
3.1
0.5
0.7
0.2
1.0
5.0
0.6
4.4
7.6
Λð1405Þ
ACP
2.4
9.6
2.7
5.1
5.1
5.5
5.1
19.5
20.6
31.9
F
0.3
1.4
0.3
0.8
<0.1
0.3
0.3
2.3
0.9
3.0
Λð1520Þ
ACP
0.3
0.9
0.6
2.9
4.3
5.0
0.7
1.2
1.3
7.6
F
1.1
1.8
<0.1
1.3
<0.1
0.6
1.8
0.6
1.7
3.6
Λð1670Þ
ACP
1.8
4.2
1.4
2.9
4.4
3.3
3.7
4.9
1.6
10.1
F
0.8
2.3
0.1
0.7
0.1
0.6
1.4
1.8
4.4
5.6
Σð1775Þ
ACP
2.5
7.8
1.7
3.1
3.4
7.0
3.8
4.7
3.7
13.8
F
0.5
1.5
0.1
0.4
0.2
0.2
1.0
0.9
3.5
4.1
Σð1915Þ
ACP
2.5
6.7
5.0
6.4
4.8
5.2
10.5
2.1
13.9
21.8
F
0.2
2.3
0.1
1.3
0.2
0.2
2.2
1.5
8.4
9.2
Λð1405Þ; Λð1520Þ I
0.2
0.6
0.1
0.1
<0.1
0.1
0.2
0.2
0.4
0.8
Λð1405Þ; Λð1670Þ I
0.3
0.9
0.1
0.5
<0.1
<0.1
1.2
1.6
0.9
2.4
Λð1405Þ; Σð1385Þ I
0.2
0.4
0.1
0.2
<0.1
0.1
0.5
0.7
1.8
2.0
Λð1405Þ; Σð1775Þ I
<0.1
0.3
<0.1
0.1
<0.1
0.1
0.4
0.3
1.1
1.2
Λð1405Þ; Σð1915Þ I
0.1
0.4
<0.1
0.1
<0.1
<0.1
0.1
0.3
0.6
0.8
Λð1520Þ; Λð1670Þ I
0.1
0.2
<0.1
<0.1
<0.1
<0.1
0.2
0.1
0.8
0.8
Λð1520Þ; Σð1385Þ I
0.7
0.9
0.2
0.2
0.2
0.1
1.6
0.9
3.6
4.2
Λð1520Þ; Σð1775Þ I
0.3
0.6
0.1
0.1
0.1
0.2
0.5
0.5
1.6
1.9
Λð1520Þ; Σð1915Þ I
<0.1
0.1
<0.1
<0.1
<0.1
<0.1
0.1
<0.1
0.1
0.2
Λð1670Þ; Σð1385Þ I
0.2
0.3
0.1
0.1
<0.1
<0.1
0.4
0.6
0.6
1.0
Λð1670Þ; Σð1775Þ I
0.1
0.1
<0.1
<0.1
<0.1
<0.1
0.1
0.1
0.2
0.3
Λð1670Þ; Σð1915Þ I
<0.1
0.1
<0.1
<0.1
<0.1
<0.1
0.3
0.1
0.4
0.5
Σð1385Þ; Σð1775Þ I
0.1
0.3
<0.1
<0.1
<0.1
<0.1
0.2
0.1
0.2
0.4
Σð1385Þ; Σð1915Þ I
0.2
0.6
<0.1
0.2
<0.1
0.1
0.6
0.2
1.0
1.3
Σð1775Þ; Σð1915Þ I
0.1
0.4
<0.1
0.1
<0.1
0.1
0.7
0.2
1.0
1.3 R ≡
fΩ−
b
fΞ−
b
× BðΩ−
b →pK−K−Þ
BðΞ−
b →pK−K−Þ
¼ ð24 21 ðstatÞ 14 ðsystÞÞ R ≡
fΩ−
b
fΞ−
b
× BðΩ−
b →pK−K−Þ
BðΞ−
b →pK−K−Þ
¼ ð24 21 ðstatÞ 14 ðsystÞÞ × 10−3; R ≡
fΩ−
b
fΞ−
b
× BðΩ−
b →pK−K−Þ
BðΞ−
b →pK−K−Þ B. Amplitude analysis The results for the CP-asymmetry parameters for each
component of the signal model are shown in Table V. No
significant CP asymmetry is observed. The fit fraction
matrix is reported in Table VI. The diagonal elements ¼ ð24 21 ðstatÞ 14 ðsystÞÞ × 10−3; ¼ ð24 21 ðstatÞ 14 ðsystÞÞ × 10−3; where both uncorrelated and correlated systematic uncer-
tainties are included. In the combined result, it is implied
that fΩ−
b =fΞ−
b , which may vary with center-of-mass energy where both uncorrelated and correlated systematic uncer-
tainties are included. In the combined result, it is implied
that fΩ−
b =fΞ−
b , which may vary with center-of-mass energy TABLE V. Results for the CP -asymmetry parameters. The
statistical uncertainties are obtained from pseudoexperiments,
while the systematic uncertainties are obtained following the
procedure described in Sec. VI. b
b
of the LHC pp collisions, is an effective value averaged
over the Run 1 and Run 2 data samples. This result is found
to be consistent with, and more precise than, the previous
measurement
[24]. No
significant
evidence
of
the
Ω−
b →pK−K−decay is found and, therefore, an upper
limit on R is calculated at 90 (95) % confidence level by
integrating the likelihood in physical region of non-neg-
ative branching fraction, Component
ACP (10−2)
Σð1385Þ
−27 34 ðstatÞ 73 ðsystÞ
Λð1405Þ
−1 24 ðstatÞ 32 ðsystÞ
Λð1520Þ
−5
9 ðstatÞ 8 ðsystÞ
Λð1670Þ
3 14 ðstatÞ 10 ðsystÞ
Σð1775Þ
−47 26 ðstatÞ 14 ðsystÞ
Σð1915Þ
11 26 ðstatÞ 22 ðsystÞ R ≡
fΩ−
b
fΞ−
b
× BðΩ−
b →pK−K−Þ
BðΞ−
b →pK−K−Þ < 62ð71Þ × 10−3: 052010-16 SEARCH FOR CP VIOLATION IN Ξ−
b →pK−K−… PHYS. REV. D 104, 052010 (2021) TABLE VI. Results for the fit fractions (diagonal elements) and interference fit fractions (off-diagonal elements) obtained from the
amplitude analysis. Identical values for the interference fit fractions in the upper triangle are omitted. All values are in units of 10−2, with
the first uncertainty being statistical and the second systematic. B. Amplitude analysis where the uncertainties are statistical, systematic and due to
the knowledge of BðΞ−
b →pK−K−Þ, respectively. B. Amplitude analysis Component
Σð1385Þ
Λð1405Þ
Λð1520Þ
Λð1670Þ
Σð1775Þ
Σð1915Þ
Σð1385Þ
11.4 4.9 7.6
Λð1405Þ
−1.3 0.8 2.0
8.1 2.7 3.0
Λð1520Þ
3.4 1.6 4.2
0.1 0.5 0.8
33.0 4.1 3.6
Λð1670Þ
−0.1 0.6 1.0
3.0 1.8 2.4
−0.1 0.4 0.8
19.5 3.2 5.6
Σð1775Þ
0.1 0.3 0.4 −0.7 0.5 1.2
1.1 1.0 1.9
−0.3 0.2 0.3
9.7 3.5 4.1
Σð1915Þ
0.6 0.6 1.3
0.3 0.3 0.8
0.1 0.1 0.2
−0.1 0.2 0.5
1.0 0.5 1.3
11.3 3.7 9.2 where the dominant uncertainty is that due to possible
SU(3)-breaking
effects
which
affect
fΞ−
b =fΛ0
b
[44]. Consequently, the values of the quasi-two-body branching
fractions are found to be correspond to the fit fractions of the respective components,
and the off-diagonal elements are the interference fit
fractions. These results are derived from the helicity
couplings that are the free parameters of the amplitude
fit. Their statistical uncertainties are evaluated from an
ensemble of pseudoexperiments, while systematic uncer-
tainties are obtained as described in Sec. VI. BðΞ−
b →Σð1385ÞK−Þ¼ð0.260.110.170.10Þ×10−6;
BðΞ−
b →Λð1405ÞK−Þ¼ð0.190.060.070.07Þ×10−6;
BðΞ−
b →Λð1520ÞK−Þ¼ð0.760.090.080.30Þ×10−6;
BðΞ−
b →Λð1670ÞK−Þ¼ð0.450.070.130.18Þ×10−6;
BðΞ−
b →Σð1775ÞK−Þ¼ð0.220.080.090.09Þ×10−6;
BðΞ−
b →Σð1915ÞK−Þ¼ð0.260.090.210.10Þ×10−6; The significance of each component in the baseline
model is evaluated using pseudoexperiments. These are
generated, each with a sample size corresponding to the
data, according to the best fit model with the component
of interest removed from the model. They are then fitted
both with the model used to generate and with the model
including the component of interest. Twice the difference
between the negative log-likelihood values obtained in
these two fits (−2Δ ln L) is used as a test statistic. A p-
value, corresponding to the probability of observing
−2Δ ln L values as large or larger than that found in the
fit to data, is found by extrapolating the tail of the
distribution obtained from the ensemble of pseudoexperi-
ments. In order to account for dominant systematic uncer-
tainties, this procedure is performed for the alternative
model that gives the smallest value of −2 ln L in fits to data. The outcome is that the Λð1520Þ and Λð1670Þ components
have p-values corresponding to 12.0σ and 6.1σ, respec-
tively. All other components have significance below 3.5σ. APPENDIX: FIT PROJECTIONS Projections of the fit result are compared to the data in
slices of mlow in Figs. 7–11. Similar projections in slices of
mhigh are shown in Figs. 12–14. 1.45
1.5
1.55
1.6
) [GeV]
−
K
p
(
low
m
0
5
10
15
20
25
Entries / (0.01 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.43, 1.6] GeV
)
−
K
p
(
low
m
1.45
1.5
1.55
1.6
) [GeV]
+
K
p
(
low
m
0
5
10
15
20
25
Entries / (0.01 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.43, 1.6] GeV
)
+
K
p
(
low
m
3.5
4
4.5
5
) [GeV]
−
K
p
(
high
m
0
2
4
6
8
10
12
14
16
18
20
Entries / (0.06 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.43, 1.6] GeV
)
−
K
p
(
low
m
3.5
4
4.5
5
) [GeV]
+
K
p
(
high
m
0
2
4
6
8
10
12
14
16
18
20
Entries / (0.06 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.43, 1.6] GeV
)
+
K
p
(
low
m
FIG. 7. Distributions of (top) mlow and (bottom) mhigh, with 1.43 < mlow < 1.60 GeV, for (left) Ξ−
b and (right) ¯Ξþ
b candidates, with
results of the fits superimposed. The total fit result is shown as the blue solid curve, with contributions from individual signal
components and from combinatorial (Comb) and cross feed (Crsfd) background shown as indicated in the legend. VIII. SUMMARY Individual groups or
members have received support from ARC and ARDC
(Australia);
AvH
Foundation
(Germany);
EPLANET,
Marie Skłodowska-Curie Actions and ERC (European
Union); A*MIDEX, ANR, IPhU and Labex P2IO, and
R´egion Auvergne-Rhône-Alpes (France); Key Research
Program of Frontier Sciences of CAS, CAS PIFI, CAS
CCEPP, Fundamental Research Funds for the Central
Universities, and Sci. & Tech. Program of Guangzhou
(China); RFBR, RSF and Yandex LLC (Russia); GVA,
XuntaGal and GENCAT (Spain); the Leverhulme Trust, the
Royal Society and UKRI (United Kingdom). fractions of Ω−
b →pK−K−and Ξ−
b →pK−K−decays is set. With the substantially larger samples that are anticipated
following the upgrade of LHCb [68,69], it will be possible
to reduce both statistical and systematic uncertainties on
CP -violation observables in three-body b -baryon decays,
and thereby test the Standard Model using the methods
pioneered in this study. ACKNOWLEDGMENTS We express our gratitude to our colleagues in the CERN
accelerator departments for the excellent performance of
the LHC. We thank the technical and administrative staff at
the LHCb institutes. We acknowledge support from CERN
and from the national agencies: CAPES, CNPq, FAPERJ
and FINEP (Brazil); MOST and NSFC (China); CNRS/
IN2P3 (France); BMBF, DFG and MPG (Germany); INFN
(Italy); NWO (Netherlands); MNiSW and NCN (Poland);
MEN/IFA (Romania); MSHE (Russia); MICINN (Spain);
SNSF and SER (Switzerland); NASU (Ukraine); STFC
(United Kingdom); DOE NP and NSF (USA). We
acknowledge the computing resources that are provided
by CERN, IN2P3 (France), KIT and DESY (Germany), VIII. SUMMARY The structure of Ξ−
b →pK−K−decays has been studied
through an amplitude analysis. This is the first amplitude
analysis of any b -baryon decay mode allowing for
CP -violation effects. The analysis uses pp collision data
recorded with the LHCb detector, corresponding to integrated
luminosities of 1 fb−1 at
ffiffiffis
p ¼ 7 TeV, 2 fb−1 at
ffiffiffis
p ¼
8 TeV and 2 fb−1 at
ffiffiffis
p ¼ 13 TeV. Due to the inclusion
of more data and significantly improving the selection
procedure compared to the previous study of this channel
[24], a yield of about 460 signal decays within the
mðpK−K−Þ signal region is obtained, with a signal to
background ratio of about 2∶1. A good description of the
data is obtained with an amplitude model containing con-
tributions from Σð1385Þ, Λð1405Þ, Λð1520Þ, Λð1670Þ,
Σð1775Þ and Σð1915Þ resonances. The CP asymmetry for
each contributing component is evaluated and no significant
CP -violation effect is observed. The Ξ−
b →Λð1520ÞK−and
Ξ−
b →Λð1670ÞK−decays are observed with significance
greater than 5σ, and their branching fractions measured,
together with those ofΞ−
b →Σð1385ÞK−, Ξ−
b →Λð1405ÞK−,
Ξ−
b →Σð1775ÞK−and Ξ−
b →Σð1915ÞK−decays. No sig-
nificant signal for Ω−
b →pK−K−decays is found, and an
upper limit on the ratio of fragmentation and branching The branching fraction of each quasi-two-body contri-
bution to the Ξ−
b →pK−K−decay, corresponding to an
intermediate resonance R, can be obtained from its fit
fraction F i, BðΞ−
b →RK−Þ ¼ BðΞ−
b →pK−K−Þ × F i:
ð29Þ ð29Þ The branching fraction of Ξ−
b →pK−K−has not been
measured directly, but the ratio of fragmentation and
branching fractions relative to the B−→KþK−K−decay
is known [24]. This can be combined with the known
values of BðB−→KþK−K−Þ [23,65,66], fΞ−
b =fΛ0
b [44] and
fΛ0
b=ðfu þ fdÞ [67], assuming that fu ¼ fd, to obtain BðΞ−
b →pK−K−Þ ¼ ð2.3 0.9Þ × 10−6; 052010-17 PHYS. REV. D 104, 052010 (2021) R. AAIJ et al. INFN (Italy), SURF (Netherlands), PIC (Spain), GridPP
(United Kingdom), RRCKI and Yandex LLC (Russia),
CSCS (Switzerland), IFIN-HH (Romania), CBPF (Brazil),
PL-GRID (Poland) and NERSC (USA). We are indebted to
the communities behind the multiple open-source software
packages on which we depend. APPENDIX: FIT PROJECTIONS 1.6
1.65
1.7
1.75
1.8
) [GeV]
−
K
p
(
l
m
0
2
4
6
8
10
12
14
16
18
Entries / (0.01 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.6, 1.8] GeV
)
−
K
p
(
low
m 1.6
1.65
1.7
1.75
1.8
) [GeV]
+
K
p
(
m
0
2
4
6
8
10
12
14
16
18
Entries / (0.01 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.6, 1.8] GeV
)
+
K
p
(
low
m Entries / (0.01 GeV) ) [GeV]
+
K
p
(
low
m
3
4
5
) [GeV]
+
K
p
(
high
m
0
2
4
6
8
10
12
14
16
18
Entries / (0.11 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.6, 1.8] GeV
)
+
K
p
(
low
m ) [GeV]
K
p
(
low
m
3
4
5
) [GeV]
−
K
p
(
high
m
0
2
4
6
8
10
12
14
16
18
Entries / (0.11 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.6, 1.8] GeV
)
−
K
p
(
low
m Entries / (0.11 GeV) FIG. 8. Distributions of (top) mlow and (bottom) mhigh, with 1.6 < mlow < 1.8 GeV, for (left) Ξ−
b and (right) ¯Ξþ
b candidates, with
results of the fits superimposed. The total fit result is shown as the blue solid curve, with contributions from individual signal
components and from combinatorial (Comb) and cross feed (Crsfd) background shown as indicated in the legend. APPENDIX: FIT PROJECTIONS 1.45
1.5
1.55
1.6
) [GeV]
+
K
p
(
low
m
0
5
10
15
20
25
Entries / (0.01 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.43, 1.6] GeV
)
+
K
p
(
low
m 1.45
1.5
1.55
1.6
) [GeV]
−
K
p
(
low
m
0
5
10
15
20
25
Entries / (0.01 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.43, 1.6] GeV
)
−
K
p
(
low
m Entries / (0.01 GeV) low
3.5
4
4.5
5
) [GeV]
+
K
p
(
high
m
0
2
4
6
8
10
12
14
16
18
20
Entries / (0.06 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.43, 1.6] GeV
)
+
K
p
(
low
m 3.5
4
4.5
5
) [GeV]
−
K
p
(
high
m
0
2
4
6
8
10
12
14
16
18
20
Entries / (0.06 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.43, 1.6] GeV
)
−
K
p
(
low
m Entries / (0.06 GeV) FIG. 7. Distributions of (top) mlow and (bottom) mhigh, with 1.43 < mlow < 1.60 GeV, for (left) Ξ−
b and (right) ¯Ξþ
b candidates, with
results of the fits superimposed. The total fit result is shown as the blue solid curve, with contributions from individual signal
components and from combinatorial (Comb) and cross feed (Crsfd) background shown as indicated in the legend. 052010-18 052010-18 PHYS. REV. APPENDIX: FIT PROJECTIONS D 104, 052010 (2021) SEARCH FOR CP VIOLATION IN Ξ−
b →pK−K−… 1.6
1.65
1.7
1.75
1.8
) [GeV]
−
K
p
(
low
m
0
2
4
6
8
10
12
14
16
18
Entries / (0.01 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.6, 1.8] GeV
)
−
K
p
(
low
m
1.6
1.65
1.7
1.75
1.8
) [GeV]
+
K
p
(
low
m
0
2
4
6
8
10
12
14
16
18
Entries / (0.01 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.6, 1.8] GeV
)
+
K
p
(
low
m
3
4
5
) [GeV]
−
K
p
(
high
m
0
2
4
6
8
10
12
14
16
18
Entries / (0.11 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.6, 1.8] GeV
)
−
K
p
(
low
m
3
4
5
) [GeV]
+
K
p
(
high
m
0
2
4
6
8
10
12
14
16
18
Entries / (0.11 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.6, 1.8] GeV
)
+
K
p
(
low
m
FIG. 8. Distributions of (top) mlow and (bottom) mhigh, with 1.6 < mlow < 1.8 GeV, for (left) Ξ−
b and (right) ¯Ξþ
b candidates, with
results of the fits superimposed. The total fit result is shown as the blue solid curve, with contributions from individual signal
components and from combinatorial (Comb) and cross feed (Crsfd) background shown as indicated in the legend. APPENDIX: FIT PROJECTIONS 1.8
1.9
2
2.1
2.2
) [GeV]
−
K
p
(
low
m
0
2
4
6
8
10
12
14
16
18
20
22
Entries / (0.01 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.8, 2.2] GeV
)
−
K
p
(
low
m
1.8
1.9
2
2.1
2.2
) [GeV]
+
K
p
(
low
m
0
2
4
6
8
10
12
14
16
18
20
22
Entries / (0.01 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.8, 2.2] GeV
)
+
K
p
(
low
m
3
4
5
) [GeV]
−
K
p
(
high
m
0
2
4
6
8
10
12
14
16
18
20
22
24
Entries / (0.11 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.8, 2.2] GeV
)
−
K
p
(
low
m
3
4
5
) [GeV]
+
K
p
(
high
m
0
2
4
6
8
10
12
14
16
18
20
22
24
Entries / (0.11 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.8, 2.2] GeV
)
+
K
p
(
low
m
FIG. 9. Distributions of (top) mlow and (bottom) mhigh, with 1.8 < mlow < 2.2 GeV, for (left) Ξ−
b and (right) ¯Ξþ
b candidates, with
results of the fits superimposed. The total fit result is shown as the blue solid curve, with contributions from individual signal
components and from combinatorial (Comb) and cross feed (Crsfd) background shown as indicated in the legend. APPENDIX: FIT PROJECTIONS 2.2
2.4
2.6
2.8
) [GeV]
−
K
p
(
low
m
0
2
4
6
8
10
12
14
16
18
20
Entries / (0.02 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[2.2, 2.8] GeV
)
−
K
p
(
low
m 2.2
2.4
2.6
2.8
) [GeV]
+
K
p
(
low
m
0
2
4
6
8
10
12
14
16
18
20
Entries / (0.02 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[2.2, 2.8] GeV
)
+
K
p
(
low
m Entries / (0.02 GeV) low
3
4
5
) [GeV]
−
K
p
(
high
m
0
2
4
6
8
10
12
14
16
Entries / (0.11 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[2.2, 2.8] GeV
)
−
K
p
(
low
m low
3
4
5
) [GeV]
+
K
p
(
high
m
0
2
4
6
8
10
12
14
16
Entries / (0.11 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[2.2, 2.8] GeV
)
+
K
p
(
low
m Entries / (0.11 GeV) FIG. 10. Distributions of (top) mlow and (bottom) mhigh, with 2.2 < mlow < 2.8 GeV, for (left) Ξ−
b and (right) ¯Ξþ
b candidates, with
results of the fits superimposed. The total fit result is shown as the blue solid curve, with contributions from individual signal
components and from combinatorial (Comb) and cross feed (Crsfd) background shown as indicated in the legend. APPENDIX: FIT PROJECTIONS 1.8
1.9
2
2.1
2.2
) [GeV]
+
K
p
(
low
m
0
2
4
6
8
10
12
14
16
18
20
22
Entries / (0.01 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.8, 2.2] GeV
)
+
K
p
(
low
m
3
4
5
) [GeV]
+
K
p
(
high
m
0
2
4
6
8
10
12
14
16
18
20
22
24
Entries / (0.11 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[1.8, 2.2] GeV
)
+
K
p
(
low
m Entries / (0.11 GeV) FIG. 9. Distributions of (top) mlow and (bottom) mhigh, with 1.8 < mlow < 2.2 GeV, for (left) Ξ−
b and (right) ¯Ξþ
b candidates, with
results of the fits superimposed. The total fit result is shown as the blue solid curve, with contributions from individual signal
components and from combinatorial (Comb) and cross feed (Crsfd) background shown as indicated in the legend. 052010-19 052010-19 PHYS. REV. D 104, 052010 (2021) R. AAIJ et al. APPENDIX: FIT PROJECTIONS 2.2
2.4
2.6
2.8
) [GeV]
−
K
p
(
low
m
0
2
4
6
8
10
12
14
16
18
20
Entries / (0.02 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[2.2, 2.8] GeV
)
−
K
p
(
low
m
2.2
2.4
2.6
2.8
) [GeV]
+
K
p
(
low
m
0
2
4
6
8
10
12
14
16
18
20
Entries / (0.02 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[2.2, 2.8] GeV
)
+
K
p
(
low
m
3
4
5
) [GeV]
−
K
p
(
high
m
0
2
4
6
8
10
12
14
16
Entries / (0.11 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[2.2, 2.8] GeV
)
−
K
p
(
low
m
3
4
5
) [GeV]
+
K
p
(
high
m
0
2
4
6
8
10
12
14
16
Entries / (0.11 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[2.2, 2.8] GeV
)
+
K
p
(
low
m
FIG. 10. Distributions of (top) mlow and (bottom) mhigh, with 2.2 < mlow < 2.8 GeV, for (left) Ξ−
b and (right) ¯Ξþ
b candidates, with
results of the fits superimposed. The total fit result is shown as the blue solid curve, with contributions from individual signal
components and from combinatorial (Comb) and cross feed (Crsfd) background shown as indicated in the legend. APPENDIX: FIT PROJECTIONS 3
3.5
4
) [GeV]
−
K
p
(
low
m
0
2
4
6
8
10
12
Entries / (0.05 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[2.8, 4.4] GeV
)
−
K
p
(
low
m
3
3.5
4
) [GeV]
+
K
p
(
low
m
0
2
4
6
8
10
12
Entries / (0.05 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[2.8, 4.4] GeV
)
+
K
p
(
low
m
3
4
5
) [GeV]
−
K
p
(
high
m
0
2
4
6
8
10
12
Entries / (0.11 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[2.8, 4.4] GeV
)
−
K
p
(
low
m
3
4
5
) [GeV]
+
K
p
(
high
m
0
2
4
6
8
10
12
Entries / (0.11 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[2.8, 4.4] GeV
)
+
K
p
(
low
m
FIG. 11. Distributions of (top) mlow and (bottom) mhigh, with mlow > 2.8 GeV, for (left) Ξ−
b and (right) ¯Ξþ
b candidates, with results of
the fits superimposed. The total fit result is shown as the blue solid curve, with contributions from individual signal components and
from combinatorial (Comb) and cross feed (Crsfd) background shown as indicated in the legend. APPENDIX: FIT PROJECTIONS 2
3
4
) [GeV]
−
K
p
(
low
m
0
2
4
6
8
10
12
14
Entries / (0.10 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[2.2, 3.15] GeV
)
−
K
p
(
high
m 2
3
4
) [GeV]
+
K
p
(
low
m
0
2
4
6
8
10
12
14
Entries / (0.10 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[2.2, 3.15] GeV
)
+
K
p
(
high
m 2
3
4
) [GeV]
+
K
p
(
low
m
0
2
4
6
8
10
12
14
Entries / (0.10 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[2.2, 3.15] GeV
)
+
K
p
(
high
m
2.2
2.4
2.6
2.8
3
) [GeV]
+
K
p
(
high
m
0
2
4
6
8
10
12
14
Entries / (0.03 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[2.2, 3.15] GeV
)
+
K
p
(
high
m Entries / (0.10 GeV) Entries / (0.03 GeV) FIG. 12. Distributions of (top) mlow and (bottom) mhigh, with mhigh < 3.15 GeV, for (left) Ξ−
b and (right) ¯Ξþ
b candidates, with results of
the fits superimposed. The total fit result is shown as the blue solid curve, with contributions from individual signal components and
from combinatorial (Comb) and cross feed (Crsfd) background shown as indicated in the legend. APPENDIX: FIT PROJECTIONS 3
3.5
4
) [GeV]
+
K
p
(
low
m
0
2
4
6
8
10
12
Entries / (0.05 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[2.8, 4.4] GeV
)
+
K
p
(
low
m 3
3.5
4
) [GeV]
−
K
p
(
low
m
0
2
4
6
8
10
12
Entries / (0.05 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[2.8, 4.4] GeV
)
−
K
p
(
low
m Entries / (0.05 GeV) ) [
]
p
(
low
3
4
5
) [GeV]
−
K
p
(
high
m
0
2
4
6
8
10
12
Entries / (0.11 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[2.8, 4.4] GeV
)
−
K
p
(
low
m ) [
]
p
(
low
3
4
5
) [GeV]
+
K
p
(
high
m
0
2
4
6
8
10
12
Entries / (0.11 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[2.8, 4.4] GeV
)
+
K
p
(
low
m FIG. 11. Distributions of (top) mlow and (bottom) mhigh, with mlow > 2.8 GeV, for (left) Ξ−
b and (right) ¯Ξþ
b candidates, with results of
the fits superimposed. The total fit result is shown as the blue solid curve, with contributions from individual signal components and
from combinatorial (Comb) and cross feed (Crsfd) background shown as indicated in the legend. 052010-20 PHYS. REV. APPENDIX: FIT PROJECTIONS 2
3
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+
K
p
(
high
m
FIG. 13. Distributions of (top) mlow and (bottom) mhigh, with 3.15 < mhigh < 3.95 GeV, for (left) Ξ−
b and (right) ¯Ξþ
b candidates, with
results of the fits superimposed. The total fit result is shown as the blue solid curve, with contributions from individual signal
components and from combinatorial (Comb) and cross feed (Crsfd) background shown as indicated in the legend. APPENDIX: FIT PROJECTIONS D 104, 052010 (2021) SEARCH FOR CP VIOLATION IN Ξ−
b →pK−K−… 2
3
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Crsfd bkgd
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)
+
K
p
(
high
m
FIG. 12. Distributions of (top) mlow and (bottom) mhigh, with mhigh < 3.15 GeV, for (left) Ξ−
b and (right) ¯Ξþ
b candidates, with results of
the fits superimposed. The total fit result is shown as the blue solid curve, with contributions from individual signal components and
from combinatorial (Comb) and cross feed (Crsfd) background shown as indicated in the legend. APPENDIX: FIT PROJECTIONS 2
3
4
) [GeV]
+
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5 fb
[3.15, 3.95] GeV
)
+
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p
(
high
m 2
3
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) [GeV]
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low
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5 fb
[3.15, 3.95] GeV
)
−
K
p
(
high
m Entries / (0.10 GeV) low
3.2
3.4
3.6
3.8
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5 fb
[3.15, 3.95] GeV
)
+
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(
high
m 3.2
3.4
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) [GeV]
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5 fb
[3.15, 3.95] GeV
)
−
K
p
(
high
m Entries / (0.03 GeV) 3.8 FIG. 13. Distributions of (top) mlow and (bottom) mhigh, with 3.15 < mhigh < 3.95 GeV, for (left) Ξ−
b and (right) ¯Ξþ
b candidates, with
results of the fits superimposed. The total fit result is shown as the blue solid curve, with contributions from individual signal
components and from combinatorial (Comb) and cross feed (Crsfd) background shown as indicated in the legend. 052010-21 PHYS. REV. D 104, 052010 (2021) R. AAIJ et al. APPENDIX: FIT PROJECTIONS 2
3
4
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5 fb
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+
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p
(
high
m ) [
]
p
(
low
4
4.5
5
5.5
) [GeV]
−
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(
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5
10
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Entries / (0.05 GeV)
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(
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1670
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(
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1775
(
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1915
(
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LHCb
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5 fb
[3.95, 5.6] GeV
)
−
K
p
(
high
m 4
4.5
5
5.5
) [GeV]
+
K
p
(
high
m
0
5
10
15
20
25
30
35
40
45
Entries / (0.05 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[3.95, 5.6] GeV
)
+
K
p
(
high
m FIG. 14. Distributions of (top) mlow and (bottom) mhigh, with mhigh > 3.95 GeV, for (left) Ξ−
b and (right) ¯Ξþ
b candidates, with results of
the fits superimposed. The total fit result is shown as the blue solid curve, with contributions from individual signal components and
from combinatorial (Comb) and cross feed (Crsfd) background shown as indicated in the legend. [1] N. Cabibbo, Unitary Symmetry, and Leptonic Decays, Phys. Rev. Lett. 10, 531 (1963). [8] R. Aaij et al. (LHCb Collaboration), First Observation of
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normalizable theory of weak interaction, Prog. Theor. Phys. 49, 652 (1973). [9] R. Aaij et al. APPENDIX: FIT PROJECTIONS 2
3
4
) [GeV]
−
K
p
(
low
m
0
10
20
30
40
50
60
70
80
90
Entries / (0.10 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[3.95, 5.6] GeV
)
−
K
p
(
high
m
2
3
4
) [GeV]
+
K
p
(
low
m
0
10
20
30
40
50
60
70
80
90
Entries / (0.10 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[3.95, 5.6] GeV
)
+
K
p
(
high
m
4
4.5
5
5.5
) [GeV]
−
K
p
(
high
m
0
5
10
15
20
25
30
35
40
45
Entries / (0.05 GeV)
Data
Fit
)
1405
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1520
(
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1670
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1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[3.95, 5.6] GeV
)
−
K
p
(
high
m
4
4.5
5
5.5
) [GeV]
+
K
p
(
high
m
0
5
10
15
20
25
30
35
40
45
Entries / (0.05 GeV)
Data
Fit
)
1405
(
)
1520
(
)
1670
(
)
1385
(
)
1775
(
)
1915
(
Comb bkgd
Crsfd bkgd
LHCb
-1
5 fb
[3.95, 5.6] GeV
)
+
K
p
(
high
m
FIG. 14. Distributions of (top) mlow and (bottom) mhigh, with mhigh > 3.95 GeV, for (left) Ξ−
b and (right) ¯Ξþ
b candidates, with results of
the fits superimposed. The total fit result is shown as the blue solid curve, with contributions from individual signal components and
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[67] R. Aaij et al. (LHCb Collaboration), Measurement of
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100, 031102(R) (2019). [59] F. James and M. R. Aaij,32 C. Abellán Beteta,50 T. Ackernley,60 B. Adeva,46 M. Adinolfi,54 H. Afsharnia,9 C. A. Aidala,86 S. Aiola,25
Z. Ajaltouni,9 S. Akar,65 J. Albrecht,15 F. Alessio,48 M. Alexander,59 A. Alfonso Albero,45 Z. Aliouche,62 G. Alkhazov,38
P. Alvarez Cartelle,55 S. Amato,2 Y. Amhis,11 L. An,48 L. Anderlini,22 A. Andreianov,38 M. Andreotti,21 F. Archilli,17
A. Artamonov,44 M. Artuso,68 K. Arzymatov,42 E. Aslanides,10 M. Atzeni,50 B. Audurier,12 S. Bachmann,17
M. Bachmayer,49 J. J. Back,56 P. Baladron Rodriguez,46 V. Balagura,12 W. Baldini,21 J. Baptista Leite,1 R. J. Barlow,62
S. Barsuk,11 W. Barter,61 M. Bartolini,24 F. Baryshnikov,83 J. M. Basels,14 G. Bassi,29 B. Batsukh,68 A. Battig,15 A. Bay,49
M. Becker,15 F. Bedeschi,29 I. Bediaga,1 A. Beiter,68 V. Belavin,42 S. Belin,27 V. Bellee,49 K. Belous,44 I. Belov,40
I. Belyaev,41 G. Bencivenni,23 E. Ben-Haim,13 A. Berezhnoy,40 R. Bernet,50 D. Berninghoff,17 H. C. Bernstein,68
C. Bertella,48 A. Bertolin,28 C. Betancourt,50 F. Betti,48 Ia. Bezshyiko,50 S. Bhasin,54 J. Bhom,35 L. Bian,73 M. S. Bieker,15
S. Bifani,53 P. Billoir,13 M. Birch,61 F. C. R. Bishop,55 A. Bitadze,62 A. Bizzeti,22,k M. Bjørn,63 M. P. Blago,48 T. Blake,56
F. Blanc,49 S. Blusk,68 D. Bobulska,59 J. A. Boelhauve,15 O. Boente Garcia,46 T. Boettcher,65 A. Boldyrev,82 A. Bondar,43
N. Bondar,38,48 S. Borghi,62 M. Borisyak,42 M. Borsato,17 J. T. Borsuk,35 S. A. Bouchiba,49 T. J. V. Bowcock,60 A. Boyer,48
C. Bozzi,21 M. J. Bradley,61 S. Braun,66 A. Brea Rodriguez,46 M. Brodski,48 J. Brodzicka,35 A. Brossa Gonzalo,56
D. Brundu,27 A. Buonaura,50 C. Burr,48 A. Bursche,72 A. Butkevich,39 J. S. Butter,32 J. Buytaert,48 W. Byczynski,48
S. Cadeddu,27 H. Cai,73 R. Calabrese,21,f L. Calefice,15,13 L. Calero Diaz,23 S. Cali,23 R. Calladine,53 M. Calvi,26,j
M. Calvo Gomez,85 P. Camargo Magalhaes,54 P. Campana,23 A. F. Campoverde Quezada,6 S. Capelli,26,j L. Capriotti,20,d
A. Carbone,20,d G. Carboni,31 R. Cardinale,24 A. Cardini,27 I. Carli,4 P. Carniti,26,j L. Carus,14 K. Carvalho Akiba,32
A. Casais Vidal,46 G. Casse,60 M. Cattaneo,48 G. Cavallero,48 S. Celani,49 J. Cerasoli,10 A. J. Chadwick,60 M. G. Chapman,54
M. Charles,13 Ph. Charpentier,48 G. Chatzikonstantinidis,53 C. A. Chavez Barajas,60 M. Chefdeville,8 C. Chen,3 S. Chen,4
A. Chernov,35 V. Chobanova,46 S. Cholak,49 M. Chrzaszcz,35 A. Chubykin,38 V. Chulikov,38 P. Ciambrone,23 M. F. Cicala,56
X. Cid Vidal,46 G. Ciezarek,48 P. E. L. Clarke,58 M. Clemencic,48 H. V. Cliff,55 J. Closier,48 J. L. Cobbledick,62 V. Coco,48
J. A. B. Coelho,11 J. Cogan,10 E. Cogneras,9 L. Cojocariu,37 P. Collins,48 T. Colombo,48 L. Congedo,19,c A. Contu,27
N. Cooke,53 G. Coombs,59 G. Corti,48 C. M. Costa Sobral,56 B. Couturier,48 D. C. Craik,64 J. Crkovská,67 M. Cruz Torres,1
R. Currie,58 C. L. Da Silva,67 S. Dadabaev,83 E. Dall’Occo,15 J. Dalseno,46 C. D’Ambrosio,48 A. Danilina,41 P. d’Argent,48 APPENDIX: FIT PROJECTIONS Roos, Minuit: A system for function
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upgrade: Technical design report, Report No. CERN-
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2017-003, 2017. [61] M. Williams, How good are your fits? Unbinned multivari-
ate goodness-of-fit tests in high energy physics, J. Instrum. 5, P09004 (2010). R. Aaij,
C. Abellán Beteta,
T. Ackernley,
B. Adeva,
M. Adinolfi,
H. Afsharnia, C. A. Aidala,
S. Aiola,
Z. Ajaltouni,9 S. Akar,65 J. Albrecht,15 F. Alessio,48 M. Alexander,59 A. Alfonso Albero,45 Z. Aliouche,62 G. Alkhazov,38 P. Alvarez Cartelle,55 S. Amato,2 Y. Amhis,11 L. An,48 L. Anderlini,22 A. Andreianov,38 M. Andreotti,21 F. Archilli,17
A. Artamonov,44 M. Artuso,68 K. Arzymatov,42 E. Aslanides,10 M. Atzeni,50 B. Audurier,12 S. Bachmann,17 y
M. Bachmayer,49 J. J. Back,56 P. Baladron Rodriguez,46 V. Balagura,12 W. Baldini,21 J. Baptista 052010-24 PHYS. REV. D 104, 052010 (2021) J. P. Grabowski,
T. Grammatico,
L. A. Granado Cardoso,
E. Grauges,
E. Graverini,
G. Graziani,
A. Grecu,
L. M. Greeven,32 P. Griffith,21,f L. Grillo,62 S. Gromov,83 B. R. Gruberg Cazon,63 C. Gu,3 M. Guarise,21 P. A. Günther,17
E. Gushchin,39 A. Guth,14 Y. Guz,44 T. Gys,48 T. Hadavizadeh,69 G. Haefeli,49 C. Haen,48 J. Haimberger,48
T. Halewood-leagas,60 P. M. Hamilton,66 J. P. Hammerich,60 Q. Han,7 X. Han,17 T. H. Hancock,63
S. Hansmann-Menzemer,17 N. Harnew,63 T. Harrison,60 C. Hasse,48 M. Hatch,48 J. He,6,b M. Hecker,61 K. Heijhoff,32
K. Heinicke,15 A. M. Hennequin,48 K. Hennessy,60 L. Henry,48 J. Heuel,14 A. Hicheur,2 D. Hill,49 M. Hilton,62 S. E. Hollitt,15
J. Hu,17 J. Hu,72 W. Hu,7 X. Hu,3 W. Huang,6 X. Huang,73 W. Hulsbergen,32 R. J. Hunter,56 M. Hushchyn,82 D. Hutchcroft,60
D. Hynds,32 P. Ibis,15 M. Idzik,34 D. Ilin,38 P. Ilten,65 A. Inglessi,38 A. Ishteev,83 K. Ivshin,38 R. Jacobsson,48 S. Jakobsen,48
E. Jans,32 B. K. Jashal,47 A. Jawahery,66 V. Jevtic,15 M. Jezabek,35 F. Jiang,3 M. John,63 D. Johnson,48 C. R. Jones,55
T. P. Jones,56 B. Jost,48 N. Jurik,48 S. Kandybei,51 Y. Kang,3 M. Karacson,48 M. Karpov,82 F. Keizer,48 M. Kenzie,56
T. Ketel,33 B. Khanji,15 A. Kharisova,84 S. Kholodenko,44 T. Kirn,14 V. S. Kirsebom,49 O. Kitouni,64 S. Klaver,32
K. Klimaszewski,36 S. Koliiev,52 A. Kondybayeva,83 A. Konoplyannikov,41 P. Kopciewicz,34 R. Kopecna,17
P. Koppenburg,32 M. Korolev,40 I. Kostiuk,32,52 O. Kot,52 S. Kotriakhova,21,38 P. Kravchenko,38 L. Kravchuk,39
R. D. Krawczyk,48 M. Kreps,56 F. Kress,61 S. Kretzschmar,14 P. Krokovny,43,v W. Krupa,34 W. Krzemien,36 W. Kucewicz,35,t
M. Kucharczyk,35 V. Kudryavtsev,43,v H. S. Kuindersma,32,33 G. J. Kunde,67 T. Kvaratskheliya,41 D. Lacarrere,48
G. Lafferty,62 A. Lai,27 A. Lampis,27 D. Lancierini,50 J. J. Lane,62 R. Lane,54 G. Lanfranchi,23 C. Langenbruch,14 J. Langer,15
O. Lantwin,50 T. Latham,56 F. Lazzari,29,q R. Le Gac,10 S. H. Lee,86 R. Lef`evre,9 A. Leflat,40 S. Legotin,83 O. Leroy,10
T. Lesiak,35 B. Leverington,17 H. Li,72 L. Li,63 P. Li,17 S. Li,7 Y. Li,4 Y. Li,4 Z. Li,68 X. Liang,68 T. Lin,61 R. Lindner,48
V. Lisovskyi,15 R. Litvinov,27 G. Liu,72 H. Liu,6 S. Liu,4 A. Loi,27 J. Lomba Castro,46 I. Longstaff,59 J. H. Lopes,2
G. H. Lovell,55 Y. Lu,4 D. Lucchesi,28,l S. Luchuk,39 M. Lucio Martinez,32 V. Lukashenko,32 Y. Luo,3 A. Lupato,62
E. Luppi,21,f O. Lupton,56 A. Lusiani,29,m X. Lyu,6 L. Ma,4 R. Ma,6 S. Maccolini,20,d F. Machefert,11 F. Maciuc,37
V. Macko,49 P. Mackowiak,15 S. Maddrell-Mander,54 O. Madejczyk,34 L. R. Madhan Mohan,54 O. Maev,38 A. Maevskiy,82
D. Maisuzenko,38 M. W. Majewski,34 J. J. Malczewski,35 S. Malde,63 B. Malecki,48 A. Malinin,81 T. Maltsev,43,v
H. Malygina,17 G. Manca,27,e G. Mancinelli,10 D. Manuzzi,20,d D. Marangotto,25,i J. Maratas,9,s J. F. Marchand,8
U. Marconi,20 S. Mariani,22,g C. Marin Benito,48 M. Marinangeli,49 J. Marks,17 A. M. Marshall,54 P. J. Marshall,60
G. Martellotti,30 L. Martinazzoli,48,j M. Martinelli,26,j D. Martinez Santos,46 F. Martinez Vidal,47 A. Massafferri,1
M. Materok,14 R. Matev,48 A. Mathad,50 Z. Mathe,48 V. Matiunin,41 C. Matteuzzi,26 K. R. Mattioli,86 A. Mauri,32
E. Maurice,12 J. Mauricio,45 M. Mazurek,48 M. McCann,61 L. Mcconnell,18 T. H. Mcgrath,62 A. McNab,62 R. McNulty,18
J. V. Mead,60 B. Meadows,65 G. Meier,15 N. Meinert,76 D. Melnychuk,36 S. Meloni,26,j M. Merk,32,80 A. Merli,25
L. Meyer Garcia,2 M. Mikhasenko,48 D. A. Milanes,74 E. Millard,56 M. Milovanovic,48 M.-N. Minard,8 A. Minotti,21 SEARCH FOR CP VIOLATION IN Ξ−
b →pK−K−… SEARCH FOR CP VIOLATION IN Ξ−
b →pK−K−… S. Ferreres Sole,32 M. Ferrillo,50 M. Ferro-Luzzi,48 S. Filippov,39 R. A. Fini,19 M. Fiorin D. S. Fitzgerald,86 C. Fitzpatrick,62 T. Fiutowski,34 A. Fkiaras,48 F. Fleuret,12 M. Fontana,13 F. Fontanelli,24,h R. Forty,48
V. Franco Lima,60 M. Franco Sevilla,66 M. Frank,48 E. Franzoso,21 G. Frau,17 C. Frei,48 D. A. Friday,59 J. Fu,25
Q. Fuehring,15 W. Funk,48 E. Gabriel,32 T. Gaintseva,42 A. Gallas Torreira,46 D. Galli,20,d S. Gambetta,58,48 Y. Gan,3 M. Gandelman,2 P. Gandini,25 Y. Gao,5 M. Garau,27 L. M. Garcia Martin,56 P. Garcia Moren B. Garcia Plana,46 F. A. Garcia Rosales,12 L. Garrido,45 C. Gaspar,48 R. E. Geertsema,32 D. Gerick,17 L. L. Gerken,15 E. Gersabeck,62 M. Gersabeck,62 T. Gershon,56 D. Gerstel,10 Ph. Ghez,8 V. Gibson,55 H. K. Gi A. Gioventù,46 P. Gironella Gironell,45 L. Giubega,37 C. Giugliano,21,48,f K. Gizdov,58 E. L. Gkougkousis,48 V. V. Gligorov,13
C. Göbel,70 E. Golobardes,85 D. Golubkov,41 A. Golutvin,61,83 A. Gomes,1,a S. Gomez Fernandez,45
F. Goncalves Abrantes,63 M. Goncerz,35 G. Gong,3 P. Gorbounov,41 I. V. Gorelov,40 C. Gotti,26 E. Govorkova,48
17
13
48
45
49
22
37 L. M. Greeven,32 P. Griffith,21,f L. Grillo,62 S. Gromov,83 B. R. Gruberg Cazon,63 C. Gu,3 M. Guarise,21 P. A. Günther,17
E. Gushchin,39 A. Guth,14 Y. Guz,44 T. Gys,48 T. Hadavizadeh,69 G. Haefeli,49 C. Haen,48 J. Haimberger,48
T. Halewood-leagas,60 P. M. Hamilton,66 J. P. Hammerich,60 Q. Han,7 X. Han,17 T. H. Hancock,63
S. Hansmann-Menzemer,17 N. Harnew,63 T. Harrison,60 C. Hasse,48 M. Hatch,48 J. He,6,b M. Hecker,61 K. Heijhoff,32
K. Heinicke,15 A. M. Hennequin,48 K. Hennessy,60 L. Henry,48 J. Heuel,14 A. Hicheur,2 D. Hill,49 M. Hilton,62 S. E. Hollitt,15
J. Hu,17 J. Hu,72 W. Hu,7 X. Hu,3 W. Huang,6 X. Huang,73 W. Hulsbergen,32 R. J. Hunter,56 M. Hushchyn,82 D. Hutchcroft,60
D. Hynds,32 P. Ibis,15 M. Idzik,34 D. Ilin,38 P. Ilten,65 A. Inglessi,38 A. Ishteev,83 K. Ivshin,38 R. Jacobsson,48 S. Jakobsen,48
E. Jans,32 B. K. Jashal,47 A. Jawahery,66 V. Jevtic,15 M. Jezabek,35 F. Jiang,3 M. John,63 D. Johnson,48 C. R. Jones,55
T. P. Jones,56 B. Jost,48 N. Jurik,48 S. Kandybei,51 Y. Kang,3 M. Karacson,48 M. Karpov,82 F. Keizer,48 M. Kenzie,56
T. Ketel,33 B. Khanji,15 A. Kharisova,84 S. Kholodenko,44 T. Kirn,14 V. S. Kirsebom,49 O. Kitouni,64 S. Klaver,32
K. Klimaszewski,36 S. Koliiev,52 A. Kondybayeva,83 A. Konoplyannikov,41 P. Kopciewicz,34 R. Kopecna,17
P. Koppenburg,32 M. Korolev,40 I. Kostiuk,32,52 O. Kot,52 S. Kotriakhova,21,38 P. Kravchenko,38 L. Kravchuk,39
R. D. Krawczyk,48 M. Kreps,56 F. Kress,61 S. Kretzschmar,14 P. Krokovny,43,v W. L. Minzoni,21,f S. E. Mitchell,58 B. Mitreska,62 D. S. Mitzel,48 A. Mödden,15 R. A. Mohammed,63 R. D. Moise,61
T. Mombächer,46 I. A. Monroy,74 S. Monteil,9 M. Morandin,28 G. Morello,23 M. J. Morello,29,m J. Moron,34 A. B. Morris,75
A. G. Morris,56 R. Mountain,68 H. Mu,3 F. Muheim,58,48 M. Mulder,48 D. Müller,48 K. Müller,50 C. H. Murphy,63
D. Murray,62 P. Muzzetto,27,48 P. Naik,54 T. Nakada,49 R. Nandakumar,57 T. Nanut,49 I. Nasteva,2 M. Needham,58 I. Neri,21
N. Neri,25,i S. Neubert,75 N. Neufeld,48 R. Newcombe,61 T. D. Nguyen,49 C. Nguyen-Mau,49,x E. M. Niel,11 S. Nieswand,14
N. Nikitin,40 N. S. Nolte,64 C. Normand,8 C. Nunez,86 A. Oblakowska-Mucha,34 V. Obraztsov,44 D. P. O’Hanlon,54
R. Oldeman,27,e M. E. Olivares,68 C. J. G. Onderwater,79 R. H. O’neil,58 A. Ossowska,35 J. M. Otalora Goicochea,2
T. Ovsiannikova,41 P. Owen,50 A. Oyanguren,47 B. Pagare,56 P. R. Pais,48 T. Pajero,63 A. Palano,19 M. Palutan,23 Y. Pan,62
G. Panshin,84 A. Papanestis,57 M. Pappagallo,19,c L. L. Pappalardo,21,f C. Pappenheimer,65 W. Parker,66 C. Parkes,62
C. J. Parkinson,46 B. Passalacqua,21 G. Passaleva,22 A. Pastore,19 M. Patel,61 C. Patrignani,20,d C. J. Pawley,80 A. Pearce,48
A. Pellegrino,32 M. Pepe Altarelli,48 S. Perazzini,20 D. Pereima,41 P. Perret,9 M. Petric,59,48 K. Petridis,54 A. Petrolini,24,h
A. Petrov,81 S. Petrucci,58 M. Petruzzo,25 T. T. H. Pham,68 A. Philippov,42 L. Pica,29,n M. Piccini,78 B. Pietrzyk,8
G. Pietrzyk,49 M. Pili,63 D. Pinci,30 F. Pisani,48 Resmi P. K,10 V. Placinta,37 J. Plews,53 M. Plo Casasus,46 F. Polci,13
M. Poli Lener,23 M. Poliakova,68 A. Poluektov,10 N. Polukhina,83,u I. Polyakov,68 E. Polycarpo,2 G. J. Pomery,54 S. Ponce,48
D. Popov,6,48 S. Popov,42 S. Poslavskii,44 K. Prasanth,35 L. Promberger,48 C. Prouve,46 V. Pugatch,52 H. Pullen,63
G. Punzi,29,n H. Qi,3 W. Qian,6 J. Qin,6 N. Qin,3 R. Quagliani,13 B. Quintana,8 N. V. Raab,18 R. I. Rabadan Trejo,10
B. Rachwal,34 J. H. Rademacker,54 M. Rama,29 M. Ramos Pernas,56 M. S. Rangel,2 F. Ratnikov,42,82 G. Raven,33
M. Reboud,8 F. Redi,49 F. Reiss,62 C. Remon Alepuz,47 Z. Ren,3 V. Renaudin,63 R. Ribatti,29 S. Ricciardi,57 K. Rinnert,60
P. Robbe,11 G. Robertson,58 A. B. Rodrigues,49 E. Rodrigues,60 J. A. Rodriguez Lopez,74 A. Rollings,63 P. Roloff,48
V. Romanovskiy,44 M. Romero Lamas,46 A. Romero Vidal,46 J. D. Roth,86 M. Rotondo,23 M. S. Rudolph,68 T. Ruf,48
J. Ruiz Vidal,47 A. Ryzhikov,82 J. Ryzka,34 J. J. Saborido Silva,46 N. Sagidova,38 N. Sahoo,56 B. Saitta,27,e M. Salomoni,48
D. Sanchez Gonzalo,45 C. Sanchez Gras,32 R. Santacesaria,30 C. Santamarina Rios,46 M. Santimaria,23 E. Santovetti,31,p
D. Saranin,83 G. Sarpis,59 M. Sarpis,75 A. Sarti,30 C. Satriano,30,o A. Satta,31 M. Saur,15 D. Savrina,41,40 H. Sazak,9
L. G. Scantlebury Smead,63 A. Scarabotto,13 S. Schael,14 M. Schiller,59 H. Schindler,48 M. Schmelling,16 B. Schmidt,48
O. Schneider,49 A. Schopper,48 M. Schubiger,32 S. Schulte,49 M. H. Schune,11 R. Schwemmer,48 B. Sciascia,23 S. Sellam,46
A. Semennikov,41 M. Senghi Soares,33 A. Sergi,24 N. Serra,50 L. Sestini,28 A. Seuthe,15 P. Seyfert,48 Y. Shang,5
D. M. Shangase,86 M. Shapkin,44 I. Shchemerov,83 L. Shchutska,49 T. Shears,60 L. Shekhtman,43,v Z. Shen,5
V. Shevchenko,81 E. B. Shields,26,j E. Shmanin,83 J. D. Shupperd,68 B. G. Siddi,21 R. Silva Coutinho,50 G. Simi,28
S. Simone,19,c N. Skidmore,62 T. Skwarnicki,68 M. W. Slater,53 I. Slazyk,21,f J. C. Smallwood,63 J. G. Smeaton,55
A. Smetkina,41 E. Smith,14 M. Smith,61 A. Snoch,32 M. Soares,20 L. Soares Lavra,9 M. D. Sokoloff,65 F. J. P. Soler,59
A. Solovev,38 I. Solovyev,38 F. L. Souza De Almeida,2 B. Souza De Paula,2 B. Spaan,15 E. Spadaro Norella,25,i P. Spradlin,59
F. Stagni,48 M. Stahl,65 S. Stahl,48 P. Stefko,49 O. Steinkamp,50,83 O. Stenyakin,44 H. Stevens,15 S. Stone,68
M. E. Stramaglia,49 M. Straticiuc,37 D. Strekalina,83 F. Suljik,63 J. Sun,27 L. Sun,73 Y. Sun,66 P. Svihra,62 P. N. Swallow,53
K. Swientek,34 A. Szabelski,36 T. Szumlak,34 M. Szymanski,48 S. Taneja,62 A. R. Tanner,54 A. Terentev,83 F. Teubert,48
E. Thomas,48 K. A. Thomson,60 V. Tisserand,9 S. T’Jampens,8 M. Tobin,4 L. Tomassetti,21,f D. Torres Machado,1
D. Y. Tou,13 M. T. Tran,49 E. Trifonova,83 C. Trippl,49 G. Tuci,29,n A. Tully,49 N. Tuning,32,48 A. Ukleja,36 D. J. Unverzagt,17
E. Ursov,83 A. Usachov,32 A. Ustyuzhanin,42,82 U. Uwer,17 A. Vagner,84 V. Vagnoni,20 A. Valassi,48 G. Valenti,20
N. Valls Canudas,85 M. van Beuzekom,32 M. Van Dijk,49 E. van Herwijnen,83 C. B. Van Hulse,18 M. van Veghel,79
R. Vazquez Gomez,46 P. Vazquez Regueiro,46 C. Vázquez Sierra,48 S. Vecchi,21 J. J. Velthuis,54 M. Veltri,22,r
A. Venkateswaran,68 M. Veronesi,32 M. Vesterinen,56 D. Vieira,65 M. Vieites Diaz,49 H. Viemann,76 X. Vilasis-Cardona,85
E. Vilella Figueras,60 A. Villa,20 P. Vincent,13 D. Vom Bruch,10 A. Vorobyev,38 V. Vorobyev,43,v N. Voropaev,38 K. Vos,80
R. Waldi,17 J. Walsh,29 C. Wang,17 J. Wang,5 J. Wang,4 J. Wang,3 J. Wang,73 M. Wang,3 R. Wang,54 Y. Wang,7 Z. Wang,50
Z. Wang,3 H. M. Wark,60 N. K. Watson,53 S. G. Weber,13 D. Websdale,61 C. Weisser,64 B. D. C. Westhenry,54 D. J. White,62
54
15
63
68
55
64
58 SEARCH FOR CP VIOLATION IN Ξ−
b →pK−K−… Krupa,34 W. Krzemien,36 W. Kucewicz,35,t
M. Kucharczyk,35 V. Kudryavtsev,43,v H. S. Kuindersma,32,33 G. J. Kunde,67 T. Kvaratskheliya,41 D. Lacarrere,48
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31 31INFN Sezione di Roma Tor Vergata, Roma, Italy 32Nikhef National Institute for Subatomic Physics, Amsterdam, Netherlands onal Institute for Subatomic Physics and VU University Amsterdam, Amsterdam, Netherlands f
y
y
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y
f
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36National Center for Nuclear Research (NCBJ) Warsaw Poland Henryk Niewodniczanski Institute of Nuclear Physics Polish Academy of Sciences, Kraków, Poland
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38Petersburg Nuclear Physics Institute NRC Kurchatov Institute (PNPI NRC KI), Gatchina, Russia
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p
,
p
47Instituto de Fisica Corpuscular, Centro Mixto Universidad de Valencia—CSIC, Valencia, Spain
48European Organization for Nuclear Research (CERN), Geneva, Switzerland 47Instituto de Fisica Corpuscular, Centro Mixto Universidad de Valencia—CSIC, Valencia, Spain
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Moscow, Russia
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76Institut für Physik, Universität Rostock, Rostock, Germany, associated to European Organization for Nuclear Research (CERN), Geneva, Switzerland
78 associated to INFN Sezione di Ferrara, Ferrara, Italy 79Van Swinderen Institute, University of Groningen, Groningen, Netherlands, associated to Nikhef National Institute for Subatomic Physics, Amsterdam, Netherlands
81 81National Research Centre Kurchatov Institute, Moscow, Russia, sociated to Institute of Theoretical and Experimental Physics NRC Kurchatov Institute (ITEP NRC KI),
M
R
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associated to Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany
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associated to Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany
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associated to European Organization for Nuclear Research (CERN), Geneva, Switzerland
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associated to INFN Sezione di Ferrara, Ferrara, Italy
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associated to Nikhef National Institute for Subatomic Physics, Amsterdam, Netherlands
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associated to Institute of Theoretical and Experimental Physics NRC Kurchatov Institute (ITEP NRC KI),
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associated to Institute of Theoretical and Experimental Physics NRC Kurchatov Institute (ITEP NRC KI),
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84National Research Tomsk Polytechnic University, Tomsk, Russia, associated to Institute of Particle Physics, Central China Normal University, Wuhan, Hubei, China associated to LPNHE, Sorbonne Universit´e, Paris Diderot Sorbonne Paris Cit´e, CNRS/IN2P3, 052010-27 052010-27 R. AAIJ et al. PHYS. REV. D 104, 052010 (2021) 49Institute of Physics, Ecole Polytechnique F´ed´erale de Lausanne (EPFL), Lausanne, Switzerland
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52Institute for Nuclear Research of the National Academy of Sciences (KINR), Kyiv, Ukraine
53University of Birmingham, Birmingham, United Kingdom
54H.H. Moscow, Russia 85DS4DS, La Salle, Universitat Ramon Llull, Barcelona, Spain, 85DS4DS, La Salle, Universitat Ramon Llull, Barcelona, Spain, associated to ICCUB, Universitat de Barcelona, Barcelona, Spain 052010-28 86University of Michigan, Ann Arbor, United States,
associated to Syracuse University, Syracuse, New York, USA 86University of Michigan, Ann Arbor, United States,
associated to Syracuse University, Syracuse, New York, USA 86University of Michigan, Ann Arbor, United States,
associated to Syracuse University, Syracuse, New York, USA aUniversidade Federal do Triângulo Mineiro (UFTM), Uberaba-MG, Brazil. b SEARCH FOR CP VIOLATION IN Ξ−
b →pK−K−… PHYS. REV. D 104, 052010 (2021) PHYS. REV. D 104, 052010 (2021) sMSU—Iligan Institute of Technology (MSU-IIT), Iligan, Philippines.
tAGH—University of Science and Technology, Faculty of Computer Science, Electronics and Telecommunications, Kraków,
Poland aUniversidade Federal do Triângulo Mineiro (UFTM), Uberaba-MG, Brazil.
b bHangzhou Institute for Advanced Study, UCAS, Hangzhou, China. angzhou Institute for Advanced Study, UCAS, Hang cUniversit`a di Bari, Bari, Italy. d dUniversit`a di Bologna, Bologna, Italy. dUniversit`a di Bologna, Bologna, Italy. eUniversit`a di Cagliari, Cagliari, Italy. f fUniversit`a di Ferrara, Ferrara, Italy. gUniversit`a di Firenze, Firenze, Italy. h gUniversit`a di Firenze, Firenze, Italy. h hUniversit`a di Genova, Genova, Italy. i hUniversit`a di Genova, Genova, Italy. i iUniversit`a degli Studi di Milano, Milano, Italy. j iUniversit`a degli Studi di Milano, Milano, Italy. j jUniversit`a di Milano Bicocca, Milano, Italy. k jUniversit`a di Milano Bicocca, Milano, Italy. k kUniversit`a di Modena e Reggio Emilia, Modena, Italy. l lUniversit`a di Padova, Padova, Italy. lUniversit`a di Padova, Padova, Italy. mScuola Normale Superiore, Pisa, Italy. mScuola Normale Superiore, Pisa, Italy. nUniversit`a di Pisa, Pisa, Italy. nUniversit`a di Pisa, Pisa, Italy. oUniversit`a della Basilicata, Potenza, Italy. oUniversit`a della Basilicata, Potenza, Italy. pUniversit`a di Roma Tor Vergata, Roma, Italy. pUniversit`a di Roma Tor Vergata, Roma, Italy. qUniversit`a di Siena, Siena, Italy. rUniversit`a di Urbino, Urbino, Italy. sMSU—Iligan Institute of Technology (MSU-IIT), Iligan, Philippines. t sMSU—Iligan Institute of Technology (MSU-IIT), Iligan, Philippines. t tAGH—University of Science and Technology, Faculty of Computer Science, Electronics and Telecommunications, Kraków,
Poland. tAGH—University of Science and Technology, Faculty of Computer Science, Electronics an
Poland. uP.N. Lebedev Physical Institute, Russian Academy of Science (LPI RAS), Moscow, Russia. vNovosibirsk State University, Novosibirsk, Russia. vNovosibirsk State University, Novosibirsk, Russia. vNovosibirsk State University, Novosibirsk, Russia. y
wDepartment of Physics and Astronomy, Uppsala University, Uppsala, Sweden. Department of Physics and Astronomy, Uppsala Un wDepartment of Physics and Astronomy, Uppsala U xHanoi University of Science, Hanoi, Vietnam. xHanoi University of Science, Hanoi, Vietnam. 052010-29
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RNA-SSPT: RNA Secondary Structure Prediction Tools
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Bioinformation
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cc-by
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Freed Ahmad, Shahid Mahboob, Tahsin Gulzar, Salah U din, Tanzeela Hanif, Hifza Ahmad &
Muhammad Afzal* Freed Ahmad, Shahid Mahboob, Tahsin Gulzar, Salah U din, Tanzeela Hanif, Hifza Ahmad &
Muhammad Afzal* Department of Bioinformatics and Biotechnology, G C University, Faisalabad, Pakistan; Muhammad Afzal - Email:
afzalarsenal@googlemail.com; *Corresponding author Received September 16, 2013; Accepted September 16, 2013; Published October 16, 2013 Abstract: The prediction of RNA structure is useful for understanding evolution for both in silico and in vitro studies. Physical methods like
NMR studies to predict RNA secondary structure are expensive and difficult. Computational RNA secondary structure prediction
is easier. Comparative sequence analysis provides the best solution. But secondary structure prediction of a single RNA sequence is
challenging. RNA-SSPT is a tool that computationally predicts secondary structure of a single RNA sequence. Most of the RNA
secondary structure prediction tools do not allow pseudoknots in the structure or are unable to locate them. Nussinov dynamic
programming algorithm has been implemented in RNA-SSPT. The current studies shows only energetically most favorable
secondary structure is required and the algorithm modification is also available that produces base pairs to lower the total free
energy of the secondary structure. For visualization of RNA secondary structure, NAVIEW in C language is used and modified in
C# for tool requirement. RNA-SSPT is built in C# using Dot Net 2.0 in Microsoft Visual Studio 2005 Professional edition. The
accuracy of RNA-SSPT is tested in terms of Sensitivity and Positive Predicted Value. It is a tool which serves both secondary
structure prediction and secondary structure visualization purposes. Key words: RNA secondary structure prediction, C#, Nussinov algorithm, dot net open access
www.bioinformation.net
Software
Volume 9(17)
RNA-SSPT: RNA Secondary Structure Prediction
Tools open access
www.bioinformation.net
Software
Volume 9(17)
RNA-SSPT: RNA Secondary Structure Prediction
Tools open access open access G, C and U) and the second line shows the dot-parenthesis
expression. structure element is known as a multi-branch loop. It is a more
complex structure that consists of several stem-loop type
structures [6]. Some other secondary structure elements like pseudoknots,
kissing hairpins and loop-bulge interactions are also present
due to complementary base pairing. These elements are
important in tertiary structure formation and are usually very
difficult to predict. Most of the RNA secondary structure
prediction tools do not allow pseudoknots in the structure and
unable to locate them. Today, there are many RNA secondary structure prediction
tools available but most of these are either web-based or run
only in UNIX environment. Table 1 (see supplementary
material) summarizes some well-known RNA secondary
structure prediction tools. Only a few are available for
Microsoft Windows as [10] “RNAstructure” [11, 12] but the
output drawing is not very impressive. The first method devised to predict RNA secondary structure
was comparative sequence analysis [7]. This method infers
base-pairs by determining canonical pairs that are common
among multiple homologous sequences. Specific pairs are
confirmed by the existence of compensating base-pair changes,
where, for example, a GC pair in one sequence is replaced by an
AU pair in another sequence. Comparative analysis is quite
forceful when a number of homologous sequences are available. Over 97% of base-pairs predicted for ribosomal RNA were
demonstrated in subsequent crystal structures. Comparative
analysis has also been used to infer tertiary structure contacts. Comparative analysis, however, requires multiple sequences,
can be time consuming and requires significant insights [8]. So, the need arises to develop the software tool, specific for
Microsoft windows users, as there are many tools available for
UNIX or other operating system users, with some graphical
enhancements. Thus the aim and objectives for developing
RNA-SSPT are: (i) To predict RNA secondary structure by just
entering a single RNA sequence or from fasta file; (ii) To
provide desktop software tool facility to a scientist in any
version of Microsoft Windows (Windows XP or later version);
(iii) To bring .NET technologies and C# language [13] in the
field of bioinformatics; (iv) Software in which there is [12]
facility to draw an already predicted structure that is in dot-
parenthesis format; (v) To find pseudoknots using Nussinov
algorithm; (vi) Software that also calculates the GC content in
the sequence and percentage of each nitrogenous base. open access (vii) A
better drawing environment than other renowned secondary
structure prediction tools; (viii) To have user friendly interface
so that every task should be performed on single click
environment. To predict the secondary structure of a single sequence, the
most popular approach is the dynamic programming. Two
algorithms have mostly been used to predict [1] secondary
structure from single sequence. These are Nussinov maximum
base pairing algorithm and Zuker free energy minimization
algorithm. Both are based on dynamic programming. Dynamic
programming is an algorithmic technique that organizes
computations to avoid re-computing values that are already
known, which can often save a great deal of time [9]. Problems
having the overlapping sub-problems property [1] are almost
always solved using dynamic programming, a catch-all term for
any algorithm in which the definition of a function is extended
as the computation proceeds. This is generally accomplished by
constructing a solution “bottom up” (e.g., progressing from
simpler to more complex cases), the goal being to solve each
sub-problem before it is needed by any other sub-problem [10]. Now free energy minimization approach is mostly used for [9]
Wagner DB, predicting secondary structure with greater
success [9, 1]. Figure 1: RNA structure: (a) primary structure, which is the
nucleotide sequence of the RNA; (b) The secondary structure,
which is the folded RNA that forms upon intra-strand base
pairing; (c) The tertiary structure, which is the three-
dimensional RNA that forms upon higher-order interactions
among the base-paired regions (P1, P2, etc.) (Lehman, N., 2010). Because RNA structure determination is often experimentally
difficult despite tremendous advances in RNA crystallography,
nuclear magnetic resonance spectroscopy, and chemical
modification, RNA structure prediction serves as an important
tool for generating hypotheses about structure-function
relationships in RNA. RNA structure prediction can be useful
for interpreting or designing mutagenesis experiments,
identifying conserved structural features and designing siRNA
strategies [3]. RNA secondary structure can be described in many formats like
dot-parenthesis format, .ct file format and RNAML (RNA
Markup Language) format etc. but most common and simple is
dot-parenthesis format which shows bases that forms base-pair
as nested parenthesis and loops, and other bases as dots. In .dp
file format, the first line describes the bases in alphabets (e.g. Background: contacts are generally stronger than tertiary structure contacts
and the formation of secondary structure occurs on a faster
timescale than tertiary structure [4]. Therefore, RNA secondary
structure can generally be predicted without knowledge of
tertiary structure. Figure 1 explains the hierarchy of RNA
structure [5]. g
RNA is not just an intermediate between genes and protein as
messenger RNA (mRNA) which is often described as a linear,
unstructured sequence, uninteresting but also for the protein
amino acid that it encodes. However, many non-coding RNAs
exist which adopt sophisticated three-dimensional structures,
and even some of these catalyze biochemical reactions [1]. RNA secondary structure consists of different structural
elements. Part of RNA where no base pairs are found remains
single stranded. When more than one base pair appears in the
form of a group of contiguous base pairs, the resulting double
stranded secondary structure is described as a stem. Bases that
are attached with stems but do not base pair form loops. Simplest
is
the
hairpin
loop which consists of
two
complementary sequences joined by some non-pairing bases. An internal loop appears between two stems and may be
symmetric or asymmetric (bulge). Another common secondary ISSN 0973-2063 (online) 0973-8894 (print)
Bioinformation 9(17): 873-878 (2013)
8
RNA folds hierarchically [2, 3]. At the first level of organization
is the primary structure, which is the sequence of nucleotides. The next level is secondary structure, the base-pairing
according to the Watson-Crick complementarity; A binds with
U, G binds with C; but also the less stable combination G with
U, called wobble base-pair. Tertiary structure is the three-
dimensional arrangement of atoms and the quaternary
structure is the interaction with other molecules, which are
often either proteins or other RNA strands. Secondary structure 873 © 2013 Biomedical Informatics open access A, Figure 1: RNA structure: (a) primary structure, which is the
nucleotide sequence of the RNA; (b) The secondary structure,
which is the folded RNA that forms upon intra-strand base
pairing; (c) The tertiary structure, which is the three-
dimensional RNA that forms upon higher-order interactions
among the base-paired regions (P1, P2, etc.) (Lehman, N., 2010). ISSN 0973-2063 (online) 0973-8894 (print)
Bioinformation 9 (17): 873-878 (2013) 874 © 2013 Biomedical Informatics open access of length 0 or 1 have no base pairs, so B(i, i) = B(i, i − 1) = 0 (by
convention, the i, i − 1 cells represent zero length sequences; the
recursion must never access an empty matrix cell) then worked
outwards on larger and larger subsequences, until reached the
upper right corner, as shown in the bottom of Figure 2.1. This
corner is B(1,N), the score of the optimal structure for the
complete sequence from i = 1 to j = N. Then, from that point,
recovered the optimal structure by tracing back the optimal
path that got us into the upper corner, one step in the structure
at a time. Figure 2: Nussinov algorithm steps: a. the four cases examined
by the dynamic programming recursion; b. The dynamic
programming algorithm in operation. (Eddy S.R. 2004) Figure 2: Nussinov algorithm steps: a. the four cases examined
by the dynamic programming recursion; b. The dynamic
programming algorithm in operation. (Eddy S.R. 2004) Simple energy minimization Maximizing the number of base pairs as described above does
not lead to good structure predictions. Better predictions can be
obtained by minimizing the free energy for an RNA sequence. Reasonable values for e at 37oC are −3, −2 and −1 kcal/mol for
base pairs C − G, A − U and G − U, respectively. Using this we
generalize the Nussinov algorithm such that the free energy of a
base pair is considered. In the algorithm e(i, j) is now used
rather than the simple +1. Function e(i, j) generate energy values
based on the base pairing found. Since the free energy of a base
pair is negative the algorithm search for the structures with
overall minimal energy. Thus the recursion formula is [17, 18]
(Please see supplementary material for equation and
explanation). Figure 2: Nussinov algorithm steps: a. the four cases examined
by the dynamic programming recursion; b. The dynamic
programming algorithm in operation. (Eddy S.R. 2004) Figure 3: Sequence Diagram of RNA_SSPT
Tools Used for Software Development:
(i) Microsoft Visual Studio 2005 Professional Edition
(ii) Microsoft .Net version 2.0
(iii) Microsoft Visual C# Algorithm Used g
Nussinov dynamic programming algorithm was used for
predicting RNA secondary structure in the development of
RNA-SSPT. For visualization of RNA secondary structure from
dot-Parenthesis expression to secondary structure, NAView.c
program [14] was converted into NAView.cs. Some help was
taken from Naview.java in the VARNA tool (a java based RNA
secondary structure display tool) by [15]. NAView.cs takes
input in the dot-parenthesis format and calculates the
coordinates of each base of input sequence. Methodology: The software developed for the prediction of RNA secondary
structure was solely based on the information of single input
RNA sequence. The development of software involved
following steps: BIOINFORMATION open access Nussinov algorithm implementation Base pair maximization is the simplest approach to predict the
secondary structure of RNA. Testing and scoring each possible
structure is numerically impossible so dynamic programming
must be used for an efficient solution. In 1978, Nussinov [16]
published a method to do so. The basic mathematical recursive
function for the matrix fill, of an RNA string containing N
nucleotides is given below: (Please see supplementary material
for equation). In the above described manner, a two dimensional array is
filled recursively. Then this matrix was traced back to find the
best optimal folding containing maximum base pairs. Figure
2(b) shows the matrix B(i, j) for a sequence GGGAAAUCC after
initialization, after the recursive fill, and after an optimal
structure with three base pairs has been traced back. Figure 3: Sequence Diagram of RNA_SSPT To run this recursion efficiently, the scores B(i, j) were tabulated
in a triangular matrix. Initialized on the diagonal; subsequences ISSN 0973-2063 (online) 0973-8894 (print)
Bioinformation 9 (17): 873-878 (2013) © 2013 Biomedical Informatics 875 Interface Design: g
Interface was designed in Microsoft Visual Studio 2005 using
“System.Windows.Forms” class. Menu strip was dragged and
dropped onto main form from tool box. Icon and Front image of
main menu was developed in Macromedia Flash 8.0. Remaining
coding was done in Microsoft Visual C#. Comparison of pseudoknots:
STRAND ID # PDB_00908 also contains pseudoknots. Its
pseudoknots were calculated by RNA-SSPT and compared with
the actually known pseudoknoted secondary structure. The
result of this comparison is shown in Figure 4. Drawing Comparison: “RNAstructure” is a windows-based software package for RNA
secondary structure prediction and analysis. The drawings of
RNA
secondary
structures
of
both
RNA-SSPT
and
RANstructure 5.1 for same secondary structures were also
compared to check whose result is graphically better Table 3
(see supplementary material). These were used to test the
accuracy of RNA-SSPT. PDB (www.pdb.org) is Protein Data
Bank and Rfam [19, 20] is database for RNA families. Discussion: RNA-SSPT is a user-friendly RNA secondary structure
prediction tool which accepts RNA input in different forms
either by keyboard input or by opening text files or fasta files. If
the user has DNA sequence, the transcription option is available
which converts DNA into RNA. It also predicts pseudoknots by
reusing the same nussinov algorithm in a unique way. It
compares the results of RNA-SSPT either in pseudoknot free
secondary structure form or in pseudoknotted structure with
known secondary structures in the database. Positive predictive value is the percentage of predicted base
pairs that are in the known structure: # of predicted pairs in known structure
Positive predicted value= × 100 Total # of pairs in the known structure Sequence Diagram: sequence can converted to RNA by using “TranscribetoRNA”. Sensitivity and Positive Predicted Values can also be calculated. A sequence diagram is a kind of interaction diagram in UML,
which shows how processes operate with one another and in
what order. It is a construct of a Message Sequence Chart. Sequence Diagram of RNA-SSPT is shown in (Figure 3). Accuracy Results:
Accuracy calculation results of different RNA sequences of
table 2 are given in the table 3. Accuracy Results: Accuracy calculation results of different RNA sequences of
table 2 are given in the table 3. Pseudoknot Prediction: Pseudoknots in secondary structure of STRAND ID #
PDB_00908 were also predicted with RNA-SSPT. For this, the
pseudoknots were removed from the actual secondary structure
and dots were used in their place. Then predicted them with
RNA-SSPT and compared with actual secondary structure. The results produced by RNA-SSPT were encouraging, there
are several areas where the program could be improved and
work could be continued in many directions. The algorithm
implemented, is not very efficient in correct prediction of
secondary structures for larger RNA sequences. The Zuker Free
Energy minimization dynamic programming algorithm [20, 21]
can be implemented in RNA-SSPT to make it globally
acceptable. As most of the applications of Zuker algorithm
works only on UNIX operating systems or are web-based, the
tool can make its place as being user friendly and windows-
based. Known Structures: STRAND [19] database provides a wide collection of known
RNA secondary structures drawn from public databases,
searchable and downloadable in a common format. Six different
types of RNA sequences were taken for the STRAND database. The detail about those sequences is given in Table 2 (see
supplementary material). These six sequences were used to
calculate the accuracy of RNA-SSPT. RNA-SSPT is developed to detect the maximum occurring of
base pairs in a single RNA sequence. Different types of RNA
sequences of known secondary structure, taken from STRAND
database, are used to test the accuracy of RNA-SSPT. The
software performed quite well for these RNA data sets and
showed good results for “Positive Predictive Value”. The
sensitivity of RNA-SSPT is much higher which confirmed that
base pair maximization approach must not be used solely to
predict the exact secondary structure of a given single RNA
sequence. BIOINFORMATION open access Accuracy Testing: Comparison of graphical outputs of both RNA-SSPT and
RNAstructure 5.1, which is also a windows-based RNA
secondary structure prediction and analysis tool, for the same
RNA sequence “GGGAACCACAUUUCAACA” is shown in
Figure 5. The accuracy of RNA-SSPT was tested by the sensitivity and
Positive predictive value [8]. Sensitivity is the percentage of
known base pairs correctly predicted: # of predicted pairs in predicted structure
Sensitivity = × 100
Total # of pairs in the known structure
Positive predictive value is the percentage of predicted base
pairs that are in the known structure:
# of predicted pairs in known structure
Positive predicted value= × 100
Total # of pairs in the known structure # of predicted pairs in predicted structure
Sensitivity = × 100
Total # of pairs in the known structure Facilities Provided: The algorithm [13, 14] used for calculating coordinates of
nucleotides is old but still used in most of RNA secondary
structure prediction and drawing tools. RNA-SSPT also
implements it, but there are some new vector based [21, 22] and
planer drawing [22] algorithms available which can be used in
RNA-SSPT to make it better than most of secondary structure
tools. RNA-SSPT is a user friendly software, providing the user to
input RNA sequence, and facilitating to open sequence in any
text file or in a fast file. It enables the user to graphically draw
RNA secondary structure by just entering its sequence and its
known secondary structure in dot-parenthesis format. It
calculates the sequence information like GC content etc. DNA ISSN 0973-2063 (online) 0973-8894 (print)
Bioinformation 9 (17): 873-878 (2013) 876 © 2013 Biomedical Informatics Acknowledgement: All the work was done and supported by department of
Bioinformatics and Biotechnology, G C University,
Faisalabad, Pakistan. All the work was done and supported by department of
Bioinformatics and Biotechnology, G C University,
Faisalabad, Pakistan. References: [1] Mathews et al. Proc Natl Acad Sci USA. 2004 101: 7287
DOI:10.1073/pnas.0401799101 Figure 4: Comparison of pseudoknots. Figure 5: Comparison of graphical outputs. In background, the
output of RNAstructure and front matter is the output of RNA-
SSPT. [1] Mathews et al. Proc Natl Acad Sci USA. 2004 101:
DOI:10.1073/pnas.0401799101
[2] Tinoco JR & Bustamante C, J Mol Biol. 1999 293, 271
[3] Schroeders J, Journal of virology. 2009 83: 6326
[4] Woodson SA, Cell Mol Life Sci. 2000 57: 796
[5] Lehman N, Wiley Interdisciplinary Reviews – RNA. 201
202
[6] http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book
boc4&part=A1119
[7] Pace NR et al. The RNA World. 1999 pp. 113-141. [8] Mathews DH, Predicting RNA secondary structure by
energy minimization: Theoretical Chemistry Accounts: Th
Computation,
and
Modeling
(Theoretica
Chimica
A
Springer
Berlin
/
Heidelberg,
2006
116:
DOI:10.1007/s00214-005-0027-7
[9] http://www.mathematica-
journal.com/issue/v5i4/columns/wagner/
[10] Mathews DH et al. Cold Spring Harb Perspect Biol. 2010
DOI: 10.1101/cshperspect.a003665
[11] Reuter & Mathews, BMC Bioinformatics. 2010 11: 129
[12] http://www.microsoft.com/net/overview.aspx
[13] Bruccoleri R & Heinrich G, Computer Applications in
Biosciences
4,
pages
167–173. 1988
10.1093/bioinformatics/4.1.167
[14] Darty K et al. Bioinformatics. 2009 25: 1974
[15] Nussinov R et al. SIAM J Appl Math. 1978 35: 62
[16] EDDY SR, Nature Biotechnology. 2004 22: 1457
[17] Nussinov R & Jacobson AB, Proc Nati Acad Sci. 1980
6309
[18] Andronescu et al. J BMC Bioinformatics. 2008 9: 340
[19] Griffiths-Jones S et al. Nucleic Acids Res. 2003 31: 439
[20] Zuker M & Stiegler P, Nucleic Acids Research. 1981 9:133
[21] Han K et al. Bioinformatics. 1999 15: 286
[22] Auber et al. Journal of Graph Algorithms and Applicat
2006 10: 329 Figure 4: Comparison of pseudoknots. Figure 5: Comparison of graphical outputs. In background, the
output of RNAstructure and front matter is the output of RNA-
SSPT. Figure 4: Comparison of pseudoknots. Figure 4: Comparison of pseudoknots. p
[2] Tinoco JR & Bustamante C, J Mol Biol. 1999 293, 271 [3] Schroeders J, Journal of virology. 2009 83: 6326 [4] Woodson SA, Cell Mol Life Sci. 2000 57: 796 [5] Lehman N, Wiley Interdisciplinary Reviews – RNA. 2010 1:
202 [6] http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=m
boc4&part=A1119 p
[7] Pace NR et al. The RNA World. 1999 pp. 113-141. BIOINFORMATION open access The salient features of present software are; the software is easy
to use and it has friendly graphical user interface. This software
is fast and works in a single click. All parts of RNA-SSPT work
properly and give effective output. It also checks input and
prompts user to enter valid sequence or secondary structure for
comparison. Overall the predictive part of RNA-SSPT is error
free. References: [8] Mathews DH, Predicting RNA secondary structure by free
energy minimization: Theoretical Chemistry Accounts: Theory,
Computation,
and
Modeling
(Theoretica
Chimica
Acta)
Springer
Berlin
/
Heidelberg,
2006
116:
160
DOI:10.1007/s00214-005-0027-7 /
[9] http://www.mathematica-
journal.com/issue/v5i4/columns/wagner/ /
[9] http://www.mathematica-
journal.com/issue/v5i4/columns/wagner/ journal.com/issue/v5i4/columns/wagner/ Figure 4: Comparison of pseudoknots. j
g
[10] Mathews DH et al. Cold Spring Harb Perspect Biol. 2010 1: 15
DOI: 10.1101/cshperspect.a003665 Methodology:
Nussinov algorithm implementation Methodology: gy
Nussinov algorithm implementation g
p
The basic mathematical recursive function for the matrix fill is g
p
The basic mathematical recursive func In the above described manner, a two dimensional array is filled recursively. Then this matrix was traced back to find the best
optimal folding containing maximum base pairs. Figure 2(b), shows the matrix B(i, j) for a sequence GGGAAAUCC after
initialization, after the recursive fill, and after an optimal structure with three base pairs has been traced back. In the above described manner, a two dimensional array is filled recursively. Then this matrix was traced back to find the best
optimal folding containing maximum base pairs. Figure 2(b), shows the matrix B(i, j) for a sequence GGGAAAUCC after
initialization, after the recursive fill, and after an optimal structure with three base pairs has been traced back. To run this recursion efficiently, the scores B(i, j) were tabulated in a triangular matrix. Initialized on the diagonal; subsequences of
length 0 or 1 have no base pairs, so B(i, i) = B(i, i − 1) = 0 (by convention, the i, i − 1 cells represent zero length sequences; the
recursion must never access an empty matrix cell) then worked outwards on larger and larger subsequences, until reached the
upper right corner, as shown in the bottom of Figure 2.1. This corner is B(1,N), the score of the optimal structure for the complete
sequence from i = 1 to j = N. Then, from that point, recovered the optimal structure by tracing back the optimal path that got us into
the upper corner, one step in the structure at a time. Simple energy minimization: Simple energy minimization: This formula was implemented in the same manner as the base pair maximization. This formula was implemented in the same manner as the base pair maximization. p
g
DOI: 10.1101/cshperspect.a003665 [11] Reuter & Mathews, BMC Bioinformatics. 2010 11: 129 [12] http://www.microsoft.com/net/overview.aspx [13] Bruccoleri R & Heinrich G, Computer Applications in the
Biosciences
4,
pages
167–173. 1988
DOI:
10.1093/bioinformatics/4.1.167 [14] Darty K et al. Bioinformatics. 2009 25: 1974 [15] Nussinov R et al. SIAM J Appl Math. 1978 35: 62 [16] EDDY SR, Nature Biotechnology. 2004 22: 1457 [17] Nussinov R & Jacobson AB, Proc Nati Acad Sci. 1980 77:
6309 [18] Andronescu et al. J BMC Bioinformatics. 2008 9: 340 [19] Griffiths-Jones S et al. Nucleic Acids Res. 2003 31: 439 [20] Zuker M & Stiegler P, Nucleic Acids Research. 1981 9:133 [20] Zuker M & Stiegler P, Nucleic Acids Resear [21] Han K et al. Bioinformatics. 1999 15: 286 [22] Auber et al. Journal of Graph Algorithms and Applications. 2006 10: 329 Figure 5: Comparison of graphical outputs. In background, the
output of RNAstructure and front matter is the output of RNA-
SSPT. Edited by P Kangueane
Citation: Ahmad et al. Bioinformation 9(17): 873-878 (2013)
License statement: This is an open-access article, which permits unrestricted use, distribution, and reproduction in any medium,
for non-commercial purposes, provided the original author and source are credited y
g
Citation: Ahmad et al. Bioinformation 9(17): 873-878 (2013)
License statement: This is an open-access article, which permits unrestricted use, distribution, and reproduction in any medium,
for non-commercial purposes, provided the original author and source are credited y
g
Citation: Ahmad et al. Bioinformation 9(17): 873-878 (2013)
License statement: This is an open-access article, which permits unrestricted use, distribution, and reproduction in any medium,
for non-commercial purposes, provided the original author and source are credited ISSN 0973-2063 (online) 0973-8894 (print)
Bioinformation 9 (17): 873-878 (2013) 877 © 2013 Biomedical Informatics BIOINFORMATION open access © 2013 Biomedical Informatics Supplementary material: Methodology:
Nussinov algorithm implementation Methodology:
Nussinov algorithm implementation ISSN 0973-2063 (online) 0973-8894 (print)
Bioinformation 9 (17): 873-878 (2013)
878
© 2013 Biomedical Inform
Table 1: Some already available RAN Tools
Program
URL
Features
RNAstructure
http://rna.urmc.rochester.edu/
RNAstructure.html
JAVA/Windows, Graphical User Interface; Command Line
Interface; C++
Class Library
Sfold
http://sfold.wadsworth.org/
Web server
UNAFold/Mfold
http://mfold.bioinfo.rpi.edu/
Web server; Command
Line Interface
Vienna RNA Package
http://www.tbi.univie.ac.at/RNA/
Web server; Command
Line Interface; C
Function Library
Table 2: Summary of Accuracy Calculation Results
Type of sequence
Sensitivity
Positive Predictive Value
Cis Regulatory
118.8
90.6
Hairpin Ribozyme
100
60.2
Group I Intron
117.9
75.4
Group II Intron
107.7
92.3
Small Nuclear RNA
100
45.8
Viral & Page
100
92.3
Average
107.4
75.6
Table 3: Different RNA types with known secondary structures
Type of RNA sequence
Sequence Length
STRAND Database ID
External Source
Cis Regulatory
101
RFA_00799
Rfam ID # RFA_00799
Hairpin Ribozyme
226
PDB_00555
PDB ID # 1M5K
Group I Intron
222
PDB_00908
PDB ID # 1ZZN
Group II Intron
70
PDB_00136
PDB ID # 1KXK
Small Nuclear RNA
66
PDB_01199
PDB ID # 2OZB
Viral & Page
34
PDB_00203
PDB ID # 1R7W © 2013 Biomedical Informatics 878
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https://openalex.org/W2600131385
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https://www.epj-conferences.org/articles/epjconf/pdf/2017/07/epjconf_ishepp2017_01008.pdf
|
English
| null |
Hyperons polarization in heavy-ion collisions
|
EPJ web of conferences
| 2,017
|
cc-by
| 3,256
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⋆e-mail: baznat@theor.jinr.ru
⋆⋆e-mail: gudima@cc.acad.md
⋆⋆⋆e-mail: sorin@theor.jinr.ru
⋆⋆⋆⋆e-mail: teryaev@theor.jinr.ru Hyperons polarization in heavy-ion collisions Mircea Baznat1,2,⋆, Konstantin Gudima1,⋆⋆, Alexander Sorin2,3,4,⋆⋆⋆, and Oleg Teryaev2,3,4,⋆⋆⋆⋆
1Institute of Applied Physics, Academy of Sciences of Moldova, MD-2028 Kishinev, Moldova
2Joint Institute for Nuclear Research, 141980 Dubna, Russia
3National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), Kashirskoe Shosse
31, 115409 Moscow, Russia
4D b
I
i
l U i
i
141980 D b
R
i Mircea Baznat1,2,⋆, Konstantin Gudima1,⋆⋆, Alexander Sorin2,3,4,⋆⋆⋆, and Oleg Teryaev2,3,4,⋆⋆⋆⋆
1Institute of Applied Physics, Academy of Sciences of Moldova, MD-2028 Kishinev, Moldova
2Joint Institute for Nuclear Research, 141980 Dubna, Russia
3National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), Kashirskoe Shosse
31, 115409 Moscow, Russia
4 4Dubna International University, 141980, Dubna, Russia Abstract. We study the structure of vorticity and hydrodynamic helicity fields in pe-
ripheral heavy-ion collisions using the kinetic Quark-Gluon Strings Model. The angular
momentum which is a source of P-odd observables is preserved within this model with
a good accuracy. We observe the formation of specific toroidal structures of vorticity
field. Their existence is mirrored in the polarization of hyperons of the percent order. The observed qualitative energy dependence of polarization was predicted earlier and is
quantified now. , 0
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EPJ Web of Conferences
Baldin ISHEPP XXIII
1008 , 0
(2017)
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EPJ Web of Conferences
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1008 DOI: 10.1051/
713801008
epjconf/201 © 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/). 1 Introduction The local violation [1] of discrete symmetries in strongly interacting QCD matter is now under in-
tensive theoretical and experimental investigations. The renowned Chiral Magnetic Effect (CME)
uses the (C)P-violating (electro)magnetic field emerging in heavy ion collisions in order to probe the
(C)P-odd effects in QCD matter. There is an interesting counterpart of this effect, Chiral Vortical Effect (CVE)[2] due to coupling
to P-odd medium vorticity leading to the induced electromagnetic and all conserved-charge currents
[3], in particular the baryonic one. Another important P-odd observable is the baryon polarizatiion. The mechanism analogous to
CVE (known as axial vortical effect, see [4] and references therein) leads to induced axial current of
strange quarks which may be converted to polarization of Λ-hyperons [3, 5, 6]. Another mechanism
of this polarization is provided by so-called thermal vorticity in the hydrodynamical approach [7]. The zeroth component of axial current and correspondent axial charge are related to hydrodynam-
ical helicity H ≡
dV(⃗v · ⃗w), © 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/). , 0
(2017)
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713801008
epjconf/201 DOI: 10.1051/
713801008
epjconf/201 being the projection of velocity ⃗v to vorticity ⃗w = curl⃗v. This quantity is manifesting the recently
discovered [5] and confirmed [8] phenomenon of the separation, i.e. its mirror behavior with the same
magnitudes but different signs in the half-spaces separated by the reaction plane. being the projection of velocity ⃗v to vorticity ⃗w = curl⃗v. This quantity is manifesting the recently
discovered [5] and confirmed [8] phenomenon of the separation, i.e. its mirror behavior with the same
magnitudes but different signs in the half-spaces separated by the reaction plane. The noncentral heavy ion collisions could naturally generate a rotation (global or local, both re-
lated to vorticity) with an angular velocity normal to the reaction plane, which is their generic qualita-
tive feature. It is natural to expect that angular momentum conservation plays an essential role in the
defining the quantitative properties of vortical effects. At the same time, it remains to be studied to
which extent the particles carrying the main part of angular momentum participate in the collisions. In the current paper we address this problem by performing the extensive numerical simulations. 2 Angular momentum conservation in the kinetic model The natural source of the P-odd observables in heavy-ion collisions is the pseudovector of angular
momentum. The question immediately emerges whether it is conserved in the course of evolution
governed by Quark-Gluon String Model (QGSM)[9, 10]. To check this we calculated the angular
momentum at various time moments of collision taking into account both the contributions of partic-
ipants and spectators. We consider the Au + Au collisions with b = 8 fm at √s = 5GeV/u typical
for future NICA collider. We observed (see figure 1) that the participants carry about 20% of angular
momentum and that the total angular momentum of participants and spectators is conserved with a
rather good accuracy. g
y
One may conclude that the angular momentum is under the good control in QGSM model We also studied correlations of angular momentum and the hydrodynamic helicity and observed
that these quantities vary in accordance with each other. In order to perform this comparison and
further calculations, velocity, vorticity and helicity were determined following the earlier suggested
[5] procedure when the respective quantities were properly averaged over events and particles within
the three-dimensional cells providing the transition from the kinetic to hydrodynamic description. p
g
y
y
p
Let us pass to the corresponding results. Let us pass to the corresponding results. Let us pass to the corresponding results. 1 Introduction We explore the distribution of angular momentum and find that the role of participant nucleons is
relatively small, albeit noticeable. We study in some detail the structure of vorticity field and compare
different approaches to polarization calculation. We observed the peculiar toroidal "tire-like" struc-
ture manifesting themselves in the polarization of hyperons. We will explore different approaches to
polarization calculation which will lead to qualitatively similar results. 3 Large-scale structures of vorticity fields We start our studies with the qualitative structure of velocity and vorticity fields. The general structure of velocity field follows the "little bang" pattern which may be quantified by
the velocity dependence allowing to extract the "little Hubble" constant. We calculated the dependence
of average cell velocity on the transverse distance ρ =
x2 + y2 and found (see figure 2) that it is
consistent with the linear "Hubble" law < v/c >= v0/c + Hρ. (1) (1) The Hubble constant H is measured in the units 10−22 sec = 30 fm/c and changes in the range
H = 0.024 ÷ 0.028 (fm/c)−1 The Hubble constant H is measured in the units 10−22 sec = 30 fm/c and changes in the range
H = 0.024 ÷ 0.028 (fm/c)−1 The Hubble constant H is measured in the units 10−22 sec = 30 fm/c and changes in the range
H = 0.024 ÷ 0.028 (fm/c)−1 H = 0.024 ÷ 0.028 (fm/c)−1 It corresponds to the "little Universe" lifetime of about 40 fm/c which is only twice larger than the
collision time. 2 2 , 0
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epjconf/201 10
-3
10
-2
10
-1
1
10
10 2
10 3
10 4
0
2.5
5
7.5
10
12.5
15
17.5
20
|MT|,h/2π
|MF|,h/2π
|MF|/|MT|
|H|/2, fm2c2
t, fm/c
Figure 1. The time dependence of the total (MT and fireball (MF) angular momenta in Planck constant units and
that of hydrodynamical helicity. 10
-3
10
-2
10
-1
1
10
10 2
10 3
10 4
0
2.5
5
7.5
10
12.5
15
17.5
20
|MT|,h/2π
|MF|,h/2π
|MF|/|MT|
|H|/2, fm2c2
t, fm/c Figure 1. The time dependence of the total (MT and fireball (MF) angular momenta in Planck constant units and
that of hydrodynamical helicity. Our key observation is that while velocity field represent the "little bang" picture, vorticity field
form the relatively thin toroidal "tire-like" structures (figure 3), which emerge in the layer (where
velocity field changes rapidly) separating the "core" and "corona" regions [11, 12] and form the sort
of vortex sheet [13]. An interesting property of these structures is that, while emerging due to angular momentum
pseudovector ⃗L in the non-central collisions they do not "remember" the production plane and possess
the cylindrical symmetry w.r.t. collisions axis z. 3 Large-scale structures of vorticity fields This may be observed (figure 4) by considering the
vortex sheet in the case of the particular direction of ⃗L along the y axis. Such behaviour may resemble cyclones appearing at femtoscopic scale. Let us now discuss the observable signatures of these nice structures. 4 Hyperon polarization We consider hyperon polarization as the observable related to vorticity and helicity [3]. We shall
concentrate mostly on Λ hyperon production, which has some advantages: they are produced in large
numbers, their polarization may be easily determined in their weak decays, and their spin is carried
by strange quark. We compare the two rather distinct methods of determining the hyperon polarization. The first
corresponds to its earlier suggested [3] and explored [5] relation to the induced axial current while the
second one follows the procedure based on the thermal vorticity [7]. 3 3 , 0
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epjconf/201 0.5
0.6
0.7
0.8
0.9
1
0
5
10
15
20
25
<|v/c|>(ρ)
ρ, fm
t= 7 fm/c, 1/H=1.4 10-22s
t=10 fm/c, 1/H=1.3
t=15 fm/c, 1/H=1.2
t=20 fm/c, 1/H=1.2
v = v0 + Hρ
Figure 2. The cell velocity dependence on the transverse distance. 0.5
0.6
0.7
0.8
0.9
1
0
5
10
15
20
25
<|v/c|>(ρ)
ρ, fm
t= 7 fm/c, 1/H=1.4 10-22s
t=10 fm/c, 1/H=1.3
t=15 fm/c, 1/H=1.2
t=20 fm/c, 1/H=1.2
v = v0 + Hρ
Figure 2. The cell velocity dependence on the transverse distance. 0.5
0.6
0.7
0.8
0.9
1
0
5
10
15
20
25
<|v/c|>(ρ)
ρ, fm
t= 7 fm/c, 1/H=1.4 10-22s
t=10 fm/c, 1/H=1.3
t=15 fm/c, 1/H=1.2
t=20 fm/c, 1/H=1.2
v = v0 + Hρ 0.5
0.6
0.7
0.8
0.9
1
0
5
10
15
20
25
<|v/c|>(ρ)
ρ, fm
t= 7 fm/c, 1/H=1.4 10-22s
t=10 fm/c, 1/H=1.3
t=15 fm/c, 1/H=1.2
t=20 fm/c, 1/H=1.2
v = v0 + Hρ
Figure 2. The cell velocity dependence on the transverse distance. Figure 2. The cell velocity dependence on the transverse distance. The first method is based on the calculation of strange axial charge The first method is based on the calculation of strange axial charge Qs
5 = Nc
2π2
d3xμ2(x)γ2ϵi jkui∂juk
= < μ2γ2 > NcH
2π2
. (2) Qs
5 = Nc
2π2
d3xμ2(x)γ2ϵi jkui∂juk
= < μ2γ2 > NcH
2π2
. Qs
5 = Nc
2π2
d3xμ2(x)γ2ϵi jkui∂juk
= < μ2γ2 > NcH
2π2
. = < μ2γ2 > NcH
2π2
. (2) (2) In [5] we used the latter equality exploring the mean-value theorem, while here the spatial variation
of strange chemical potential μ is taken into account. 4 Hyperon polarization To do so, the description of kinetic distribution
functions by the correspondent equilibrium equation was performed, providing the matching of kinetic
and thermodynamical descriptions. The average polarization can be estimated by dividing Qs
5 (2) by
the number of Λ’s, assuming that the pseudovector of axial current is proportional to the pseudovector
of polarization, Qs
5 ∼< ΠΛ,lab
0
>. Selecting the axial charge related to the particles in the definite
rapidity or transverse momentum interval, the respective dependence of polarization may be also
obtained. As the axial charge should be related to the zeroth component of hyperon polarization in laboratory
frame Πlab
0 , the transformation to hyperon rest frame should be performed. Taking into account that 4 , 0
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epjconf/201 Figure 3. The vortex sheet. Figure 4. The vortex sheet for the particular direction of angular momentum. Baldin ISHEPP XXIII Figure 3 The vortex sheet Figure 3 The vortex sheet Figure 3. The vortex sheet. Figure 4. The vortex sheet for the particular direction of angular momentum. Figure 4. The vortex sheet for the particular direction of angular momentum. Figure 4. The vortex sheet for the particular direction of angular momentum. 5 5 , 0
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713801008
epjconf/201 polarization pseudovector should be directed along y axis (as it has to be collinear to ⃗L pseudovector),
one get
ΠΛ ΠΛ,lab
0
= ΠΛ
0 py
MΛ
,
(3) ΠΛ,lab
0
= ΠΛ
0 py
MΛ
, (3) so that the rest frame polarization can be obtained as so that the rest frame polarization can be obtained as < ΠΛ
0 > = Qs
5 <
MΛ
NΛ py
> . (4) (4) The possible violation of positivity (|ΠΛ| ≥1) or even the divergence is due to the fact, that hyperons
with zero y component of the momentum should not have the zeroth component of polarization and
therefore should not contribute to Qs
5. To avoid this problem one may instead attribute the factor py/M
to each hyperon in the denominator of (4). Nevertheless the comparison (see figure 5) of various
approaches shows the similar scale and rapidity dependence of polarization. Figure 5. The rapidity dependence of polarization in helicity-based approach. Figure 5. The rapidity dependence of polarization in helicity-based approach. 4 Hyperon polarization Another approach to polarization is based on the so-called thermal vorticity [7]. To provide the
comparison we calculated (see figure 6) the thermal vorticity field and respective polarization. While
a scale of the polarization in the thermal vorticity-based approach is several times larger, its rapidity
dependence, surprisingly enough, appear to be similar in these rather distinct approaches. Another approach to polarization is based on the so-called thermal vorticity [7]. To provide the
comparison we calculated (see figure 6) the thermal vorticity field and respective polarization. While
a scale of the polarization in the thermal vorticity-based approach is several times larger, its rapidity
dependence, surprisingly enough, appear to be similar in these rather distinct approaches. The current STAR@RHIC measurements of polarization [14] manifested the decrease with energy
which is naturally explained in the anomalous mechanism but also happens in the thermal vorticity
mechanism. To quantify the predictions, we calculated the energy dependence in the anomalous
mechanism, covering the experimental points (see figure 7). 6 , 0
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713801008
epjconf/201 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
0
20
40
60
80
100
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
(1/NΛ)dNΛ/dy
|Π0|,%
y-ycm
y-ycm
t= 5fm/c
t=20fm/c
t= 5 x6
t=10 x4
t=15 x2
t=20fm/c
Figure 6. The rapidity dependence of polarization in thermal vorticity-based approach. The magnitudes of
polarization at different times are rescaled by the identified factors. 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
0
20
40
60
80
100
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
(1/NΛ)dNΛ/dy
|Π0|,%
y-ycm
y-ycm
t= 5fm/c
t=20fm/c
t= 5 x6
t=10 x4
t=15 x2
t=20fm/c
Figure 6
The rapidity dependence of polarization in thermal vorticity based approach
The Figure 6. The rapidity dependence of polarization in thermal vorticity-based approach. The magnitudes of
polarization at different times are rescaled by the identified factors. 5 Conclusions and Outlook We investigated vorticity and hydrodynamical helicity in noncentral heavy-ion collisions in the frame-
work of the kinetic Quark-Gluon String Model. We have confirmed our earlier observation that the
vorticity field is predominantly localized in a relatively thin layer (2÷3 fm) on the boundary between
the participant and spectator nucleons and observed that it is forming the specific toroidal structures,
which might be considered as vortex sheets with the unexpected cylindrical symmetry. They look as
cyclones appearing at femtoscopic scale. The vorticity and helicity fields are manifested in the Λ hyperon polarization. We performed its
detailed calculations including the simulations of the strange chemical potential. We found that the
polarization magnitude might reach a percent level. The comparison with the very different approach 7 , 0
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713801008
epjconf/201 10
-2
10
-1
0
2
4
6
8
10
12
14
<Π0Λ >
sNN1/2, GeV
Data STAR
DCM-QGSM
Au + Au, b=8 fm
Figure 7. The energy dependence of polarization in anomaly-based approach compared with experimental STAR
points. 10
-2
10
-1
0
2
4
6
8
10
12
14
<Π0Λ >
sNN1/2, GeV
Data STAR
DCM-QGSM
Au + Au, b=8 fm Figure 7. The energy dependence of polarization in anomaly-based approach compared with experimental STAR
points. exploring the thermal vorticity leads to qualitatively similar results, although the polarization scale is
several times larger. exploring the thermal vorticity leads to qualitatively similar results, although the polarization scale is
several times larger. Acknowledgements This work was supported in part by the Russian Foundation for Basic Research, Grant No. 14-01-
00647. 8 8 8 , 0
(2017)
138
EPJ Web of Conferences
Baldin ISHEPP XXIII
1008 DOI: 10.1051/
713801008
epjconf/201 References [1] K. Fukushima, D.E. Kharzeev and H.J. Warringa, Phys. Rev. D 78, 074033 (2008). [arXiv:0808.3382 [hep-ph]] harzeev and A. Zhitnitsky, Nucl. Phys. A 797, 67 (2007). [arXiv:0706.1026 [hep-ph]] [2] D. Kharzeev and A. Zhitnitsky, Nucl. Phys. A 797, 67 (2007). [arXiv:0706.1026 [hep-ph]]
[3] O. Rogachevsky, A. Sorin and O. Teryaev, Phys. Rev. C 82, 054910 (2010). [arXiv:1006.1331
[hep-ph]] alaydzhyan, Phys. Rev. D 89 (10), 105012 (2014). [arXiv:1403.1256 [hep-th]] [4] T. Kalaydzhyan, Phys. Rev. D 89 (10), 105012 (2014). [arXiv:1403.1256 [hep-th]]
[5] M
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iv:1301.7003 [nucl-th]] [arXiv:1301.7003 [nucl-th]]
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[nucl-th]] [8] O. Teryaev and R. Usubov, Phys. Rev. C 92 (1), 014906 (2015) [9] V. D. Toneev, N. S. Amelin, K. K. Gudima and S. Y. Sivoklokov, Nucl. Phys. A 519, 463C
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1541 (1991) [11] J. Aichelin and K. Werner, Phys. Rev. C 79, 064907 (2009). [Phys. Rev. C 81, 029902 (2010)]. [arXiv:0810.4465 [nucl-th]] [12] J. Steinheimer and M. Bleicher, Phys. Rev. C 84, 024905 (2011). [arXiv:1104.3981 [hep-ph]]
[13] M I B
K K G di
A S S
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C 93 (3) 031902 (2016) [12] J. Steinheimer and M. Bleicher, Phys. Rev. C 84, 024905 (2011). [arXiv:1104.3981 [hep-ph]]
[13] M. I. Baznat, K. K. Gudima, A. S. Sorin and O. V. Teryaev, Phys. Rev. C 93 (3), 031902 (2016). doi:10.1103/PhysRevC.93.031902. [arXiv:1507.04652 [nucl-th]] [13] M. I. Baznat, K. K. Gudima, A. S. Sorin and O. References V. Teryaev, Phys. Rev. C 93 (3), 031902 (2016). doi:10.1103/PhysRevC.93.031902. [arXiv:1507.04652 [nucl-th]] [14] M. Lisa [for STAR Collaboration]. Talk at QCD Chirality Workshop, UCLA, February 2016 [14] M. Lisa [for STAR Collaboration]. Talk at QCD Chirality Workshop, UCLA, February 2016 9
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Tks5-dependent formation of circumferential podosomes mediates cell-cell fusion
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From rheumatic diseases to cancer - role of autoantibodies as
diagnostic biomarkers Since it was shown that synovial fibroblasts (SF) are not only effector cells
responding to inflammatory stimuli, but appear endogenously activated
and potentially involved into spreading the disease [1], we searched for
the epigenetic modifications leading to the activated phenotype of these
cells. g
Department of Molecular and Experimental Medicine, The Scripps Research
Institute, La Jolla, CA 92037, USA g
Department of Molecular and Experimental Medicine, The Scripps Research
Institute, La Jolla, CA 92037, USA Arthritis Research & Therapy 2012, 14(Suppl 1):O2 Epigenetics in its scientific definition “is the study of all heritable and
potentially reversible changes in genome function that do not alter the
nucleotide sequence within the DNA”, but might be considered in simpler
terms as the regulation of gene expression. Rheumatology has pioneered in the study of autoantibodies by showing
that they are not only involved in pathogenesis but are also highly useful as
diagnostic biomarkers. The diagnostic biomarker aspect of autoimmunity
has gained increasing importance in cancer and many of the insights gained
in Rheumatology have contributed to understanding the significance of
autoantibodies in cancer. Epigenetic modifications include: Acetylation, Acetylation, Methylation, Methylation, Features of autoantibodies in rheumatic disorders: In rheumatic
diseases no individual autoantibody-antigen system has sufficient
combination of sensitivity and specificity to serve as a useful diagnostic
biomarker. Instead, several antigen-antibody systems constructed as
profiles of biomarkers are highly effective in distinguishing one disorder
from another. In lupus, anti-double strand DNA and anti-Sm distinguishes
it from scleroderma, where the profile is anti-DNA topoisomerase 1 and
anti-centromere proteins. The autoantigensare cell components involved
in universal and basic gene expression pathways, such as Sm in precursor
mRNA splicing and DNA topoisomerase 1 in DNA replication and
transcription [1]. Phosphorylation, Sumoylation, Sumoylation, miRs or microRNAs. Our laboratory is studying these processes and we have found that RASF
reside in a hyperacetylated synovial tissue and appear hypomethylated
[2]. Hypomethylation leads to the activated phenotype of RASF which is
characterized by the production of matrix-degrading enzymes and of
potent chemokines induced by Toll-like receptor signalling. Current
strategies are designed to methylate these cells to deactivate and
“normalise” them again. miRs are about 20 nucleotide long smallRNAs acting to destroy specific
mRNA. Features of autoantibodies in cancer: Autoantibodies in cancer target
intracellular molecules referred to as TAAs (tumor-associated antigens). As
in rheumatic disorders, no individual autoantibody-antigen system has
sensitivity and specificity to serve as a stand-alone diagnostic marker [2]. © 2012 various authors, licensee BioMed Central Ltd. All articles published in this supplement are 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. MEETING ABSTRACTS
Open Access Open Access Proceedings of the 8th Global Arthritis
Research Network (GARN) Meeting and 1st
Bio-Rheumatology International Congress (BRIC)
T k
J
14 16 N
b
2011 These abstracts are available online at http://arthritis-research.com/supplements/14/S1 O1
Future direction of pathogenesis and treatment for rheumatic disorders
Steffen Gay 2. Karouzakis E, et al: Epigenetic control in rheumatoid arthritis synovial
fibroblasts. Nat Rev Rheumatol 2009, 5:266-72. 3. Brock M, et al: Interleukin-6 modulates the expression of the bone
morphogenic protein receptor type II through a novel STAT3-microRNA
cluster 17/92 pathway. Circ Res 2009, 104:1184-91. Arthritis Research & Therapy 2012, 14(Suppl 1):O1 y
4. Stanczyk J, et al: Altered expression of miRNA-203 in rheumatoid arthritis
synovial fibroblasts and its role in fibroblast activation. Arthritis Rheum
2011, 63:373-81. After the breakthrough in the treatment of rheumatoid arthritis and
numerous related disorders with biological therapies targeting TNFa at
the Kennedy Institute in London 5. Willyard C: The saving switch. Nat Med 2010, 16:16-8. 5. Willyard C: The saving switch. Nat Med 2010, 16:16-8. Millions of patients have tremendously benefitted. However, we cannot
cure these diseases yet and have to search for additional therapeutic
targets. ORAL PRESENTATIONS Most interestingly, epigenetic therapy is also on the horizon [5]. References Most interestingly, epigenetic therapy is also on the horizon [5].
References Most interestingly, epigenetic therapy is also on the horizon [5]. References 1. Lefèvre S, et al: Synovial fibroblasts spread rheumatoid arthritis to
unaffected joints. Nat Med 2009, 15:1414-20. O1
Future direction of pathogenesis and treatment for rheumatic disorders
Steffen Gay
Department of Rheumatology, University Hospital, Gloriastrasse 25, CH-8091
Zurich, Switzerland
E-mail: Steffen.Gay@usz.ch
Arthritis Research & Therapy 2012, 14(Suppl 1):O1 1. Lefèvre S, et al: Synovial fibroblasts spread rheumatoid arthritis to
unaffected joints. Nat Med 2009, 15:1414-20. O2 O2
From rheumatic diseases to cancer - role of autoantibodies as
diagnostic biomarkers
Eng M Tan
Department of Molecular and Experimental Medicine, The Scripps Research
Institute, La Jolla, CA 92037, USA
Arthritis Research & Therapy 2012, 14(Suppl 1):O2 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 From rheumatic diseases to cancer - role of autoantibodies as
diagnostic biomarkers Most tumors show multiple antibody specificities and with panels of TAA-
anti-TAAs (analogous toprofiles) the cumulative sensitivity and specificity
reaches diagnostic significance. Different tumorigenesis pathways are In the race to identify specific miRs as novel targets we have identified
for example, that interleukin-6 modulates the expression of the Bone
Morphogenic Protein Receptor Type II through a novel STAT3microRNA
cluster 17/92 pathway, which helps to explain the loss of the BMPR2 in
the vascular cells in pulmonary hypertension [3]. Moreover, miR-203 is
regulating the production of IL-6 [4]. Page 2 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 References 1. Tan EM: Antinuclear antibodies: diagnostic markers for autoimmune
diseases and probes for cell biology. Adv Immunol 1989, 44:93-151. Oligodendropathy and astrocytopathy in demyelinating disorders:
Neuromyelitisoptica (NMO) was previously considered to be a variant of MS
but is now recognized as an astrocytopathy and secondary demyelinating
event mimicking MS characteristics occurring due to autoantibody mediated
mechanisms. Advancement of molecular biology makes it possible to
differentiate MS by measuring abnormal autoantibody to aquaporin 4 (water
channel). Interestingly, collagen diseases coexist more frequently with NMO
than with MS. B cell depletion therapy with Rituximab has showed the same
benefits, although, plasma exchange therapy is more effective with NMO
than with MS. 2. Tan EM, Zhang J: Autoantibodies to tumor-associated antigens: reporters
from the immune system. Immunol Rev 2008, 222:328-340. 2. Tan EM, Zhang J: Autoantibodies to tumor-associated antigens: reporters
from the immune system. Immunol Rev 2008, 222:328-340. 3. Robles AI, Harris CC: Clinical outcomes and correlates of TP53 mutations
and cancer. Cold Spring Harb Perspect Biol 2010, 2(3):a001016, Review. 4. Levine AJ, Oren M: The first 30 years of p53: growing ever more
complex. Nat Rev Cancer 2009, 9:749-758. 5. Efeyan A, Serrano M: p53: guardian of the genome and policeman of the
oncogenes. Cell Cycle 2007, 6:1006-1010. 6. SooHoo L, Chan EK: Cloning and characterization of a novel 90 kDa
‘companion’ autoantigen of p62 overexpressed in cancer. Oncogene
2002, 21:5006-5015. TNF therapy and demyelinating event: A report indicates that adverse
events such as the demyelinating lesion in the brain, optic neuritis, and
neuropathy occurred after treatment with anti-TNF alpha therapy in
collagen disease, and TNF antagonizing therapy showed worsening in a
clinical trial with MS. Pathogenesis of these events such as primary or
secondary demyelination are still in enigma. 7. Juntilla MR, Puustinen P, Niemela M, et al: CIP2A inhibits PP2A in human
malignancies. Cell 2007, 130:51-62. In this presentation, I will decode the temporal and spatial demyelinating
processes in collagen diseases and show practical approaches and
treatments. O3
Etiology and molecular pathogenesis of RA; how can we best use
European initiatives to advance our knowledge? Lars Klareskog
Rheumatology Unit, Department of Medicine, Karolinska Institutet/Karolinska
University Hospital, 171 76 Stockholm, Sweden
Arthritis Research & Therapy 2012, 14(Suppl 1):O3 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Background: Acute isolated neurological syndromes, such as optic
neuropathy or transverse myelopathy, may cause diagnostic problems
since they can be the first presentations in a number of demyelinating
disorders including multiple sclerosis (MS) and collagen diseases. However, clinical presentation and lesions evidenced by magnetic
resonance imaging may be similar. Collagen disease coexists in
demyelinating disorders and frequently various collagen disease related
autoantibodies are positive in daily practice. activated in similar cell-type tumors from the same organ and are the
driving mechanisms behind the autoantibody response. The immune
responses are directed to products of oncogenes and tumor-suppressor
genes such as p53 and other proteins that regulate and modulate the
functions of p53 [3-5]. Protein phosphatase 2A (PP2A) is an important tumor suppressor
protein. It is a serine/threonine phosphatase and is a trimeric complex. The B subunit is recruited from several intracellular proteins and the
type of B subunit determines the substrate of its tumor suppressor
activity. One of the B subunits, p90, was identified in our laboratory
with autoantibody from a patient with hepatocellular carcinoma [6]. It
was found to co-immunoprecipitate with other subunits of PP2A [7]
and was shown to function as an inhibitor of the tumor-suppressor
activity of PP2A. Hence, the algorithm to overcome these diagnostic and therapeutic
issues should be clarified. B cell immunity in demyelinating disorders: In primary demyelinating
disease, MS, a renewed interest in the role of humoral immunity in the
pathophysiology has been investigated because oligoclonalIgG band in
the CSF and increased intrathecalIgG synthesis are used as an auxiliary
diagnosis measure. Moreover, in the secondary progressive MS, meningeal
B-cell follicles are associated with early onset of the disease and severe
cortical pathology. B cell but not plasma cell depletion therapy with single
treatment by Rituximab in MS showed reduced inflammatory brain lesions
and clinical relapses. The immune system is capable of sensing dysregulation of tumorigenesis
pathways. The goal of continuing research is in developingTAA-anti-TAAs
for detecting cancer in individual patients and profiles which are
common to specific types of tumors. References 1. Franciotta D, Salvetti M: B cells and multiple sclerosis. Lancet Neurol 2008,
7:852-8. 2. Bernatsky S, Renoux C: Demyelinating events in rheumatoid arthritis after
drug exposures. Ann Rheum Dis 2010, 69:1691-3. Arthritis Research & Therapy 2012, 14(Suppl 1):O3 Understanding etiology and molecular pathogenesis of rheumatoid
arthritis is key to the development of precise prevention and curative
therapy for this disease. Recent progress on how genes and environment
interact in causing immune reactions that may induce arthritis in humans
as well as in mice, have provided a conceptual basis for the development
of new prevention and treatment strategies which need to be different for
different subsets of RA. In order to bring this emerging knowledge to the
level where basic and clinical academic science can collaboratewitj
industry for rapid development of the potential new therapies, there is a
need for closer collaboration between basic and clinical scientists from
many centers, and for increased collaboration between industry and
academia in translational medicine. O5 The role of mRNA degradation in immunity and inflammation
Shizuo Akira O6
Recent advances in understanding of various chronic pain mechanisms
through lysophosphatidic acid (LPA) receptor signaling
Hiroshi Ueda
Division of Molecular Pharmacology and Neuroscience, Nagasaki University
Graduate School Biomedical Sciences, Nagasaki 852-8521, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O6 O6
Recent advances in understanding of various chronic pain mechanisms
through lysophosphatidic acid (LPA) receptor signaling
Hiroshi Ueda Division of Molecular Pharmacology and Neuroscience, Nagasaki University
Graduate School Biomedical Sciences, Nagasaki 852-8521, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O6 We recently found that the IKK complex controls Il6 mRNA stability by
phosphorylating Regnase-1 in response to IL-1R/TLR stimulation. Phosphorylated Regnase-1 underwent ubiquitination and degradation. Regnase-1 re-expressed in IL-1R/TLR-activated cells exhibited delayed
kinetics, and Regnase-1 mRNA was found to be negatively regulated by
Regnase-1 itself via a stem-loop region present in the Regnase-1 3’
untranslated region. These data demonstrate that the IKK complex
phosphorylates not only IkBalpha, activating transcription, but also
Regnase-1, releasing the “brake” on Il6 mRNA expression. Lysophosphatidic acid (LPA) receptor (LPA1) signaling plays the key role
in initiation of nerve injury-induced neuropathic pain [1-4]. LPA, which
is produced in the spinal cord following the sciatic nerve injury causes
a calpain-mediated demyelination of dorsal root fibers and sprouting
through
LPA1
receptor,
leading
to
an
induction
of
synaptic
reorganization underlying allodynia. The LPA1 signaling also initiates
the up-regulation of Cava2δ1 in DRG, leading to an enhancement of
spinal pain transmission underlying hyperalgesia. Similar LPA1-mediated
chronic abnormal pain and underlying mechanisms are observed in
mouse models with Meth-A sarcoma surrounding sciatic nerve (cancer
model) or with chemotherapy (paclitaxel). Central neuropathic pain
following spinal nerve injury is now recently found to include the LPA1-
mediated mechanisms. In contrast, (arthritic) inflammatory pain
following Complete Freund Adjuvant treatment fails to show the
involvement of LPA1 signaling. Thus it seems that many models of
neuropathic pain, but not inflammatory pain model include LPA1-
mediated mechanisms. The role of mRNA degradation in immunity and inflammation
Shizuo Akira O8 O8
Death receptor-induced apoptosis signalling - essential guardian
against autoimmune disease
Andreas Strasser1*, Lorraine A O’Reilly1, Philipp Jost1, Thomas Kaufmann1,
Stephanie Grabow1, Elizabeth Kruse1, Lin Tai1, Mark Smyth2, Philippe Bouillet1
1The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade,
Parkville, Victoria 3050, Australia; 2The Peter MacCallum Cancer Centre, St
Andrews Place, East Melbourne, Victoria 3000, Australia
E-mail: strasser@wehi.edu.au
Arthritis Research & Therapy 2012, 14(Suppl 1):O8 Recent studies revealed that another subtype LPA3 receptor plays a crucial
role in neuropathic pain mechanisms in terms of LPA biosynthesis. Nerve
injury and intrathecal administration of LPA increased the levels of
lysophosphatidylcholine (LPC) and LPA in the spinal dorsal horn and dorsal
root with peaks at 1 - 2 h. We obtained the evidence for in vitro LPA
biosynthesis in spinal dorsal horn and dorsal root as well as in vivo one. In
these studies we successfully identified the species of LPC and LPA
molecules by use of Mass Spectrometery. Major species are the molecules
with lipid chain 16:0, 18:0 or 18:1, and their contents were all time-
dependently increased by nerve injury. Interestingly, there was an LPA-
induced amplification of LPA biosynthesis through an activation of LPA3
receptor and microglia. The microglial involvement was found to play key
roles as an initiation of neuropathic pain mechanisms including LPA3-
mediated amplification of LPA biosynthesis. The FasL/Fas system is critical for deletion of autoreactive and antigen-
activated T and B cells. Accordingly, mutations in these proteins result in
lymphadenopathy and autoimmunity in gld and lpr mutant mice, which
lack functional FasL or Fas, respectively. Upon antigenic stimulation of
T cells, FasL is sythesised, directed to and stored in secretory lysosomes
followed by extrusion at the immunological synapse where it is rapidly
downregulated by a metalloprotease, shedding the extracellular portion
(sFasL) to prevent non-specific killing. It is unclear whether the pathology
observed in gld mutant mice is due to the loss of the membrane-bound
or the secreted form of FasL or both. We have produced a panel of mutant FasL knock-in mice to address this
question. In the first mutant strain the cytoplasmic and trans-membrane
domains of FasL were replaced with the signal peptide from G-CSF. Research platform for fibromyalgia in Japan
Kusuki Nishioka Japan College of Fibromyalgia Investigation, Japan Rheumatism Foundation,
Institute of Medical Science, Tokyo Medical University, Tokyo, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O5 Epidemiology: Fibromyalgia (FM) is found worldwide, with an estimated
prevalence of 1% to 4% of the general population. In Japan epidemiological
surveillance showed number of the patient up to1.66% in population-based
studies in 2003, following 2.04% by internet surveillance in 2011. 3,500
Japanese patients with our FM data base, frequent age of onset is 35 to
55 years, estimated prevalence of FM, reaching 40% in woman of age 30 to
50 years old. FM occurs in children and adolescents, although only a few
epidemiologic studies. In Europe, both the EU-funded framework programs and the EU and
industry funder Innovative Medicine Initiative (IMI) funder programs in
rheumatology are geared to accomplishing these goals. This presentation
will be concerned both with the scientific basis of these programs and with
a descriptions of the challenges and potential promises that these new
collaborative programs offer to rheumatology. Pathogenesis: FM was initially one the kind for inflammatory muscle pain. Following this concept FM was classified so called soft-tissue rheumatism. Now FM is recognized CNS sensitization following functional pain caused by
neuroendocrine system and stress. The abundant neuroendocrine and pain
regulation descending pathway and sensitization of pain receptor target
molecules such as a2δ ligand, LPA and collapsing response mediator, where
currently protein (CRMP) family have been revealed. O4
Clinically isolated syndrome in collagen diseases; approaches and
treatments
Kazumasa Yokoyama*, Nobutaka Hattori
Department of Neurology, Juntendo University School of Medicine, Hongo,
Tokyo, 113-8421, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O4 O4
Clinically isolated syndrome in collagen diseases; approaches and
treatments
Kazumasa Yokoyama*, Nobutaka Hattori
Department of Neurology, Juntendo University School of Medicine, Hongo,
Tokyo, 113-8421, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O4 O4 New diagnosis, provisional ACR 2010 criteria: 1990, ACR proposed FM
criteria based on 18 tender point sites on digital palpation with exclusive
differential diagnosis. In 2010, provisional criteria is score based on total
score of severity for pain and somatic symptom (2010 ACR criteria). Based Page 3 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 The role of mRNA degradation in immunity and inflammation
Shizuo Akira Activated T cells from these mutant mice can produce cytoplasmic but no
membrane bound FasL and, interestingly, they are defective in FasL-
mediated cytotoxic function and undergo significantly less activation-
induced cell death upon re-stimulation with anti-CD3 antibodies than wt
T cells. The extent of these defects is similar to that seen in FasL mutant gld
T cells. With age these FasL mutant knock-in mice develop lymphadenopathy
and splenomegaly and CD3+B220+CD4-CD8- T cells accumulate, similarly to
what has been observed in gld and lpr mutant mice. In contrast to gld mice,
the FasL mutant knock-in mice on the C57BL/6 background develop
haemopoietic tumours and reticular cell sarcomas, suggesting that while The role of mRNA degradation in immunity and inflammation
Shizuo Akira FDA approved of pregabalin in FM by double-blind, multicenter and
randomized study. Both studies enrolled patients with a diagnosis of FM
using the ACR criteria. Each of these studies showed a significant
reduction in pain compared with placebo. In addition, improvement
demonstrated based on FIQ. In Japan, this clinical trial has been
developed. Sooner or later, excellent result will be revealed. Laboratory of Host Defense, WPI Immunology Frontier Research Center,
Osaka University, 3-1 Yamadaoka, Suita City, Osaka 565-0871, Japan
E-mail: sakira@biken.osaka-u.ac.jp
Arthritis Research & Therapy 2012, 14(Suppl 1):O7 Laboratory of Host Defense, WPI Immunology Frontier Research Center,
Osaka University, 3-1 Yamadaoka, Suita City, Osaka 565-0871, Japan
E-mail: sakira@biken.osaka-u.ac.jp
Arthritis Research & Therapy 2012, 14(Suppl 1):O7 In other medication, gabapentin practical efficacy for reduced pain with
FM patient. Several anti-dispersants (SSTIs, SNRIs. TCAs) NSAIDs, muscle
relaxant, anti epileptics and pilocarpine hydrochloride also reduced the
pain and an associated symptom. Based on with multivariant statistical
analysis based on 3,500 patients, we will present several associated
somatic symptoms influencing on drug response for pain and prognosis
with FM. The innate immune system is an evolutionally conserved host defense
mechanism against pathogens. Innate immune responses are initiated by
pattern recognition receptors (PRRs), which recognize specific structures
of microorganisms. Among them, Toll-like receptors (TLRs) are capable of
sensing organisms ranging from bacteria to fungi, protozoa and viruses,
and play a major role in innate immunity. Individual TLRs recognize
different microbial components, and give rise to different patterns in
gene expression. In conclusion, FM is one the most important scientific field to understand
the pain neurology and rheumatology in near. We are now focusing on the role of genes induced in response to TLR
stimulation, particularly the genes that are rapidly induced in a MyD88-
dependent manner within 30 min after LPS stimulation. Among them, we
have recently identified a novel gene named Zc3h12a which has a CCCH-
type zinc finger domain. The knockout mice developed spontaneous
autoimmune diseases accompanied by splenomegaly and lymphadenopathy. Subsequent studies showed that Zc3h12a is a nuclease involved in
destabilization of IL-6 and IL-12mRNA. We renamed it Regulatory RNase-1
(Regnase-1) based on the function. 4.
Ueda H: Lysophosphatidic acid as the initiator of neuropathic pain. Biol
Pharm Bull 2011, 34:1154-1158, Review. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 on 2010 ACR criteria, we proposed the assessment of FM severity termed
“FAS31”. Here, we would like propose the assessment of FM severity
score termed FAS31. O7 O7
The role of mRNA degradation in immunity and inflammation
Shizuo Akira
Laboratory of Host Defense, WPI Immunology Frontier Research Center,
Osaka University, 3-1 Yamadaoka, Suita City, Osaka 565-0871, Japan
E-mail: sakira@biken.osaka-u.ac.jp
Arthritis Research & Therapy 2012, 14(Suppl 1):O7 1.
Inoue M, Rashid MH, Fujita R, Contos JJ, Chun J, Ueda H: Initiation of
neuropathic pain requires lysophosphatidic acid receptor signaling. Nat
Med 2004, 10:712-718. O10 Autoimmune arthritis caused by altered thymic T-cell selection due to a
mutation of the ZAP-70 gene 3. Huang DCS, Hahne M, Schroeter M, Frei K, Fontana A, Villunger A,
Newton K, Tschopp J, Strasser A: Activation of Fas by FasL induces
apoptosis by a mechanism that cannot be blocked by Bcl-2 or Bcl-xL. Proc Natl Acad Sci USA 1999, 96:14871-14876. g
Yoshinaga Ito1*, Shimon Sakaguchi1,2 1Department of Experimental Pathology, Institute for Frontier Medical
Sciences, Kyoto University, Kyoto, Japan; 2Laboratory of Experimental
Immunology, WPI Immunology Frontier Research Center, Osaka University,
Suita, Japan 1Department of Experimental Pathology, Institute for Frontier Medical
Sciences, Kyoto University, Kyoto, Japan; 2Laboratory of Experimental
Immunology, WPI Immunology Frontier Research Center, Osaka University,
Suita, Japan 4. Kaufmann T, Tai L, Ekert PG, Huang DCS, Norris F, Lindemann RK,
Johnstone RW, Dixit VM, Strasser A: The pro-apoptotic BH3-only protein
Bid is dispensable for DNA damage- and replicative stress-induced
apoptosis or cell cycle arrest. Cell 2007, 129:423-433. Arthritis Research & Therapy 2012, 14(Suppl 1):O10 SKG mouse is a murine model of autoimmune arthritis. A spontaneous
point mutation of the gene encoding an SH2 domain of the ζ-associated
protein of 70 kDa gene (ZAP-70), a key signal transduction molecule in
T cells, causes chronic autoimmune arthritis in SKG mice that resembles
human RA in many aspects. Altered signal transduction from T-cell antigen
receptor through the aberrant ZAP-70 changes the thresholds of T cells to
thymic selection, leading to the positive selection of otherwise negatively
selected autoimmune T cells. p p
y
5. Hughes PD, Belz GT, Fortner KA, Budd RC, Strasser A, Bouillet P: Fas and
Bim cooperate in shutdown of chronic immune responses and
prevention of autoimmunity. Immunity 2008, 28:197-205. 6. Kaufmann T, Jost P, Pellegrini M, Puthalakath H, Gugasyan R, Gerondakis S,
Cretney E, Smyth M, Silke J, Hakem R, Bouillet P, Mak T, Dixit VM, Strasser A
Fatal hepatitis mediated by TNFa requires caspase-8 and involves the
BH3-only proteins Bid and Bim. Immunity 2009, 30:56-66. 7. Jost PJ, Grabow S, Gray D, McKenzie MD, Nachbur U, Huang DCS, Bouillet P,
Thomas HE, Borner C, Silke J, Strasser A, Kaufmann T: XIAP acts as a switch
between type I and type II Fas-induced apoptosis signalling. Nature 2009,
460:1035-1039. Based on the finding that the skg-mutation of ZAP-70 causes autoimmune
arthritis, we then examined how attenuated TCR signaling affects the
spectrum of autoimmune diseases. References 1. Inoue M, Rashid MH, Fujita R, Contos JJ, Chun J, Ueda H: Initiation of
neuropathic pain requires lysophosphatidic acid receptor signaling. Nat
Med 2004, 10:712-718. 2. Ueda H: Molecular mechanisms of neuropathic pain-phenotypic switch and
initiation mechanisms. Pharmacol Ther 2006, 109:57-77, Review. 3. Ueda H: Peripheral mechanisms of neuropathic pain - involvement of
lysophosphatidic acid receptor-mediated demyelination. Mol Pain 2008,
4:11, Review. 4. Ueda H: Lysophosphatidic acid as the initiator of neuropathic pain. Biol
Pharm Bull 2011, 34:1154-1158, Review. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Page 4 of 54 membrane-bound FasL is the guardian against autoimmunity, secreted FasL
may play a critical role in tissue damage and tumour suppression. References Translational clinical trials of these cancer vaccine and adoptive T cell
therapy are now on-going. Translational clinical trials of these cancer vaccine and adoptive T cell
therapy are now on-going. An open innovation to promote fusion of different fields of science and
technology played an essential role in our development of cancer
immunotherapy. membrane-bound FasL is the guardian against autoimmunity, secreted FasL
may play a critical role in tissue damage and tumour suppression. References Translational clinical trials of these cancer vaccine and adoptive T cell
therapy are now on-going. An open innovation to promote fusion of different fields of science and
technology played an essential role in our development of cancer
immunotherapy. An open innovation to promote fusion of different fields of science and
technology played an essential role in our development of cancer
immunotherapy. 1. Strasser A, Harris AW, Huang DCS, Krammer PH, Cory S: Bcl-2 and Fas/APO-
1 regulate distinct pathways to lymphocyte apoptosis. EMBO J 1995,
14:6136-6147. 2. Newton K, Harris AW, Bath ML, Smith KGC, Strasser A: A dominant
interfering mutant of FADD/MORT1 enhances deletion of autoreactive
thymocytes and inhibits proliferation of mature T lymphocytes. EMBO J
1998, 17:706-718. O9
Cancer immunotherapy; integration of T cell biology with nanogel- and
vector-technology in translational research Molecular definition of cancer specific antigens recognized by T cells
opened an approach to develop cancer specific immunotherapy. Through
a series of key findings in cancer immunology, for development of
effective therapy major effort has been directed to how to induce T cells
with fine specificity, sufficient quantity and high quality in hosts. O11 y
y
y
We intended to integrate immunobiological strategy of T cells with two
technologies, nanogel technology and retroviral vector technology for
translational research of cancer immunotherapy. Cholesterol-bearing
hydrophobizedpullulan (CHP), physically cross-linked nanogels by self-
assembly, form nanoparticle complex with protein in water.We found that
antigen protein with multiple T cell epitopes, when complexed with CHP,
was efficiently transported to lymph nodes and well captured by antigen
presenting cells such as dendritic cells and macrophages leading to cross
presentation. Hence, CHP-antigen protein complex may become excellent
cancer vaccine to induce both CD8+ killer T cells and CD4+ helper T cells of
high quality. O11
Anti-Fas IgM monoclonal antibody (anti-Fas mAb) effect on
haemophilic arthropathy (HA) synoviocytes
Serena Guiducci1*, Eloisa Romano1, Claudia Ceccarelli1, Daniela Melchiorre1,
Mirko Manetti2, Anna F Milia2, Lidia Ibba Manneshi2, Kusuki Nishioka3,
Marco Matucci Cerinic1
1Dept Medicine, Division of Rheumatology, University of Florence, Florence,
Italy; 2Dept Anatomy, Histology and Forensic Medicine, University of
Florence, Florence, Italy; 3Dept Rheumatology, Institute of Medical Science,
Tokyo Medical University, Tokyo, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O11 O10 In a set of mice with the mutation, the
amount of ZAP-70 protein as well as its tyrosine phosphorylation upon TCR
stimulation decreased from +/+, skg/+, skg/skg, to skg/−mice in a stepwise
manner. The reduction resulted in graded alterations of thymic positive
and negative selection of self-reactive T cells and Foxp3+ natural regulatory
T cells (Tregs) and their respective functions. Consequently, skg/−mice
spontaneously developed autoimmune arthritis even in a microbially clean
environment, whereas skg/skg mice required stimulation through innate
immunity for disease manifestation. After Treg depletion, organ-specific
autoimmune diseases, especially autoimmune gastritis, predominantly
developed in +/+, at a lesser incidence in skg/+, but not in skg/skg BALB/c
mice, which suffered from other autoimmune diseases, especially
autoimmune arthritis. In correlation with this change, gastritis-mediating TCR
transgenic T cells were positively selected in +/+, less in skg/+, but not in
skg/skg BALB/c mice. Similarly, on the genetic background of diabetes-prone
NOD mice, diabetes spontaneously developed in +/+, at a lesser incidence
in skg/+, but not in skg/skg mice, which instead succumbed to arthritis. Thus,
the graded attenuation of TCR signaling alters the repertoire and the
function of autoimmune T cells and natural Tregs in a progressive manner. It
also changes the dependency of disease development on environmental
stimuli. These findings collectively provide a model of how genetic anomaly
of T cell signaling contributes to the development of autoimmune disease. 8. O’Reilly LA, Tai L, Lee L, Kruse EA, Grabow S, Fairlie WD, Haynes NM,
Tarlinton DM, Zhang J-G, Belz GT, Smyth MJ, Bouillet P, Robb L, Strasser A:
Membrane-bound but not secreted Fas ligand is essential for Fas-
induced apoptosis and prevention of autoimmunity and cancer. Nature
2009, 461:659-663. 9. Strasser A, Jost P, Nagata S: The many roles of FasL-Fas signaling in the
immune system. Immunity 2009, 30:180-192. 9. Strasser A, Jost P, Nagata S: The many roles of FasL-Fas signaling in the
immune system. Immunity 2009, 30:180-192. O9 O9
Cancer immunotherapy; integration of T cell biology with nanogel- and
vector-technology in translational research
Hiroshi Shiku
Dept. of Cancer Vaccine and Immuno-Gene Therapy, Mie University
Graduate School of Medicine, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O9 O9
Cancer immunotherapy; integration of T cell biology with nanogel- and
vector-technology in translational research
Hiroshi Shiku
Dept. of Cancer Vaccine and Immuno-Gene Therapy, Mie University
Graduate School of Medicine, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O9 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Disuse osteoporosis, which occurs commonly in prolonged bed rest and
immobilization, is becoming a major problem in modern societies;
however, the molecular mechanisms underlying unloading-driven bone
loss have not been fully elucidated. Bone adjusts its shape and strength
against mechanical stress. Osteocytes are the most abundant cells in
bone and comprise the communication system through the processes
and canaliculi throughout bone. The osteocyte network is considered to
be an ideal mechanosensor and mechanotransduction system. We found
that overexpression of BCL2 in osteoblasts reduces the number of
osteocyte processes, probably due to the function of Bcl2 that modulates
cytoskeletal reorganization, and induces the apoptosis of osteocytes, in
which the transgene expression was reduced, presumably caused by an
insufficient supply of oxygen, nutrients, and survival factors due to the
reduced osteocyte processes. Our BCL2 transgenic mouse with
accumulated dead osteocytes is a useful model to analyze the function of
osteocytes, because a repair process, which replaces dead osteocytes with
new osteocytes by bone resorption and formation, was not evident in the
mice irrespective of the massive accumulation of dead osteocytes Aim: To evaluate the effects of anti-Fas mAb on HA synoviocytes and its
capacity of inducing apoptosis analysing caspase 3 activity. Methods: HA synoviocytes were incubated with IgM 1000 ng/ml (control),
TNFalpha 10 ng/ml, FGF 10 ng/ml, CH11 100 ng/ml (positive control of
apoptosis) with or without anti-Fas mAb at different concentrations (from
0,1 to 1000 ng/ml) for 24 h. RA and healthy synoviocytes were used as
controls. To measure cell proliferation/citotoxicity, the WST-1 assay has
been performed. Caspase 3 activity has been evaluated with ELISA kit and
western blot. Results: Anti-Fas mAb induced a citotoxic effect in HA (p < 0,001 for any
dose), healthy (p < 0,001 at 100 and 1000 ng/ml) and RA synoviocytes (p <
0,05 for any dose) reaching a maximum effect at 1000 ng/ml. After
stimulation with anti-Fas mAb combined with TNFalpha, there was a
citotoxic effect on healthy (p < 0,001 at 10, 100, 1000 ng/ml anti-Fas mAb),
RA (p < 0,001 for any dose) and HA synoviocytes (p < 0,005 at 1, 10, 100
and 1000 ng/ml anti-Fas mAb). After stimulation with anti-Fas mAb
combined with FGF, there was a citotoxic effect on healthy, RA and HA
synoviocytes (p < 0,001 for any dose). Anti-Fas IgM monoclonal antibody (anti-Fas mAb) effect on
haemophilic arthropathy (HA) synoviocytes
1*
1
1 Serena Guiducci1*, Eloisa Romano1, Claudia Ceccarelli1, Daniela Melchiorre1,
Mirko Manetti2, Anna F Milia2, Lidia Ibba Manneshi2, Kusuki Nishioka3,
Marco Matucci Cerinic1 1Dept Medicine, Division of Rheumatology, University of Florence, Florence,
Italy; 2Dept Anatomy, Histology and Forensic Medicine, University of
Florence, Florence, Italy; 3Dept Rheumatology, Institute of Medical Science,
Tokyo Medical University, Tokyo, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O11 Intrinsic weakness of insufficiency in number of cancer specific T cells in
hosts, prompted us to develop adoptive T cell therapy withlymphocytes
engineered to possess cancer specificity. For this purpose, we developed
novel retroviral vectors to highly express exogenously transduced cancer
specific T cell receptor (TCR), yet suppressing expression of endogenous
polyclonal TCR. This approach allowed us to prepare T cells with finer
specificity of expressed TCR. In addition, use of RetroNectin®, a recombinant
fragment of fibronectin opened a way to ex vivo prepare T cells of sufficient
quantity and good quality for clinical use. Background: Haemophilic arthropathy (HA), which shares some clinical
and biological injury characteristics with rheumatoid arthritis (RA), is
characterized by chronic proliferative synovitis and cartilage destruction. Anti-Fas mAb specifically targets the Fas molecule, which is expressed
and activated on the cell surface of inflammatory synovial cells and plays
a key role for induction of apoptosis. Caspases are the final executioners
of apoptosis and their activation requires proteolytic processing of
inactive zymogen into activated fragments. Page 5 of 54 Page 5 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Bone 2012, 50:409-419. The interaction between the immune and skeletal systems has long been
acknowledged, but molecular mechanisms linking the two systems have
not been demonstrated until recently. Investigation into autoimmune
arthritis as well as the various bone phenotypes found in mice deficient in
immunomodulatory molecules has highlighted the importance of the
dynamic interplay between the two systems and brought about a rapid
evolution of the field of osteoimmunology [1]. In bone loss in autoimmune
arthritis, IL-17-producing helper T (TH17) cells play a major role by inducing
RANKL [2]. Maintenance and mobilization of hematopoietic cells are
regulated by bone cells. In addition to cellular interactions via cytokines,
the immune and skeletal systems share various molecules, including
transcription factors, signaling molecules and membrane receptors. RANKL
stimulates osteoclastogenesis through NFATc1 in cooperation with
immunoglobulin-like receptors. Here I will discuss emerging topics in
osteoimmunology including the mechanisms underlying bone cell
communication: osteocyte RANKL [3] and inhibition of bone formation by
osteoclast Sema4D [4]. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Caspase 3 levels were increased in
HA synoviocytes after anti-Fas mAb treatment in a dose-dependent
manner, even after co-stimulation with TNFalpha (p < 0,001 for
any stimulus). CH11 induced an increase of caspase 3 levels in HA
synoviocytes more than RA synoviocytes. Western blot showed that HA
synoviocytes had higher levels of activated caspase 3 compared to
RA synoviocytes after stimulation with Anti-Fas mAb, CH11 and co-
stimulation with TNFalpha. y
We searched for the molecules responsible for disuse osteoporosis using
BCL2 transgenic mice. Pyruvate dehydrogenase kinase isozymes (Pdk1,
Pdk2, Pdk3, and Pdk4) are negative regulators of pyruvate dehydrogenase
complex (PDC), which converts pyruvate to acetyl-CoA in the
mitochondria, linking glycolysis to the energetic and anabolic functions of
the tricarboxylic acid (TCA) cycle. Pdk4 was upregulated in femurs and
tibiae of wild-type mice but not of BCL2 transgenic mice after tail
suspension. Bone in Pdk4-/- mice developed normally and was maintained. At unloading, however, bone mass was reduced due to enhanced
osteoclastogenesis and Rankl expression in wild-type mice but not in
Pdk4-/- mice. Osteoclast differentiation of Pdk4-/- bone marrow-derived
monocyte/macrophage lineage cells (BMMs) in the presence of M-CSF and
RANKL was suppressed, and osteoclastogenesis was impaired in the
coculture of wild-type BMMs and Pdk4-/- osteoblasts, in which Rankl
expression and promoter activity were reduced. Further, introduction of
Pdk4 into Pdk4-/- BMMs and osteoblasts enhanced osteoclastogenesis and
Rankl expression and activated Rankl promoter. These findings indicate
that upregulation of Pdk4 expression in osteoblasts and bone marrow cells
after unloading is, at least in part, responsible for the enhancement of
osteoclastogenesis and bone resorption after unloading [1]. R f Conclusion: Anti-Fas mAb has a dose-dependent citotoxic effect on HA
synoviocytes, even when associated with TNFalpha and FGF. Anti-Fas mAb is
effective in increasing caspase 3 levels in HA synoviocytes in a dose-
dependent manner. HA synoviocytes show higher levels of activated
caspase 3 compared to RA synoviocytes. Our results suggest that anti-Fas
IgM mAb may favour the induction of apoptosis in HA synoviocytes. O12
Overview of osteoimmunology: What’s happened? And what’s
going on? Hiroshi Takayanagi
Tokyo Medical and Dental University, ERATO, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O12 1. Wang Y, Liu W, Masuyama R, Fukuyama R, Ito M, Zhang Q, Komori H,
Murakami T, Moriishi T, Miyazaki T, Kitazawa R, Yoshida CA, Kawai Y, Izumi S,
Komori T: Pyruvate dehydrogenase kinase 4 induces bone loss at
unloading by promoting osteoclastogenesis. O13
Regulation of bone mass at unloaded condition by osteocyte network
Toshihisa Komori
Department of Cell Biology, Unit of Basic Medical Sciences, Nagasaki
University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki
852-8588, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O13 O14 O14
Assessment of histological alterations in cartilage and extracellular
matrix driven by collagen-induced arthritis in Macaca fascicularis
Norio Amizuka1*, Hiromi Hongo1, Muneteru Sasaki1, Tomoka Hasegawa1,
Paulo Henrique Luiz de Freitas2, Hiroshi Mori3, Minqi Li1
1Dept of Develop Biol of Hard Tissue, Hokkaido University, Sapporo, Japan;
2Dept of Oral/Maxillofacial Surg, Dr Mário Gatti Municipal Hospital, Campinas,
Brazil; 3Pharmacological Evaluation Section, Ono Pharmaceutical Co Ltd,
Osaka, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O14 Assessment of histological alterations in cartilage and extracellular
matrix driven by collagen-induced arthritis in Macaca fascicularis
Norio Amizuka1*, Hiromi Hongo1, Muneteru Sasaki1, Tomoka Hasegawa1,
Paulo Henrique Luiz de Freitas2, Hiroshi Mori3, Minqi Li1
1Dept of Develop Biol of Hard Tissue, Hokkaido University, Sapporo, Japan;
2Dept of Oral/Maxillofacial Surg, Dr Mário Gatti Municipal Hospital, Campinas,
Brazil; 3Pharmacological Evaluation Section, Ono Pharmaceutical Co Ltd,
Osaka, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O14 Arthritis Research & Therapy 2012, 14(Suppl 1):O14 Background: Arthritis is characterized by progressive cartilage erosion,
inflammation of adjoining soft tissues and collapse of subchondral bone
due to enhanced osteoclastic resorption. Human joints are complex
structures formed by synovial tissues, articular cartilage and subchondral
bone tissue. Believing on the similarities of normal joints in humans and
monkeys, we have employed a model of collagen-induced arthritis in
Macaca fascicularis (or crab-eating monkey) in an attempt to evaluate the
histological alterations caused by such condition in the extracellular matrix
of the articular cartilage. Skeletal involvement in the pathogenesis and outcomes of rheumatoid
arthritis and osteoarthritis
Jean-Pierre Pelletier Bone remodeling is a frequently observed phenomenon in musculoskeletal
diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA). The level
of imbalance between bone resorption/deposition is responsible for the
morphological changes osteopenia/bone erosion/osteosclerosis observed in
these arthritic conditions. Background: Active rheumatoid arthritis (RA) is characterized by
continuous progression of the inflammatory process, eventually affecting
the majority of joints. Thus far, molecular and cellular pathways of disease
progression are largely unknown. One of the key players in this destructive
scenario are synovial fibroblasts (SF) which actively attach to, invade into
and degrade articular cartilage. As RASF are able to migrate in vitro, the
current series of experiments were designed to evaluate the potential of
RASF to spread the disease in vivo in the SCID mouse model of RA. In RA, increased osteoclastic activity is responsible for the development of
focal osteopenia/erosion and systemic osteoporosis. The increased
osteoclast activity in RA has been demonstrated to be linked to a
dysregulation of pathways including cell-cell interactions, cytokines, and the
receptor activator of nuclear factor B (RANK)/RANK ligand (RANKL) system. Recent studies have shown that joint erosion in RA is linked to a decrease in
long-term physical function. Methods: Healthy human cartilage was co-implanted subcutaneously into
SCID mice together with RASF. At the contralateral flank, simulating an
unaffected joint, cartilage was implanted without cells. To analyze the
route of migration of RASF, the cells were injected subcutaneously,
intraperitoneally or intravenously before or after implantation of cartilage. In addition, whole RA synovium and normal human cartilage were
implanted separately in order to analyze the effects of matrix and other
cells on the migratory behavior of RASF. To evaluate potential influences of
wound healing, either the primary RASF-containing implant or the
contralateral implant without RASF, respectively, was inserted first,
followed by implantation of the corresponding other implant after 14 days. After 60 days, implants, organs and blood were removed and analyzed. For Under OA conditions, the subchondral bone is the site of numerous dynamic
morphological changes. These changes are associated with a number of
local abnormal biochemical pathways related to the altered metabolism of
osteoblasts and osteoclasts. At the early stages of the disease process,
increased bone loss and resorption is observed with subchondral bone
associated with local production of catabolic factors including cathepsin K
and MMP-13. Moreover, OA osteoblasts present an abnormal phenotype
resulting in increased production of growth hormones and catabolic factors. References References
1. Nat Rev Immunol. 2007, 7:292-304. 2. Nat Rev Rheumatol. 2009, 5:667-76. 3. Nat Med. 2011, 17:1231-34. 4. Nat Med. 2011,17:1473-80. 1. Nat Rev Immunol. 2007, 7:292-304. 2. Nat Rev Rheumatol. 2009, 5:667-76. 3. Nat Med. 2011, 17:1231-34. 4. Nat Med. 2011,17:1473-80. O13 Materials and methods: Intermediate phalangeal proximal joints of six
Macaca fascicularis suffering from collagen-induced arthritis were extracted
and fixed with 4% paraformaldehyde solution. Samples were also taken
from disease-free animals as controls. Tissues were embedded in paraffin or
epoxy resin for histochemical and ultrastructural observations. Paraffin
sections were used for alkaline phosphatase (ALP), tartrate-resistant acid Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Page 6 of 54 Figure 1(abstract O14) Figure 1(abstract O14) phosphatase (TRAP), cathepsin K, MMP-1, type II collagen, CTX-II (fragmented
type II collagen) and fibronectin staining assessments. the detection of human cells, immunohisto- and -cytochemistry were
performed with species-specific antibodies. Results: Control monkeys showed faint immunoreactivity against
cathepsin K and MMP-1 in cells covering the articular cartilage and
synovial tissues, indicating physiological levels of collagenous degradation. In arthritic animals, more intense cathepsin K and MMP-1 staining was
observed in similar locations. ALP-positive osteoblasts and TRAP-reactive
osteoclasts were abundant at the subchondral bone in arthritic samples,
while control ones depicted fewer osteoclasts and weakly-stained ALP-
positive osteoblasts, suggesting stimulated bone turnover in the arthritic
group. Interestingly, a thick cell layer covered the articular cartilage with
arthritis, and cellular debris overlaid this thick cell layer; nonetheless,
articular chondrocytes seemed intact (Figure 1). In arthritic joints, the
synovial tissues displayed cellular debris in abundance. CTX-II was seen in
the superficial layer of the articular cartilage in arthritic samples, but it was
virtually absent in the control group. Fibronectin also accumulated on the
surface of the arthritic cartilage. Results: RASF not only invaded and degraded the co-implanted cartilage,
they also migrated to and invaded into the contralateral cell free
implanted cartilage. Injection of RASF led to a strong destruction of the
implanted cartilage, particularly after subcutaneous and intravenous
application. Interestingly, implantation of whole synovial tissue also
resulted in migration of RASF to the contralateral cartilage in one third of
the animals. With regard to the route of migration, few RASF could be
detected in spleen, heart and lung, mainly located in vessels, most likely
resulting from an active movement to the target cartilage via the
vasculature. References With respect to functional aspects, growth factors and
adhesion molecules appear to influence significantly the migratory
behavior of the synovial fibroblasts. Conclusions: The results support the hypothesis that the clinically
characteristic phenomenon of inflammatory spreading from joint to joint
is mediated, at least in part, by a transmigration of activated RASF,
regulated by growth factors and adhesion molecules. Conclusion: Based on the evidence provided, it is possible that matrix
degradation starts not from the adjacent subchondral bone, but from the
most superficial region of the arthritic cartilage. Acknowledgements: Supported by a grant of the German Research
Foundation (DFG). O16 O15
Evidence for synovial fibroblasts spreading rheumatoid arthritis
Ulf Müller-Ladner1*, Stefanie Lefèvre1, Birgit Zimmermann1, Ingo H Tarner1,
Robert Dinser1, Thomas Pap2, Steffen Gay3, Elena Neumann1
1Dept Internal Medicine and Rheumatology, JLU Giessen, Kerckhoff-Clinic,
Bad Nauheim, 61231 Germany; 2Div Mol Med of Musculoskeletal Tissue,
University Hospital Münster, Germany; 3Ctr Exp Rheumatology, Zürich Center
for Integrative Human Physiology, USZ, Zürich, 8006 Switzerland
Arthritis Research & Therapy 2012, 14(Suppl 1):O15 O16
Skeletal involvement in the pathogenesis and outcomes of rheumatoid
arthritis and osteoarthritis
Jean-Pierre Pelletier
University of Montreal and University of Montreal Hospital Centre (CHUM),
Montreal, Quebec, Canada
Arthritis Research & Therapy 2012, 14(Suppl 1):O16 O16
Skeletal involvement in the pathogenesis and outcomes of rheumatoid
arthritis and osteoarthritis
Jean-Pierre Pelletier
University of Montreal and University of Montreal Hospital Centre (CHUM),
Montreal, Quebec, Canada
Arthritis Research & Therapy 2012, 14(Suppl 1):O16 Fcg receptor targeting in RA
* 1Department of Experimental Immunology, Institute of Development, Aging
and Cancer, Tohoku University, Sendai 980-8575, Japan; 2Department of
Immunology, Kanazawa Medical University, Ishikawa 920-0293, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O17 p
We found that the expression of C-type lectin receptor (CLR) genes was
augmented in the affected joints of these models using DNA microarrays. Dendritic cell immunoreceptor (DCIR) is one of such CLRs with a
carbohydrate recognition domain in their extracellular carboxy terminus and
an ITIM in its intracellular amino terminus. Because human shared syntenic
locus containing the Dcir gene is linked to several autoimmune diseases
including RA and SLE, we have generated Dcir KO mice to examine the roles
of this gene in the immune system. We found that aged Dcir KO mice
spontaneously developed sialadenitis and enthesitis associated with
elevated serum autoantibodies [2]. DCs were excessively expanded in Dcir
KO mice after aging. Dcir KO mouse-derived bone marrow cells (BMCs)
differentiated into DCs more efficiently than did wild-type BMCs upon
treatment with GM-CSF, owing to enhanced STAT-5 phosphorylation. These
findings indicate that DCIR is crucial for maintaining the homeostasis of the
immune system, suggesting that Dcir is one of novel targets for the
treatment of RA. The activation threshold of cells in the immune system is often tuned by
cell surface molecules. Among these, Fc receptors expressed on various
hematopoietic cells constitute critical elements for activating or down-
modulating immune responses. IgGFc receptors (FcgRs) were originally identified as B cell surface
molecules. For more than 40 years, FcgRs have continued to attract the
interest of many basic researchers and clinicians due to their intriguing
IgG binding ability, which provides a critical link between the humoral
and cellular branches of the immune system. Several activating-type FcgRs, which associate with homodimeric Fc
receptor common g subunits, are crucial for the onset and exacerbation of
inflammatory diseases. In contrast, a unique inhibitory FcgR, FcgRIIB, plays a
critical role in keeping immune cells silent. Murine models for allergic
responses and autoimmune diseases including RA illustrate the
indispensable roles of activating-type FcgRs and the inhibitory FcgRIIB in
the initiation and suppression of inflammation, respectively [1-5]. We have also found that the expression of Muratin1, which encodes
uncharacterized and secreted protein, is specifically up-regulated in affected
joins of both models. Interestingly, the development of collagen-induced
arthritis was markedly exacerbated in Muratin1 KO mice. Skeletal involvement in the pathogenesis and outcomes of rheumatoid
arthritis and osteoarthritis
Jean-Pierre Pelletier In addition, factors such as osteoprotegerin (OPG) and RANKL have been Page 7 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 1.
Nakamura A, Kubo T, Takai T: Fc receptor targeting in the treatment of
allergy, autoimmune diseases and cancer. Adv Exp Med Biol 2008,
640:220-233. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 6. Anthony RM, Kobayashi T, Wermeling F, Ravetch JV: Intravenous
gammaglobulin suppresses inflammation through a novel TH2 pathway. Nature 2011, 475:110-113. found to be expressed and modulated over time in human OA subchondral
bone. Their synthesis varies from being reduced in early OA to being
increased in the late stages of the disease. This finding may explain that in
the early stages of OA, bone remodeling favors resorption and in the more
advanced stages of the disease, bone formation is predominant. Therapeutic targets for rheumatoid arthritis: lessons from animal
models
1 2*
1
1
1 Yoichiro Iwakura1,2*, Shinobu Saijo1, Susumu Nakae1, Noriyuki Fujikado1,
Harumichi Ishigame1, Masanori Murayama1
1Center for Experimental Medicine and Systems Biology, Institute of Medical
Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan; 2Core
Research for Evolutional Science and Technology (CREST), Japan Science and
Technology Agency, Saitama 332-0012, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O18 The paradigms regarding the role of bone lesions in arthritic diseases raise a
number of important questions. A comprehensive understanding of the
factors that contribute to these changes will provide us with better
knowledge of the pathophysiology of the diseases and the role of these
structural alterations in patient symptoms and prognosis, as well as guiding
the development of new therapeutic strategies. We have generated two RA models, human T-cell leukemia virus type I
(HTLV-I) transgenic mice and IL-1 receptor antagonist (Ra)-deficient (KO)
mice, to elucidate the pathogenic mechanisms of the disease. Both
models spontaneously developed arthritis closely resembling that of RA
in humans. We found that TNF-, but not IL-6-, deficiency suppressed
development of arthritis in IL-1Ra KO mice, while IL-6 but not TNF was
involved in the HTLV-I transgenic mouse model [1]. IL-17 was important
in both models. These observations suggest that pathogenic roles of IL-6
and TNF are different and both TNF, IL-6, and IL-17 are good targets for
therapeutics. O17
Fcg receptor targeting in RA
Toshiyuki Takai1*, Akira Nakamura1,2, Akiko Tobinai1, Shota Endo1,
Masanori Inui1
1Department of Experimental Immunology, Institute of Development, Aging
and Cancer, Tohoku University, Sendai 980-8575, Japan; 2Department of
Immunology, Kanazawa Medical University, Ishikawa 920-0293, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O17 O18 Magnetic resonance imaging (MRI) studies in knee OA patients have shown
that the subchondral bone is frequently the site of signal alterations-bone
marrow lesions (BML) - indicative of a great variety of morphological
changes. BML and cartilage loss have been linked in several studies. Moreover, studies have identified, in OA patients, a number of risk factors
for total knee replacement including BMLs. O18
Therapeutic targets for rheumatoid arthritis: lessons from animal
models
Yoichiro Iwakura1,2*, Shinobu Saijo1, Susumu Nakae1, Noriyuki Fujikado1,
Harumichi Ishigame1, Masanori Murayama1
1Center for Experimental Medicine and Systems Biology, Institute of Medical
Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan; 2Core
Research for Evolutional Science and Technology (CREST), Japan Science and
Technology Agency, Saitama 332-0012, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O18 3.
Takai T: Roles of Fc receptors in autoimmunity. Nat Rev Immunol 2002,
2:580-592. 4.
Yuasa T, Kubo S, Yoshino T, Ujike A, Matsumura K, Ono M, Ravetch JV,
Takai T: Deletion of Fcg receptor IIB renders H-2b mice susceptible to
collagen-induced arthritis. J Exp Med 1999, 189:187-194. Fcg receptor targeting in RA
* I would like to
discuss the roles of Muratin-1 in the development of arthritis. The ultimate goals of FcgR research are to accomplish our understanding of
this molecular family and to delineate novel therapeutic strategies toward
the conquest of allergic and autoimmune diseases, infectious diseases,
immunodeficiency, transplantation-associated immune disorders, and
malignant tumors. Although many lines of evidence indicate that a part of
the intravenous Ig (IVIg)-mediated anti-inflammatory effects can be
attributable to the blocking of activating-type FcgRs, recent studies have
pointed out an indispensable role of FcgRIIB in therapeutic benefits of IVIg in
several murine models of inflammatory diseases including RA [6]. In this
session, we will give a brief summary of recent knowledge on antibody
biomedicine including IVIgto you, in light of exploiting FcgRs as potential
therapeutic targets for various inflammatory diseases, along with the
comparison withnon-FcgR-mediated mechanisms of IVIg. 2.
Nakamura A, Nukiwa T, Takai T: Deregulation of peripheral B-cell
development in enhanced severity of collagen-induced arthritis in
FcgammaRIIB-deficient mice. J Autoimmun 2003, 20:227-236. References 1. Iwakura Y, Nakae S, Saijo S, Ishigame H: The roles of IL-17A in
inflammatory immune responses and host defense against pathogens. Immunol Rev 2008, 226:57-79. 1. Iwakura Y, Nakae S, Saijo S, Ishigame H: The roles of IL-17A in
inflammatory immune responses and host defense against pathogens. Immunol Rev 2008, 226:57-79. 2. Fujikado N, Saijo S, Yonezawa T, Shimamori K, Ishii A, Sugai S, Kotaki H,
Sudo K, Nose M, Iwakura Y: Dcir deficiency causes development of
autoimmune diseases in mice due to excess expansion of dendritic
cells. Nat Med 2008, 14:176-180. 2. Fujikado N, Saijo S, Yonezawa T, Shimamori K, Ishii A, Sugai S, Kotaki H,
Sudo K, Nose M, Iwakura Y: Dcir deficiency causes development of
autoimmune diseases in mice due to excess expansion of dendritic
cells. Nat Med 2008, 14:176-180. O21 Regulation of inflammatory immune responses leading to the
development of bone destructive autoimmune disease rheumatoid
arthritis by IL-27
1*
1 2
1
1 2 Regulation of inflammatory immune responses leading to the
development of bone destructive autoimmune disease rheumatoid
arthritis by IL-27
1*
1 2
1
1 2 Takayuki Yoshimoto1*, Mingli Xu1,2, Izuru Mizuguchi1, Yukino Chiba1,2,
Sadahiro Kamiya3, Masanori Matsui4, Shiva Shahrara5, Junichiro Mizuguchi2
1Department of Immunoregulation, Institute of Medical Science, Tokyo
Medical University, Tokyo 160-8402, Japan; 2Department of Immunology,
Tokyo Medical University, Tokyo 160-8402, Japan; 3Departments of Clinical
Sciences, Josai International University, Chiba 283-8555, Japan; 4Department
of Microbiology, Saitama Medical University, Saitama 350-0495, Japan;
5Department of Medicine, Northwestern University Feinberg School of
Medicine, Chicago, IL 60611, USA
Arthritis Research & Therapy 2012, 14(Suppl 1):O21 evaluated by Alizarin red staining. TGF-ß1 levels were determined by ELISA. Results: DKK2 expression and production were elevated in OA Ob
compared to normal whereas DKK1 was similar. Rspo2 expression was
reduced in OA Ob whereas Rspo1 was similar. TGF-ß1mRNA expression and
protein levels were high in OA Ob. TGF-b1 stimulated DKK2 expression and
production in Ob whereas it inhibited Rspo2 expression. cWnt signaling was
reduced in OA compared to normal Ob. This inhibition was due in part to
elevated DKK2 levels and to reduced Rspo-2 levels since correcting DKK2 by
siRNA or the addition of Rspo-2 increased cWnt signaling using the TOPflash
reporter assay. These treatments also increased ß-catenin levels in OA Ob. Mineralization of OA Ob was reduced compared to normal Ob and was also
corrected in part by inhibiting DKK2 or by Rspo2 addition. Both elevated
DKK2 and reduced Rspo2 levels contributed to abnormal expression of bone
markers by OA Ob. IL-27, a member of the IL-6/IL-12 family of cytokines, induces early helper T
(Th)1 differentiation and generation of cytotoxic T cells and IL-10-producing
type 1 regulatory T cells, while it suppresses the production of inflammatory
cytokines and inhibits Th2 and Th17 differentiation [1,2]. The receptor
activator of NF-kB ligand (RANKL), which is expressed by not only
osteoblasts but also activated T cells, plays an important role in bone-
destructive disease rheumatoid arthritis (RA). Recently, IL-17-producing Th17
cells were identified as the exclusive osteoclastogenic T-cell subset. This is
because Th17 cells express RANKL, and that IL-17 not only induces RANKL
expression on osteoblasts, but also increases the production of various
inflammatory molecules. O21 We recently
investigated the mechanistic role of IL-27 in the pathogenesis of CIA and
found that local injection of adenoviral IL-27 transcript into the ankles of CIA
mice attenuates joint inflammation, synovial lining thickness, bone erosion
and leukocyte migration [4]. IL-27 reduced the production of IL-1b and IL-6,
and suppressed Th17 cell differentiation as well as IL-17 downstream target
genes, which leads to decreased IL-17-mediated monocyte recruitment and
angiogenesis possibly through the reduction of neutrophil and monocyte
chemokines. We also elucidated that IL-27 inhibits cell surface expression of
RANKL on naive CD4+ T cells activated by T cell receptor ligation and
secretion of its soluble RANKL as well [5]. The inhibitory effect was mediated
in part by STAT3 but not by STAT1 or IL-10. In differentiated Th17 cells, IL-27
much less but significantly inhibited the RANKL expression after
re-stimulation. Fas ligand (CD 178) and its receptor Fas (CD 95) are members of the TNF
superfamily of ligands and receptors involved in the activation of apoptosis. Our research group demonstrated that Fas and Fas ligand were expressed
during osteoblast and osteoclast differentiation, and their expression may
be modified by various cytokines. The lack of functional Fas signaling in
murine models leads to altered endochondral ossification, increase of the
bone mass in adult mice, and resistance to ovariectomy-induced bone loss. We also showed that mice with a Fas gene knockout lose less bone during
antigen-induced arthritis. These changes seem to be, at least in part,
mediated by increased expression of osteoprotegerin (OPG), another
member of the TNF superfamily, which acts as a decoy receptor for receptor
activator for nuclear factor B (RANK) ligand (RANKL). The bone phenotype
of mice lacking Fas signaling may be related to the immunological
disturbance rather than intrinsic bone disorder. To address this question at
molecular level, we performed a set of parabiotic experiments in mice with
non-functional Fas ligand mutation (gld mice). Mice were kept in parabiosis
for 1 to 4 weeks, and for 2 weeks after separation from 4-week parabiosis. We also analyzed OPG levels in the peripheral blood of patients with
autoimmune lymphoproliferative syndrome (ALPS). Joined circulation
between gld and wild-type mice led to increased expression of bone
protective OPG in the wild-type animal, both at the gene and protein level
at 4 weeks of parabiosis. This effect was sustained even after the separation
of parabiotic mice. O21 It was previously reported that IL-27 is detected in
RA synovial membranes and that treatment with IL-27 attenuated
inflammatory responses in collagen-induced arthritis (CIA), one of mouse RA
models. Conclusions: These studies demonstrate that elevated antagonist or
reduced agonist levels of cWnt signalling interfere in normal Ob function
and lead to abnormal mineralization. Since these are secreted soluble
proteins, this could lead to potential new avenues of treatment of OA to
correct their abnormal bone phenotype and mineralization. O20 O20
Understanding the role of Fas-Fas ligand system in bone
Ana Marusic1*, Natasa Kovacic2, Ivan Kresimir Lukic3, Vedran Katavic2,
Danka Grcevic4
1Department of Anatomy and Department of Research in Biomedicine and
Health, University of Split School of Medicine, Split, Croatia; 2Department of
Anatomy, University of Zagreb School of Medicine, Zagreb, Croatia; 3Partek
Inc, Zagreb, Croatia; 4Department of Physiology and Immunology, University
of Zagreb School of Medicine, Zagreb, Croatia
Arthritis Research & Therapy 2012, 14(Suppl 1):O20 Understanding the role of Fas-Fas ligand system in bone
1*
2
3 Understanding the role of Fas-Fas ligand system in bone
Ana Marusic1*, Natasa Kovacic2, Ivan Kresimir Lukic3, Vedran Katavic2,
Danka Grcevic4 1Department of Anatomy and Department of Research in Biomedicine and
Health, University of Split School of Medicine, Split, Croatia; 2Department of
Anatomy, University of Zagreb School of Medicine, Zagreb, Croatia; 3Partek
Inc, Zagreb, Croatia; 4Department of Physiology and Immunology, University
of Zagreb School of Medicine, Zagreb, Croatia
Arthritis Research & Therapy 2012, 14(Suppl 1):O20 1Department of Anatomy and Department of Research in Biomedicine and
Health, University of Split School of Medicine, Split, Croatia; 2Department of
Anatomy, University of Zagreb School of Medicine, Zagreb, Croatia; 3Partek
Inc, Zagreb, Croatia; 4Department of Physiology and Immunology, University
of Zagreb School of Medicine, Zagreb, Croatia
Arthritis Research & Therapy 2012, 14(Suppl 1):O20 We have been investigating the role of IL-27 in the regulation of
inflammatory responses leading to the development of bone destructive
autoimmune disease. We first demonstrated that osteoclastogenesis from
bone marrow cells induced by soluble RANKL is inhibited by IL-27 with
reduced multinucleated cell numbers [3]. Then, other group further clarified
that IL-27 directly acts on osteoclast precursor cells and suppresses RANKL-
mediated osteoclastogenesis through STAT1-dependent inhibition of c-Fos,
leading to amelioration of the inflammatory bone destruction. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 cWnt signaling was
reduced in OA compared to normal Ob. This inhibition was due in part to
elevated DKK2 levels and to reduced Rspo-2 levels since correcting DKK2 by
siRNA or the addition of Rspo-2 increased cWnt signaling using the TOPflash
reporter assay. These treatments also increased ß-catenin levels in OA Ob. Mineralization of OA Ob was reduced compared to normal Ob and was also
corrected in part by inhibiting DKK2 or by Rspo2 addition. Both elevated
DKK2 and reduced Rspo2 levels contributed to abnormal expression of bone
markers by OA Ob. O21 At the same time, double-negative T lymphocytes
transferred from gld into wild-type member of a parabiotic pair rapidly
vanished from the periphery of both gld and control mice in parabiosis. Patients with ALPS had increased OPG mRNA level in peripheral blood
mononuclear cells, as assessed by real-time PCR, in comparison to age- and Taken together, these results suggest that IL-27 regulates inflammatory
immune responses leading to the development of bone destructive
autoimmune disease through multiple mechanisms as described above
(Figure 1), and that IL-27 may be a promising target for therapeutic
intervention to control disease in RA patients. O19 O19 Abnormal osteogenesis in osteoarthritis: gone with the Wnt? Daniel Lajeunesse*, Thomas F Chan, Aline Delalandre, Jean-Pierre Pelletier,
Johanne Martel-Pelletier, Élie Abed
Unité de recherche en Arthrose, CRCHUM, Université de Montréal, Québec,
Canada, H2L 4M1
h
h
h
l 1. Nakamura A, Kubo T, Takai T: Fc receptor targeting in the treatment of
allergy, autoimmune diseases and cancer. Adv Exp Med Biol 2008,
640:220-233. ,
Unité de recherche en Arthrose, CRCHUM, Université de Montréal, Québec,
Canada, H2L 4M1 Unité de recherche en Arthrose, CRCHUM, Université de Montréal, Québec,
Canada, H2L 4M1 2. Nakamura A, Nukiwa T, Takai T: Deregulation of peripheral B-cell
development in enhanced severity of collagen-induced arthritis in
FcgammaRIIB-deficient mice. J Autoimmun 2003, 20:227-236. Arthritis Research & Therapy 2012, 14(Suppl 1):O19 3. Takai T: Roles of Fc receptors in autoimmunity. Nat Rev Immunol 2002,
2:580-592. Background: Clinical and in vitro studies suggest that subchondral bone
sclerosis due to abnormal osteoblast (Ob) functions, is involved in the
progression and/or onset of osteoarthritis (OA). Human OA subchondral Ob
show a differentiated phenotype, however they fail to mineralize normally. The canonical Wnt/b-catenin signaling pathway (cWnt) plays a key role in
osteogenesis by promoting the differentiation and mineralization of Ob. 4. Yuasa T, Kubo S, Yoshino T, Ujike A, Matsumura K, Ono M, Ravetch JV,
Takai T: Deletion of Fcg receptor IIB renders H-2b mice susceptible to
collagen-induced arthritis. J Exp Med 1999, 189:187-194. 5. Takai T, Ono M, Hikida M, Ohmori H, Ravetch JV: Augmented humoral and
anaphylactic responses in FcgRII-deficient mice. Nature 1996, 379:346-349. 5. Takai T, Ono M, Hikida M, Ohmori H, Ravetch JV: Augmented humoral and
anaphylactic responses in FcgRII-deficient mice. Nature 1996, 379:346-349. Page 8 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 sex-matched controls. These findings show that bone and immune changes
are uncoupled during Fas ligand deficiency. Under the assumption that OPG
also acts as a molecular brake in the immune system, downregulation of
OPG in gld mice during parabiosis with wild-type mice could be considered
as a molecular marker of remission. Increased expression of OPG in children
with ALPS leads to the hypothesis that a similar mechanism might be at
play in humans. Dickkopfs (DKKs) are potent antagonists whereas R-spondins (Rspo) are
newly described agonists that play key roles in cWnt signalling. However,
the regulation of DKKs and Rspos in OA Ob remains unknown. g
p
Materials and methods: We prepared primary human subchondral Ob
using the sclerotic medial portion of the tibial plateaus of OA patients
undergoing knee arthroplasty, or from tibial plateaus of normal individuals
at autopsy. DKK1, DKK2, SOST and Rspo-1 and -2 expression and production
were evaluated by qRT-PCR and WB analysis. The regulation of their
expression was determined in response to transforming growth factor-ß1
(TGF-ß1) and as a function of the growth of OA Ob. Selective inhibition was
performed using siRNA techniques. cWnt signaling was evaluated by
measuring target gene expression using the TOPflash Tcf/lef luciferase
reporter assay and intracellular ß-catenin levels by WB. Mineralization was
l
t d b Ali
i
d t i i
TGF ß1 l
l
d t
i
d b ELISA Materials and methods: We prepared primary human subchondral Ob
using the sclerotic medial portion of the tibial plateaus of OA patients
undergoing knee arthroplasty, or from tibial plateaus of normal individuals
at autopsy. DKK1, DKK2, SOST and Rspo-1 and -2 expression and production
were evaluated by qRT-PCR and WB analysis. The regulation of their
expression was determined in response to transforming growth factor-ß1
(TGF-ß1) and as a function of the growth of OA Ob. Selective inhibition was
performed using siRNA techniques. cWnt signaling was evaluated by
measuring target gene expression using the TOPflash Tcf/lef luciferase
reporter assay and intracellular ß-catenin levels by WB. Mineralization was
evaluated by Alizarin red staining. TGF-ß1 levels were determined by ELISA. Results: DKK2 expression and production were elevated in OA Ob
compared to normal whereas DKK1 was similar. Rspo2 expression was
reduced in OA Ob whereas Rspo1 was similar. TGF-ß1mRNA expression and
protein levels were high in OA Ob. TGF-b1 stimulated DKK2 expression and
production in Ob whereas it inhibited Rspo2 expression. 1.
Yoshimoto T, Morishima N, Okumura M, Chiba Y, Onishi K, Mizoguchi I,
Mizuguchi J: Interleukin-27: biological properties and application to
immunotherapy of cancer. Trends Cancer Res 2008, 4:93-103.
2.
Morishima N, Mizoguchi I, Okumura M, Chiba Y, Xu M, Shimizu M,
Matsui M, Mizuguchi J, Yoshimoto T: A pivotal role for interleukin-27 in
CD8+T cell functions and generation of cytotoxic T lymphocytes.
J Biomed Biotechnol 2010, 2010:605483. O23 O23
Synoviolin meets metabolic disorders
Naoko Yagishita1*, Satoko Aratani2, Daisuke Hasegawa1, Yoshihisa Yamano1,
Kusuki Nishioka2, Toshihiro Nakajima2,3
1Institute of Medical Science, St Marianna University School of Medicine,
Kawasaki, Kanagawa 216-8512, Japan; 2Institute of Medical Science, Tokyo
Medical University, Shinjuku-ku, Tokyo 160-8402, Japan; 3Bayside Misato
Medical Center, Kochi 781-0112, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O23 O22
Postnatal Syk deletion in mice clarifies the function of Syk in an anti-
collagen antibody-induced arthritis model
Naoko Ozaki1,2*, Shinobu Suzuki2, Hiromitsu Hara1, Hiroki Yoshida1
1Division of Molecular and Cellular Immunoscience, Department of
Biomolecular Sciences, Faculty of Medicine, Saga University, Nabeshima,
Saga, 849-8501, Japan; 2Department of Molecular & Cellular Biology, Kobe
Pharma Research Institute, Nippon Boehringer Ingelheim Co Ltd, Minatojima-
Minamimachi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O22 References Pickens SR, Volin MV, Mandelin AM II, Matsui M, Yoshimoto T, Shahrara S:
Local expression of IL-27 ameliorates collagen induced arthritis. Arthritis
Rheum in press. 5. Kamiya S, Okumura M, Chiba Y, Fukawa T, Nakamura C, Nimura N,
Mizuguchi J, Wada S, Yoshimoto T: IL-27 suppresses RANKL expression in
CD4+T cells in part through STAT3. Immunol Lett 2011, 138:47-53. 5. Kamiya S, Okumura M, Chiba Y, Fukawa T, Nakamura C, Nimura N,
Mizuguchi J, Wada S, Yoshimoto T: IL-27 suppresses RANKL expression in
CD4+T cells in part through STAT3. Immunol Lett 2011, 138:47-53. References 1. Yoshimoto T, Morishima N, Okumura M, Chiba Y, Onishi K, Mizoguchi I,
Mizuguchi J: Interleukin-27: biological properties and application to
immunotherapy of cancer. Trends Cancer Res 2008, 4:93-103. 2. Morishima N, Mizoguchi I, Okumura M, Chiba Y, Xu M, Shimizu M,
Matsui M, Mizuguchi J, Yoshimoto T: A pivotal role for interleukin-27 in
CD8+T cell functions and generation of cytotoxic T lymphocytes. J Biomed Biotechnol 2010, 2010:605483. 1. Yoshimoto T, Morishima N, Okumura M, Chiba Y, Onishi K, Mizoguchi I,
Mizuguchi J: Interleukin-27: biological properties and application to
immunotherapy of cancer. Trends Cancer Res 2008, 4:93-103. 2. Morishima N, Mizoguchi I, Okumura M, Chiba Y, Xu M, Shimizu M,
Matsui M, Mizuguchi J, Yoshimoto T: A pivotal role for interleukin-27 in
CD8+T cell functions and generation of cytotoxic T lymphocytes. J Biomed Biotechnol 2010, 2010:605483. 1. Yoshimoto T, Morishima N, Okumura M, Chiba Y, Onishi K, Mizoguchi I,
Mizuguchi J: Interleukin-27: biological properties and application to
immunotherapy of cancer. Trends Cancer Res 2008, 4:93-103. 2. Morishima N, Mizoguchi I, Okumura M, Chiba Y, Xu M, Shimizu M,
Matsui M, Mizuguchi J, Yoshimoto T: A pivotal role for interleukin-27 in
CD8+T cell functions and generation of cytotoxic T lymphocytes. J Biomed Biotechnol 2010, 2010:605483. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Page 9 of 54 Figure 1(abstract O21) IL-27 regulates inflammatory immune responses leading to the development of bone destructive autoimmune disease
RA through multiple mechanisms. Figure 1(abstract O21) IL-27 regulates inflammatory immune responses leading to the development of bone destructive autoimmune disease
RA through multiple mechanisms. 3. Kamiya S, Nakamura C, Fukawa T, Ono K, Ohwaki T, Yoshimoto T, Wada S:
Effects of IL-23 and IL-27 on osteoblasts and osteoclasts: inhibitory
effects on osteoclast differentiation. J Bone Miner Metab 2007, 25:277-285. arthritis development in CAIA, as demonstrated by using muMT mice which
lack B cells. On the other hand, Syk-deficient macrophages produced less
MCP-1 and IL-6 than Syk-sufficient cells after FcR ligation, which can account
for the absence of a pronounced accumulation of neutrophils and
macrophages in the joints of iSyk KO mice. Our results demonstrate that Syk
in macrophages is likely a key player in antibody-induced arthritis,
mediating the release of pro-inflammatory cytokines and chemokines after
macrophages bind anti-collagen antibody, and indicate that Syk is a
promising target for arthritis therapy. ,
4. O24 O24
IL-17, synoviolin and rheumatoid arthritis chronicity
Pierre Miossec
Department of Immunology and Rheumatology, Hôpital Edouard Herriot,
University of Lyon, France
Arthritis Research & Therapy 2012, 14(Suppl 1):O24 As for the treatment of RA, biological agents are approved for clinical use,
and these drugs have dramatically changed the treatment of RA during the
past decade. However, in some cases patients fail to respond to the biologic
treatment or adverse effects develop such as; an increased risk of infections. It was reported that elevated Synoviolin levels were identified in circulating
monocytes and were associated with nonresponse to infliximab treatment. Moreover, these agents are associated with high costs and discomfort
arising from subcutaneous or intravenous administration. Thus, there is a
clear need for the development of cheaper, orally administrated therapies
with fewer side effects. Then, we successfully discovered Synoviolin
inhibitors. We are now proceeding with the optimization of small
compounds, and we hope our research will lead to the development of a
new therapy for RA and serve as an example of the therapeutic benefit of
developing E3 ligase inhibitors. Background: The use of cytokine inhibitors has been a major progress in
the treatment of chronic inflammation. However, not all patients respond
and response will be often lost when treatment is stopped. These clinical
aspects indicate that other cytokines might be involved and we focus here
on the role of IL-17. In addition, the chronic nature of joint inflammation
may contribute to reduced response and enhanced chronicity. We had
previously observed that patients not responding well to TNF inhibition had
higher blood expression of synoviolin, an E3 ubiquitin ligase previously
shown to be implicated in synovial hyperplasia in human and mouse
rheumatoid arthritis (RA). Therefore we studied the capacity of IL-17 to
regulate synoviolin in human RA synoviocytes and in chronic reactivated
streptococcal cell wall (SCW)-induced arthritis. In addition, to clarify the physiological function of Synoviolin in adult, we
recently generate synoviolin conditional knockout mice using tamoxifen
inducible Cre transgenic mice under CAG promoter. In today’s session, I’d
like to introduce the preliminary data of synoviolin conditional knockout
mice. Materials and methods: Chronic reactivated SCW-induced arthritis was
examined in IL-17R deficient and wild-type mice. Synoviolin expression was
analysed by real-time RT-PCR, Western Blot or immunostaining in RA
synoviocytes and tissue, and p53 assessed by Western Blot. Postnatal Syk deletion in mice clarifies the function of Syk in an anti-
collagen antibody-induced arthritis model
1 2*
2
1
1 PLoS One 2010, 5:e13590. 4. Hasegawa D, Fujii R, Yagishita N, et al: E3 ubiquitin ligase synoviolin is
involved in liver fibrogenesis. PLoS One 2010, 5:e13590. its biological functions [3]. Therefore Synoviolin regulates, not only apoptosis
in response to ER stress, but also a p53-dependent apoptotic pathway. These studies indicate that Synoviolin is involved in overgrowth of synovial
cells through its anti-apoptotic effects. Further analysis showed that
Synoviolin is also involved in fibrosis among the multiple processes [4]. Therefore, it was suggested that Synoviolin is thought to be a candidate for
pathogenic factor for arthropathy through its involvement of multiple
processes. Postnatal Syk deletion in mice clarifies the function of Syk in an anti-
collagen antibody-induced arthritis model
1 2*
2
1
1 Naoko Ozaki1,2*, Shinobu Suzuki2, Hiromitsu Hara1, Hiroki Yoshida1
1Division of Molecular and Cellular Immunoscience, Department of
Biomolecular Sciences, Faculty of Medicine, Saga University, Nabeshima,
Saga, 849-8501, Japan; 2Department of Molecular & Cellular Biology, Kobe
Pharma Research Institute, Nippon Boehringer Ingelheim Co Ltd, Minatojima-
Minamimachi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O22 Rheumatoid arthritis (RA) is consists of multiple processes such as chronic
inflammation, overgrowth of synovial cells, joint destruction and fibrosis. To
clarify the mechanism of outgrowth of synovial cells, we carried out
immunoscreening using anti-rheumatoid synovial cell antibody, and cloned
‘Synoviolin’ [1]. Synoviolin is endoplasmic reticulum (ER)-resident E3
ubiquitin ligases, and is involved in ER-associated degradation (ERAD). Synoviolin is highly expressed in synoviocytes of patients with RA. Overexpression of synoviolin in transgenic mice leads to advanced
arthropathy caused by reduced apoptosis of synoviocytes [1]. We postulate
that the hyperactivation of the ERAD pathway by overexpression of
synoviolin results in prevention of ER-stress-induced apoptosis leading to
synovial hyperplasia [2]. In addition, Synoviolin ubiquitinates and sequesters
the tumor suppressor p53 in the cytoplasm, thereby negatively regulating Spleen tyrosine kinase (Syk) is a cytoplasmic protein expressed mainly in
immune cells including macrophages and neutrophils and is associated with
receptors containing an immunoreceptor tyrosine-based activation motif
(ITAM), such as Fcg receptors. As Syk-mediated signaling plays an important
role in activation of immune responses, to investigate whether specific
interruption of Syk-mediated signaling can affect the development of
rheumatoid arthritis (RA), we used tamoxifen-induced conditional Syk-KO
mice (iSyk KO) to evaluate the importance of Syk on disease development. Using a collagen antibody-induced arthritis model (CAIA), iSyk KO mice
showed significantly attenuated disease severity compared to Syk non-
deleted mice (Figure 1). Although iSyk KO mice contained reduced B cell
numbers after deletion of Syk in adulthood, B cells are not required for Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Page 10 of 54 Figure 1(abstract O22) Arthritis development in iSyk KO mice. Arthritis was induced by i.p. administration of anti-collagen Ab followed by LPS. Arthritis score was monitored. *; P < 0.001, **; P < 0.01. Figure 1(abstract O22) Arthritis development in iSyk KO mice. Arthritis was induced by i.p. administration of anti-collagen Ab followed by LPS. Arthritis score was monitored. *; P < 0.001, **; P < 0.01. 4. Hasegawa D, Fujii R, Yagishita N, et al: E3 ubiquitin ligase synoviolin is
involved in liver fibrogenesis. O24 Apoptosis was
detected by annexin V/ propidium iodide staining, SS DNA apoptosis ELISA
kit or TUNEL staining and proliferation by PCNA staining. IL-17 receptor A
(IL-17RA), IL-17 receptor C (IL-17-RC) or synoviolin inhibition were achieved
by small interfering RNA (siRNA) or neutralizing antibodies. 2.
Yagishita N, Yamasaki S, Nishioka K, et al: Synoviolin, protein folding and
the maintenance of joint homeostasis. Nat Clin Pract Rheumatol 2008,
4:91-97. 3.
Yamasaki S, Yagishita N, Sasaki T, et al: Cytoplasmic destruction of p53 by
the endoplasmic reticulum-resident ubiquitin ligase ‘Synoviolin’. EMBO J
2007, 26:113-122. O27 O27
Directed induction of chondrogenic cells from mouse dermal fibroblast
culture
Noriyuki Tsumaki
Center for iPS Cell Research and Application, Kyoto University, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O27 O27
Directed induction of chondrogenic cells from mouse dermal fibroblast
culture
Noriyuki Tsumaki
Center for iPS Cell Research and Application, Kyoto University, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O27 O27
Directed induction of chondrogenic cells from mouse dermal fibroblast
culture
Noriyuki Tsumaki
Center for iPS Cell Research and Application, Kyoto University, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O27 Noriyuki Tsumaki 1. Rodriguez A, et al: Genome Res 2004, 14:1902-10. 2. Tuddenham L, et al: FEBS Lett 2006, 580:4214-7. 3. Tardif G, et al: BMC Musculoskelet Disord 2009, 10:148-58. 4. Miyaki S, et al: Arthritis Rheum 2009, 60:2723-30. 5. Shimizu E, et al: J Biol Chem 2010, 285:9616-26. 6. Miyaki S, et al: Genes Dev 2010, 24:1173-85. 7. Glasson SS, et al: Nature 2005, 434:644-8. 8. Pais H, et al: RNA 2010, 16:489-94. 9. Valdes AM, et al: Arthritis Rheum 2010, 62:2347-52. Repair of cartilage injury with hyaline cartilage has been a challenging clinical
problem. Articular cartilage damage sometimes heals with fibrocartilage,
which is different from hyaline cartilage. Fibrocartilage is a type of scar tissue
that expresses types I and II collagen. In contrast, hyaline cartilage does not
express type I collagen. When aiming to induce hyaline chondrogenic cells
directly from dermal fibroblasts, in addition to activation of cartilage-specific
matrix genes, elimination of expression of type I collagen is needed for
generation of hyaline cartilage. Otherwise, the presence of type I collagen
impairs cartilage extracellular matrix architecture, which leads to formation of
fibrocartilage. The generation of induced pluripotent stem cells has provided
a tool for reprogramming dermal fibroblasts to an undifferentiated state by
ectopic expression of reprogramming factors. We found that retroviral
expression of two reprogramming factors (c-Myc and Klf4) and one
chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly
from adult dermal fibroblast cultures. Induced cells expressed marker genes
for chondrocytes but not fibroblasts; the promoters of type I collagen genes
were extensively methylated. Transduction of c-Myc, Klf4, and SOX9
produced two types of cells: chondrogenically reprogrammed cells and
partially reprogrammed intermediate cells. Chondrogenically reprogrammed
cells generated stable homogenous hyaline cartilage-like tissue without
tumor formation when subcutaneously injected into nude mice. Hyaline
cartilage-like tissue expressed type II collagen but not type I collagen. References 1. Amano T, Yamasaki S, Yagishita N, et al: Synoviolin/Hrd1, an E3 ubiquitin
ligase, as a novel pathogenic factor for arthropathy. Genes Dev 2003,
17:2436-2449. 2. Yagishita N, Yamasaki S, Nishioka K, et al: Synoviolin, protein folding and
the maintenance of joint homeostasis. Nat Clin Pract Rheumatol 2008,
4:91-97. Results: IL-17 induced sustained synoviolin expression in RA synoviocytes. Sodium nitroprusside (SNP)-induced RA synoviocyte apoptosis was
associated with reduced synoviolin expression and was rescued by IL-17
treatment with a corresponding increase in synoviolin expression. IL-17RC or
IL-17RA RNA interference increased SNP-induced apoptosis, and decreased
IL-17-induced synoviolin. IL-17 rescued RA synoviocytes from apoptosis 3. Yamasaki S, Yagishita N, Sasaki T, et al: Cytoplasmic destruction of p53 by
the endoplasmic reticulum-resident ubiquitin ligase ‘Synoviolin’. EMBO J
2007, 26:113-122. Page 11 of 54 Page 11 of 54 Page 11 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
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http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 TRAP-positive osteoclasts and ALP-positive osteoblasts were
observed in BMP-2-disks preceding the onset of calcification for one week. OPG and soluble RANK inhibited BMP-2-induced osteoclast formation but
not the appearance of ALP-positive cells in OPG-deficient mice. We then
examined how osteoblasts are involved in osteoclastogenesis other than
RANKL expression, using RANKL-deficient mice. RANKL-deficient mice
showed severe osteopetrosis due to loss of osteoclasts. Injection of RANKL
into RANKL-deficient mice induced many osteoclasts in bone but not soft
tissues [2]. These results suggest that osteoblasts determine the place of
osteoclastogenesis from haemopoietic stem cells in bone. We next
explored roles of osteoclasts in ectopic bone formation induced by BMP
using op/op and c-fos-deficient osteopetrotic mice. The ectopic bones
formed in op/op mice showed extremely rough surfaces, whereas those in
wild-type mice showed smooth ones. Bone mineral density of BMP-
induced ectopic bone in op/op mice was about 2-times higher than that in
wild-type mice. TRAP-positive osteoclasts exhibit in outer of the ectopic
bone in the wild-type mice. In op/op mice, although osteoclasts strongly
exhibit in inside of the BMP-induced ectopic bone, TRAP-positive
osteoclasts did not exhibit in outer of the BMP-induced ectopic bone. Furthermore, the accentuation of the BMP-induced ectopic bone formation
did not exist in osteopetrotic c-Fos-deficient mice. In c-Fos-deficient mice,
which are completely osteoclasts deficiency, the accentuation of the BMP-
induced ectopic bone formation did not exist. Furthermore, there is no
RANK-positive osteoclast progenitors in bone derived from c-Fos-deficient
mice. These results suggest that RANK-positive osteoclast progenitors are
positively regulate the signal of bone formation. In summary, osteoclastic
bone resorption directly activates osteoblast function and osteoclasts are
involved in normal bone morphogenesis. R f induced by synoviolin knockdown. IL-17 and TNF had additive effects on
synoviolin expression and protection against apoptosis induced by
synoviolin knowndown. In IL-17R deficient mice, a decrease in arthritis
severity was characterized by increased synovial apoptosis, reduced
proliferation and a marked reduction in synoviolin expression. A distinct
absence of synoviolin expressing germinal centres in IL-17R deficient mice
contrasted with synoviolin positive B cells and Th17 cells in synovial
germinal centre-like structures. g
Conclusions: IL-17 induction of synoviolin may contribute in part to RA
chronicity by prolonging the survival of RA synoviocytes and immune
cells in germinal centre reactions. These results extend the role of IL-17
to synovial hyperplasia. O25 In osteoarthritis (OA), despite major progress regarding the identification
and roles of catabolic mediators, further knowledge about factors regulating
their expression is needed. In this line of thought, one recently identified
class of molecules, the microRNA (miRNA), has been found to add another
level of regulation to gene expression by down-regulating its target genes. miRNAs are 20-23 nucleotides (nt)-long single-stranded non-coding RNA
molecules that act as transcriptional repressors by binding to the 3’
untranslated region (UTR) of the target messenger RNA. Recently, miR-140
has emerged as being implicated in OA by modulating genes involved in
the pathogenesis of this disease. The miRNA-140 gene is located between
exons 16 and 17 in one intron of the WW domain containing the E3
ubiquitin protein ligase 2 (WWP2) gene [1]. The miR-140, originally found in
cartilage [2], has recently been linked more specifically to the OA process
[3,4]. The miRNA-140 decreases the expression of some genes known to
play detrimental roles in OA cartilage. Those genes include histone
deacetylase 4 (HDAC4) [2,5], ADAMTS-5 [6,7], Smad3 [8,9], and IGFBP5 [3]. On human chondrocytes, the expression level of miR-140 was found to be
significantly decreased in OA compared to normal [3,4], thus favouring an
increased expression of its target genes and consequently a role in OA
progression. Interestingly, further investigation of the transcriptional
regulation of miR-140 showed that in human OA chondrocytes miR-140 also
has a WWP2-independent regulation. This occurs through the miR-140
intronic regulatory sequence in which the transcription factor NFAT3 acts
directly and NFAT5 indirectly through the growth factor TGF-b1/Smad3. These data are of importance as they can provide a new basis for the
rationalization of a therapeutic strategy for this disease. References 1. Mizoguchi T, et al: Identification of cell cycle-arrested quiescent
osteoclast precursors in vivo. J Cell Biol 2009, 184:541-554. 2. Yamamoto Y, et al: Osteoblasts provide a suitable microenvironment for
the action of receptor activator of nuclear factor-kappaB ligand. Endocrinology 2006, 147:3366-3374. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 osteoblasts, is a soluble decoy receptor for RANKL. Deficiency of OPG in
mice induces osteoporosis caused enhanced bone resorption. Elevated
osteoblastic activity was suppressed by bisphosphonate administration in
OPG-deficient mice. These results suggest that bone formation is
accurately coupled with bone resorption. Collagen sponge disks containing
BMP-2 were implanted into the dorsal muscle pouches in OPG-deficient
mice. TRAP-positive osteoclasts and ALP-positive osteoblasts were
observed in BMP-2-disks preceding the onset of calcification for one week. OPG and soluble RANK inhibited BMP-2-induced osteoclast formation but
not the appearance of ALP-positive cells in OPG-deficient mice. We then
examined how osteoblasts are involved in osteoclastogenesis other than
RANKL expression, using RANKL-deficient mice. RANKL-deficient mice
showed severe osteopetrosis due to loss of osteoclasts. Injection of RANKL
into RANKL-deficient mice induced many osteoclasts in bone but not soft
tissues [2]. These results suggest that osteoblasts determine the place of
osteoclastogenesis from haemopoietic stem cells in bone. We next
explored roles of osteoclasts in ectopic bone formation induced by BMP
using op/op and c-fos-deficient osteopetrotic mice. The ectopic bones
formed in op/op mice showed extremely rough surfaces, whereas those in
wild-type mice showed smooth ones. Bone mineral density of BMP-
induced ectopic bone in op/op mice was about 2-times higher than that in
wild-type mice. TRAP-positive osteoclasts exhibit in outer of the ectopic
bone in the wild-type mice. In op/op mice, although osteoclasts strongly
exhibit in inside of the BMP-induced ectopic bone, TRAP-positive
osteoclasts did not exhibit in outer of the BMP-induced ectopic bone. Furthermore, the accentuation of the BMP-induced ectopic bone formation
did not exist in osteopetrotic c-Fos-deficient mice. In c-Fos-deficient mice,
which are completely osteoclasts deficiency, the accentuation of the BMP-
induced ectopic bone formation did not exist. Furthermore, there is no
RANK-positive osteoclast progenitors in bone derived from c-Fos-deficient
mice. These results suggest that RANK-positive osteoclast progenitors are
positively regulate the signal of bone formation. In summary, osteoclastic
bone resorption directly activates osteoblast function and osteoclasts are
involved in normal bone morphogenesis. References osteoblasts, is a soluble decoy receptor for RANKL. Deficiency of OPG in
mice induces osteoporosis caused enhanced bone resorption. Elevated
osteoblastic activity was suppressed by bisphosphonate administration in
OPG-deficient mice. These results suggest that bone formation is
accurately coupled with bone resorption. Collagen sponge disks containing
BMP-2 were implanted into the dorsal muscle pouches in OPG-deficient
mice. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 O25
Implication of microRNA-140 in osteoarthritis
Johanne Martel-Pelletier
University of Montreal and Osteoarthritis Research Unit, Notre-Dame
Hospital, CRCHUM, Montreal, Quebec, Canada
Arthritis Research & Therapy 2012, 14(Suppl 1):O25 O27 On the
other hand, partially reprogrammed intermediate cells expressed type I O26 O26
Osteoclastic bone resorption directly activates osteoblast function
Nobuyuki Udagawa
Matsumoto Dental University, Shiojiri, Nagano, 399-0781, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O26 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 In rheumatoid arthritis (RA), targeting the vasculature may be beneficial
to control the disease. Endothelial cells lining blood vessels are involved
in a variety of functions in inflammation, including recruitment of
leukocytes and cellular adhesion, antigen presentation, coagulation,
cytokine production and angiogenesis. Angiogenesis, the growth of new
vessels, is important for the proliferation of the rheumatoid synovial
tissue pannus where these vessels also serve as a conduit for cells
entering the inflamed synovium from the blood. In rheumatoid arthritis (RA), targeting the vasculature may be beneficial
to control the disease. Endothelial cells lining blood vessels are involved
in a variety of functions in inflammation, including recruitment of
leukocytes and cellular adhesion, antigen presentation, coagulation,
cytokine production and angiogenesis. Angiogenesis, the growth of new
vessels, is important for the proliferation of the rheumatoid synovial
tissue pannus where these vessels also serve as a conduit for cells
entering the inflamed synovium from the blood. collagen and produced tumor when injected into nude mice. Induced
chondrogenic cells did not undergo pluripotent state during induction from
dermal fibroblast culture, as time-lapse observation did not detect GFP
reporter expression during induction from dermal fibroblasts prepared from
transgenic mice in which GFP is inserted into the Nanog locus. These results
suggest that chondrogenic cells induced by this approach are free from a
risk of teratoma formation which associates with cells prepared through
generation of iPS cells followed by redifferentiation into the target cell type. The dox-inducible induction system demonstrated that induced cells are able
to respond to chondrogenic medium by expressing endogenous Sox9 and
maintain chondrogenic potential after substantial reduction of transgene
expression. This approach could lead to the preparation of hyaline cartilage
directly from skin, without going through pluripotent stem cells, in future
regenerative medicine. y
We have shown before that the endothelial adhesion molecule E-selectin,
in soluble form, mediates angiogenesis via its endothelial receptor sialyl
Lewisx on adjacent endothelium [1]. We have used human RA synovial
tissues to produce an antibody detecting related molecules, Lewisy/H-5-2,
which are mainly known as blood group antigens but are also found on
endothelium in select organs such as skin, lymph node and synovium, but
not most other endothelium. This antigen is rapidly upregulated on
endothelium in vitro in response to stimuli such as tumor necrosis factor-
alpha, that is present in the RA joint. Additionally, this antigen is
upregulated on RA vs. O28 A systems approach reveals that the musculoskeletal tissues
development and homeostasis network
Hiroshi Asahara1,2,3,4 1Department of Systems BioMedicine, Tokyo Medical and Dental University,
Bunkyo-ku, Tokyo, 113-8519, Japan; 2Department of Systems BioMedicine,
National Research Institute for Child Health and Development, Setagaya-ku,
Tokyo, 157-8535, Japan; 3The Scripps Research Institute, La Jolla, CA, 92037,
USA; 4JST, CREST, Chiyoda-ku, Tokyo, 102-0075, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O28 1Department of Systems BioMedicine, Tokyo Medical and Dental University,
Bunkyo-ku, Tokyo, 113-8519, Japan; 2Department of Systems BioMedicine,
National Research Institute for Child Health and Development, Setagaya-ku,
Tokyo, 157-8535, Japan; 3The Scripps Research Institute, La Jolla, CA, 92037,
USA; 4JST, CREST, Chiyoda-ku, Tokyo, 102-0075, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O28 y
Fucosyl transferases (fut1 and fut2) are enzymes that control the synthesis of
Lewisy/H-5-2. We have examined fut1 deficient mice to determine if
fucosylation is important in angiogenesis and arthritis. Fut1 gene deficient
mouse endothelial cells did not form endothelial sprouts on Matrigel in vitro
to the same extent as wild type mouse endothelial cells. Moreover, the fut1
gene deficient mice were resistant to the development of angiogenesis in
the Matrigel plug and sponge granuloma angiogenesis models in vivo. In
terms of arthritis development, the Lewisy/H-5-2 gene deficient mice were
resistant to development of K/BxN arthritis. Moreover, the harvested joints
of these mice had decreased monocyte chemoattractant protein-1/CCL2
and interleukin-1 compared to wild type littermates, indicating that some
inflammatory mediators were downregulated when fut1 was absent. These
experiments suggest that futs may be important in the development of
angiogenesis and inflammatory arthritis and that they may serve as novel
targets in RA therapy. Materials and methods: We created a whole-mount in situ hybridization
database, termed EMBRYS http://embrys.jp/embrys/html/MainMenu.html,
containing expression data of 1520 transcription factors and cofactors
expressed in E9.5, E10.5, and E11.5 mouse embryos –a highly dynamic stage
of skeletal myogenesis. This approach implicated 43 genes in regulation of
embryonic myogenesis, including a transcriptional repressor, the zinc-finger
protein RP58 (also known as Zfp238) [1]. p
p
Results: Knockout and knockdown approaches confirmed an essential role
for RP58 in skeletal myogenesis. Cell-based high-throughput transfection
screening revealed that RP58 is a direct MyoD target. Microarray analysis
identified two inhibitors of skeletal myogenesis, Id2 and Id3, as targets for
RP58-mediated repression. References 1. Yokoyama S, Ito Y, Ueno-Kudoh H, Shimizu H, Uchibe K, Albini S,
Mitsuoka K, Miyaki S, Kiso M, Nagai A, Hikata T, Osada T, Fukuda N,
Yamashita S, Harada D, Mezzano V, Kasai M, Puri PL, Hayashizaki Y, Okado H,
Hashimoto M, Asahara H: A systems approach reveals that the
myogenesis genome network is regulated by the transcriptional
repressor RP58. Dev Cell 2009, 17:836-848. 1. Yokoyama S, Ito Y, Ueno-Kudoh H, Shimizu H, Uchibe K, Albini S,
Mitsuoka K, Miyaki S, Kiso M, Nagai A, Hikata T, Osada T, Fukuda N,
Yamashita S, Harada D, Mezzano V, Kasai M, Puri PL, Hayashizaki Y, Okado H,
Hashimoto M, Asahara H: A systems approach reveals that the
myogenesis genome network is regulated by the transcriptional
repressor RP58. Dev Cell 2009, 17:836-848. Rheumatoid arthritis (RA) affects approximately 0.5% of the world
population, yet the mechanisms underlying the development and
progression of RA remain poorly understood. We are investigating the role
of citrullinated fibrinogen as a pathogenic antigen in RA. Using arthritis
antigen arrays we demonstrate that citrullinated fibrinogen is one of the
earliest targets of the autoantibody response in RA, with autoantibodies
against citrullinated fibrinogen appearing up to 10 years prior to the
development of clinical arthritis. We further demonstrate that approximately
50% of CCP+ RA patients possess circulating immune complexes containing
citrullinated fibrinogen, and that citrullinated fibrinogen containing immune
complexes are deposited in human RA synovial tissues. To determine
whether citrullinated fibrinogen can induce inflammatory arthritis in mice,
we immunized mice with citrullinated fibrinogen and demonstrated that an
inflammatory arthritis results and that both T cells and serum can transfer
arthritis to naïve mice. Fibrinogen is an endogenous ligand for the innate
immune receptor TLR4, and to determine whether citrullination might alter
the ability of fibrinogen to bind TLR4 we performed in vitro macrophage
stimulation assays with native and citrullinated fibrinogen. We found that
citrullinated fibrinogen was ten-fold more potent than native fibrinogen at
stimulating macrophage TNF release. Further, macrophage derived from
mice deficient for TLR4 or MyD88 did not produce TNF in response to
citrullinated fibrinogen. Thus, our results demonstrate a novel mechanism
by which anti-citrullinated protein antibodies (ACPA) specifically targeting
citrullinated fibrinogen may directly stimulate macrophage TNF production,
via co-ligation of TLR4 and Fc-gamma-R. Our findings demonstrate a role for 2. Osteoclastic bone resorption directly activates osteoblast function
Nobuyuki Udagawa Osteoclasts, the multinucleated cells that resorb bone, originate from cell
cycle-arrested quiescent osteoclast precursors [1]. Mesenchymal
osteoblastic cells are involved in osteoclast differentiation. Osteoclast
precursors express RANK (a receptor of RANKL), recognize RANKL
expressed by osteoblasts through cell-cell interaction and differentiate into
osteoclasts in the presence of M-CSF. OPG, produced mainly by Page 12 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
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http://arthritis-research.com/supplements/14/S1 normal synovial endothelial cells, and in soluble
form is upregulated in RA synovial fluid vs. osteoarthritic synovial fluid. In
soluble form, Lewisy/H-5-2 mediates angiogenesis, cell adhesion via
intercellular adhesion molecule-1, and monocyte recruitment. O28 Consistently, MyoD-dependent activation of the
myogenic program is impaired in RP58 null fibroblasts and downregulation
of Id2 and Id3 rescues MyoD’s ability to promote myogenesis in these cells. Conclusions: Our combined, multi-system approach reveals a MyoD-
activated regulatory loop relying on RP58-mediated repression of muscle
regulatory factor inhibitors. We applied our systems approaches to other
locomotive tissues research including cartilage and tendon, and revealed
novel molecular network regulating joint cartilage development and
homeostasis via microRNA-140 [2,3] and tendon development by Mkx [4]. References Results: Knockout and knockdown approaches confirmed an essential role
for RP58 in skeletal myogenesis. Cell-based high-throughput transfection
screening revealed that RP58 is a direct MyoD target. Microarray analysis
identified two inhibitors of skeletal myogenesis, Id2 and Id3, as targets for
RP58-mediated repression. Consistently, MyoD-dependent activation of the
myogenic program is impaired in RP58 null fibroblasts and downregulation
of Id2 and Id3 rescues MyoD’s ability to promote myogenesis in these cells. g
py
Reference
1. Koch AE, et al: Nature 376:517-519. Reference 1. Koch AE, et al: Nature 376:517-519. O30 Conclusions: Our combined, multi-system approach reveals a MyoD-
activated regulatory loop relying on RP58-mediated repression of muscle
regulatory factor inhibitors. We applied our systems approaches to other
locomotive tissues research including cartilage and tendon, and revealed
novel molecular network regulating joint cartilage development and
homeostasis via microRNA-140 [2,3] and tendon development by Mkx [4]. References Citrullination of fibrinogen: generation of neoepitopes and
enhancement of immunostimulatory properties
William H Robinson*, Jeremy Sokolove
VA Palo Alto Health Care System, Palo Alto, CA 94304 and the Division of
Immunology and Rheumatology, Stanford University School of Medicine,
Stanford, CA 94305, USA
Arthritis Research & Therapy 2012, 14(Suppl 1):O30 From discovery of RANKL to clinical application of anti-human RANKL
antibody From discovery of RANKL to clinical application of anti-human RANKL
antibody Hisataka Yasuda1*, Yuriko Furuya2, Kohji Uchida2
1Bioindustry Division, Oriental Yeast Co Ltd, Tokyo, Japan; 2Nagahama
Institute for Biochemical Science, Oriental Yeast Co Ltd, Shiga, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O33 Osteoporosis is a common bone disease characterized by reduced bone
and increased risk of fracture. In postmenopausal women osteoporosis
results from bone loss attributable to estrogen deficiency. Receptor
activator of nuclear factor-B ligand (RANKL) is a pivotal osteoclast
differentiation factor [1]. Discovery of RANKL has opened a new era in the
understanding of mechanisms in osteoclast differentiation over the last
decade. The discovery also results in the development of a fully human
anti-RANKL neutralizing monoclonal antibody (called denosumab) and
denosumab has been approved for the treatment of osteoporosis in
Europe and the US. Systemic lupus erythematosus (SLE) is a multisystem chronic inflammatory
disease that affects many organs, and the immunological disorders are
accompanied by autoantibody production. Recent case-control association
study revealed that polymorphisms in the Egr-2 influence SLE susceptibility
in humans. Interestingly, adoptive transfer of CD4+CD25-LAG3+ Tregs from
MRL/+ mice suppressed autoantibody production and the progression of
nephritis in MRL/lpr lupus prone mice. In contrast, CD4+CD25+ Tregs from
MRL/+ mice exhibited no significant therapeutic effect upon transfer to
MRL/lpr mice. These results indicate that CD4+CD25-LAG3+ Tregs play key
roles in the regulation of humoral immunity by the strong suppressive
activity for B cell antibody production. Here I report a novel rapid bone loss model with GST-RANKL as the first
topic [2]. Pharmacologic studies of candidates for the treatment of
osteoporosis with this model can be done in short periods such as 3 days
and a couple of weeks although it took several months in the conventional
methods with ovariectomized(OVX)-rats. This model also is useful for the
rapid analyses in the functions of osteoclasts in vivo. The RANKL-induced
bone loss model is the simplest, fastest, and easiest of all osteoporosis
models and could be a gold standard in the evaluation of novel drug
candidates for osteoporosis as well as OVX. O32
Innate and adaptive immune responses to dead and dying cells
Keith B Elkon1,2*, Yueng Peng1
1Division of Rheumatology, University of Washington, Seattle, WA 98195,
USA; 2Department of Immunology, University of Washington, Seattle, WA
98195, USA
Arthritis Research & Therapy 2012, 14(Suppl 1):O32 Osteopetrosis is generally caused by failure of osteoclast-mediated
resorption of skeleton. From discovery of RANKL to clinical application of anti-human RANKL
antibody There are a numerous mouse models of
osteopetrosis without osteoclasts, including c-fos deficient mice, op/op
mice, RANKL-deficient mice and RANK-deficient mice. As the second topic I
report a mouse model of osteopetrosis induced by a denosumab-like anti-
mouse neutralizing monoclonal RANKL antibody [3]. One injection of the
antibody increased bone mass markedly with remarkable decrease in
osteoclast surface and number after two weeks. In addition, osteoblast
surface, mineral apposition rate, and bone formation rate were also
reduced markedly. These results are consistent with the recent report
treating human RANKL-knock in mice with denosumab [4]. These inducible
models of osteoporosis and osteopetrosis using normal mice exhibit
exactly mirror images in terms of change in bone mass and are quite
useful to accelerate research on osteoclast biology as well as bone
metabolism in vivo. Under steady state conditions, billions of dead and dying cells are
removed by extrusion from epithelial surfaces as well as by phagocytosis. Cells such as macrophages and dendritic cells have specialized receptors
that directly recognize altered protein or lipids on apoptotic cells or
opsonins that bind to the dying cell. Once engulfed, phagosomes
containing apoptotic cells are rapidly acidified and the contents degraded
by proteases and nucleases in lysozymes. During necrosis, cellular
material is released prior to engulfment and extracellular nucleases as
well as intracellular sensors dictate the inflammatory potential of the
cellular debris. The outcome may be release of TNF-a, IL-1-b or interferon
(IFN)-a depending upon the type of phagocyte, molecular nature of the
cellular particle and the intracellular sensor engaged. In conclusion, the discovery of OPG/RANKL/RANK system guided us to
reveal the mechanism regulating osteoclast differentiation and activation. The past decade has witnessed significant progress in the development
of the RANKL antibody as a pharmaceutical agent. This is a story from a
discovery of RANKL to clinical application of anti-human RANKL antibody. References In addition to responses by cells of the innate immune system, we have
recently defined a link between processing of apoptotic cells and their
debris to T cell activation [1]. MFG-E8 is an opsonin (bridging protein)
that binds to phosphatidylserine on apoptotic cells and facilitates their
removal through interaction with integrins on phagocytes. Mice deficient
in MFG-E8 develop lupus like autoimmunity associated with accumulation
of apoptotic cells in vivo. O31
Novel regulatory T cells controlling antibody production and systemic
autoimmunity
* These observations suggest that in addition to altering the rate of
clearance of apoptotic cells, MFG-E8 deficiency promotes immune
responses to self antigens by altered intracellular processing leading to
enhanced antigen presentation. Thus, handling of dead and dying cells
impacts both innate and adaptive immune responses to self antigens. Reference y
Kazuhiko Yamamoto*, Tomohisa Okamura, Keishi Fujio
Department of Allergy and Rheumatology, The University of Tokyo Graduate
School of Medicine, Tokyo, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O31 1. Peng Y-F, Elkon KB: Autoimmunity in MFG-E8-deficient mice is associated
with altered trafficking and enhanced cross-presentation of apoptotic
cell antigens. J Clin Invest 2011, 121:2221-2241. Regulatory T cells (Tregs) are engaged in the maintenance of immunological
self-tolerance and immune homeostasis. IL-10 has an important role in
maintaining the normal immune state. We showed that IL-10-secreting
Tregs can be delineated in normal mice as CD4+CD25-Foxp3- T cells that
express lymphocyte activation gene-3 (LAG-3), an MHC class II-binding CD4
homolog. CD4+CD25-LAG3+ Tregs characteristically express early growth
response gene-2 (Egr-2), a key molecule for anergy induction. Retroviral
gene transfer of Egr-2 converts naïve CD4+ T cells into IL-10-secreting and
LAG-3-expressing Tregs. Moreover, CD4+CD25-LAG3+ Tregs show B cell-
dependent development. CD4+CD25-LAG3+ Tregs, but not CD4+CD25+
Tregs, strongly suppressed the antibody production in B cells co-cultured
with helper T cells. Thus, IL-10-secreting Egr-2+LAG3+CD4+ Tregs are closely
related to B cells and can be exploited for the treat ment of autoimmune
diseases. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 ovalbumin (OVA) reactive OT-I CD8 T cells caused accelerated diabetes in
MFG-E8-/- RIP-mOVA mice and skin disease in kmOVA transgenic mice. The enhanced CD8 T cell response was attributed to increased cross-
presentation by dendritic cells (DCs) associated with increased detection
of antigen peptide MHCI complexes. Investigation of intracellular
trafficking revealed that, whereas intact apoptotic cells ingested by wild
type DC rapidly fused with lysosomes, in the absence of MFG-E8, smaller
apoptotic cell fragments persisted in endosomal compartments and failed
to fuse with lysosomes. ovalbumin (OVA) reactive OT-I CD8 T cells caused accelerated diabetes in
MFG-E8-/- RIP-mOVA mice and skin disease in kmOVA transgenic mice. The enhanced CD8 T cell response was attributed to increased cross-
presentation by dendritic cells (DCs) associated with increased detection
of antigen peptide MHCI complexes. Investigation of intracellular
trafficking revealed that, whereas intact apoptotic cells ingested by wild
type DC rapidly fused with lysosomes, in the absence of MFG-E8, smaller
apoptotic cell fragments persisted in endosomal compartments and failed
to fuse with lysosomes. citrullination both in creating neoantigens targeted by the adaptive immune
response in RA as well as by increasing the potency of fibrinogen as an
endogenous innate immune ligand. These results provide insights into the
mechanisms by which anti-citrulline autoimmunity, and specifically the
citrullination of fibrinogen, may contribute to both the onset and
propagation of inflammation in RA. citrullination both in creating neoantigens targeted by the adaptive immune
response in RA as well as by increasing the potency of fibrinogen as an
endogenous innate immune ligand. These results provide insights into the
mechanisms by which anti-citrulline autoimmunity, and specifically the
citrullination of fibrinogen, may contribute to both the onset and
propagation of inflammation in RA. O31
Novel regulatory T cells controlling antibody production and systemic
autoimmunity
Kazuhiko Yamamoto*, Tomohisa Okamura, Keishi Fujio
Department of Allergy and Rheumatology, The University of Tokyo Graduate
School of Medicine, Tokyo, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O31 References Miyaki S, Sato T, Inoue A, Otsuki S, Ito Y, Yokoyama S, Kato Y, Takemoto F,
Nakasa T, Yamashita S, Takada S, Lotz MK, Ueno-Kudo H, Asahara H:
MicroRNA-140 plays dual roles in both cartilage development and
homeostasis. Genes Dev 2010, 24:1173-1185. 2. Miyaki S, Sato T, Inoue A, Otsuki S, Ito Y, Yokoyama S, Kato Y, Takemoto F,
Nakasa T, Yamashita S, Takada S, Lotz MK, Ueno-Kudo H, Asahara H:
MicroRNA-140 plays dual roles in both cartilage development and
homeostasis. Genes Dev 2010, 24:1173-1185. 3. Miyaki S, Nakasa T, Otsuki S, Grogan SP, Higashiyama R, Inoue A, Kato Y,
Sato T, Lotz MK, Asahara H: MicroRNA-140 is expressed in differentiated
human articular chondrocytes and modulates interleukin-1 responses. Arthritis Rheum 2009, 60:2723-2730. 3. Miyaki S, Nakasa T, Otsuki S, Grogan SP, Higashiyama R, Inoue A, Kato Y,
Sato T, Lotz MK, Asahara H: MicroRNA-140 is expressed in differentiated
human articular chondrocytes and modulates interleukin-1 responses. Arthritis Rheum 2009, 60:2723-2730. 4. Ito Y, Toriuchi N, Yoshitaka T, Ueno-Kudoh H, Sato T, Yokoyama S, Nishida K,
Akimoto T, Takahashi M, Miyaki S, Asahara H: The Mohawk homeobox
gene is a critical regulator of tendon differentiation. Proc Natl Acad Sci U
S A 2010, 107:10538-10542. 4. Ito Y, Toriuchi N, Yoshitaka T, Ueno-Kudoh H, Sato T, Yokoyama S, Nishida K,
Akimoto T, Takahashi M, Miyaki S, Asahara H: The Mohawk homeobox
gene is a critical regulator of tendon differentiation. Proc Natl Acad Sci U
S A 2010, 107:10538-10542. 4. Ito Y, Toriuchi N, Yoshitaka T, Ueno-Kudoh H, Sato T, Yokoyama S, Nishida K,
Akimoto T, Takahashi M, Miyaki S, Asahara H: The Mohawk homeobox
gene is a critical regulator of tendon differentiation. Proc Natl Acad Sci U
S A 2010, 107:10538-10542. O29 O29
Angiogenesis in rheumatoid arthritis: the role of fut 1
Alisa Erika Koch
Department of Veteran’s Affairs, Ann Arbor, MI USA and University of
Michigan, MI, USA
Arthritis Research & Therapy 2012, 14(Suppl 1):O29 Page 13 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 From discovery of RANKL to clinical application of anti-human RANKL
antibody J Bone Miner Res 2009, 24:182-195. O34 Microparticles as antigenic targets in human and murine SLE
Anirudh Ullal1*, David S Pisetsky1,2
1Duke University Medical Center, Durham, NC, USA; 2Durham VAMC,
Durham, NC, USA
Arthritis Research & Therapy 2012, 14(Suppl 1):O34 p
g
g
Subsequently and surprisingly, TNF-a induced a tolerant state in
macrophages, with diminished cytokine production on lipopolysaccharide
(LPS) challenge and protection from LPS-induced lethality. TNF-a-induced
cross-tolerization was mediated by coordinate action of two inhibitory
mechanisms, suppression of LPS-induced signaling and chromatin
remodeling. Mechanistically, TNF-a-induced cross-tolerance was
distinguished from TLR-induced tolerance by strong dependence on the
nuclear kinase GSK3, which suppressed chromatin accessibility and
promoted rapid termination of NF-gB signaling by augmenting negative
feedback by A20 and IgBa. These results reveal an unexpected
homeostatic function of TNF-a and provide a GSK3-mediated mechanism
for preventing prolonged and excessive inflammation. This homeostatic
mechanism may be compromised during RA synovitis, possibly by
hypomorphic alleles of TNFAIP3 (encodes A20) or by cytokines that
suppress A20 expression or antagonize its function. These data suggest
that augmenting homeostatic functions and signals and thereby
rebalancing the pro- versus anti-inflammatory profile of TNF-a may
represent an efficacious alternative therapeutic approach to suppress
chronic inflammation. Microparticles are small membrane-bound vesicles that are released from
activated and dying cells by a blebbing process. These particles circulate in
the blood and display potent pro-inflammatory and pro-thrombotic
activities. In addition, particles are an important source of extracellular
DNA and RNA and may participate in the transfer of informational nucleic
acids. Because microparticles contain DNA as well as other nuclear
antigens, we have investigated their ability to bind to anti-DNA and other
anti-nuclesome antibodies that characterize the prototypic autoimmune
disease systemic lupus erythematosus (SLE). For this purpose, we
generated microparticles from HL-60, Jurkat and THP-1 cells induced to
undergo apoptosis in vitro. Using FACS analysis to assess antibody binding,
we showed that particles can bind some but not all monoclonal anti-DNA
and anti-nucleosome antibodies from MRL-lpr/lpr and NZB/NZWF1 lupus
mice. For the monoclonal anti-DNA, DNase treatment reduced binding. Like the monoclonal antibodies, patient plasma also bound to the particles
although this activity was not directly correlated with levels of anti-DNA
antibodies as measured by an ELISA. To determine whether particles
circulating in the blood of patients can represent immune complexes,
FACS analysis was performed on particles isolated from patient plasma. From discovery of RANKL to clinical application of anti-human RANKL
antibody We observed that older MFG-8-/- mice
spontaneously developed a dermatitis associated with CD8 T cell
infiltration and striking activation of effector memory CD8 T cells. T cell
responses to both exogenous and endogenous apoptotic cell associated
antigens were enhanced in MFG-E8 deficient mice and transfer of 1. Yasuda H, Shima N, Nakagawa N, et al: Osteoclast differentiation factor is
a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is
identical to TRANCE/RANKL. Proc Natl Acad Sci USA 1998, 95:3597-3602. 1. Yasuda H, Shima N, Nakagawa N, et al: Osteoclast differentiation factor is
a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is
identical to TRANCE/RANKL. Proc Natl Acad Sci USA 1998, 95:3597-3602. 2. Tomimori Y, Mori K, Koide M, et al: Evaluation of pharmaceuticals with a
novel 50-hour animal model of bone loss. J Bone Miner Res 2009,
24:1194-1205. Page 14 of 54 Page 14 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 3. Furuya Y, Mori K, Ninomiya T, et al: Increased bone mass in mice after a
single injection of an anti-RANKL neutralizing antibody: evidence for a
bone anabolic effect of PTH in mice with few osteoclasts. J Biol Chem
2011 in press. 3. Furuya Y, Mori K, Ninomiya T, et al: Increased bone mass in mice after a
single injection of an anti-RANKL neutralizing antibody: evidence for a
bone anabolic effect of PTH in mice with few osteoclasts. J Biol Chem
2011 in press. expression to RA synovial macrophages. The acute inflammatory response
to TNF-a subsided after several hours and was followed by an IFN response
characterized by sustained expression of STAT1 and downstream target
genes. TNF-a-mediated induction of an IFN response was mediated by IFN-b
and was sensitive to inhibition by Jak inhibitors. Concomitantly TNF-a
induced a state of macrophage resistance to the homeostatic cytokines
IL-10 and IL-27. Microarray analysis demonstrated that sustained TNF-a
signaling induced expression of novel genes not appreciated to be
‘TNF-inducible’, but are highly expressed in RA synovial macrophages. Induction of an IFN response and abrogation of homeostatic cytokine
signaling was also observed in RA synovial macrophages and likely
contributes to the pathogenic actions of TNF-a during arthritis. 4. Kostenuik PJ, Nguyen HQ, McCabe J, et al: Denosumab, a fully human
monoclonal antibody to RANKL, inhibits bone resorption and increases
BMD in knock-in mice that express chimeric (murine/human) RANKL. From discovery of RANKL to clinical application of anti-human RANKL
antibody These studies indicated that, while the total levels of microparticles in the
blood of patients with SLE did not differ significantly from those of normal
controls, the number of IgG-positive particles was significantly elevated
using a R-phycoerythrin-labeled anti-human IgG (g-chain specific) reagent. In this study, the number of IgG-positive particles was correlated with
levels of anti-DNA. In similar studies with plasma from MRL-lpr/lpr and
NZB/NZWF1 mice, we showed that the total levels of particles were
increased compared to those of BALB/c control mice and that the number
of particles that stained with an anti-IgG reagent was also increased. Furthermore, plasma of mice could bind to particles generated in vitro
from apoptotic cells. Together, these findings indicate that microparticles
can express antigenically active DNA in an accessible form, either because
of a surface location or particle permeability. Furthermore, they
demonstrate that microparticles can form immune complexes and that at
least some of the immune complexes in the blood in SLE contain particles. Current studies are characterizing the immune properties of these
complexes and their potential role in pathogenicity. Overall, the data reveal novel signals and functions of TNF-a and that are
likely operative during chronic inflammation and RA synovitis. Targeted
inhibition of these non-traditional functional components of the TNF-a
response may be efficacious in alleviating chronic inflammation while
preserving acute TNF-a responses and host defense against infections. O36
Synovial fibroblasts display an uncontrolled inflammatory and tissue
destructive response to TNF-a
George D Kalliolias1,2*, Janice Chen1, Galina Grigoriev1, Lionel B Ivashkiv1,3
1Research Department, Arthritis and Tissue Degeneration Program, Hospital
for Special Surgery, New York, NY, 10021, USA; 2Medicine, Weill Cornell
Medical College, New York, NY, 10021, USA; 3Graduate Program in
Immunology and Microbial Pathogenesis, Weill Cornell Graduate School of
Medical Sciences, New York, NY, 10021, USA
Arthritis Research & Therapy 2012, 14(Suppl 1):O36 Synovial fibroblasts display an uncontrolled inflammatory and tissue
destructive response to TNF-a
1 2*
1
1
1 3 George D Kalliolias1,2*, Janice Chen1, Galina Grigoriev1, Lionel B Ivashkiv1,3
1Research Department, Arthritis and Tissue Degeneration Program, Hospital
for Special Surgery, New York, NY, 10021, USA; 2Medicine, Weill Cornell
Medical College, New York, NY, 10021, USA; 3Graduate Program in
Immunology and Microbial Pathogenesis, Weill Cornell Graduate School of
Medical Sciences, New York, NY, 10021, USA
Arthritis Research & Therapy 2012, 14(Suppl 1):O36 George D Kalliolias1,2*, Janice Chen1, Galina Grigoriev1, Lionel B Ivashkiv1,3
1Research Department, Arthritis and Tissue Degeneration Program, Hospital
for Special Surgery, New York, NY, 10021, USA; 2Medicine, Weill Cornell
Medical College, New York, NY, 10021, USA; 3Graduate Program in
Immunology and Microbial Pathogenesis, Weill Cornell Graduate School of
Medical Sciences, New York, NY, 10021, USA
A th iti R
h & Th
2012 14(S
l 1) O36 Arthritis Research & Therapy 2012, 14(Suppl 1):O36 Background: Synovial fibroblasts are key players in the pathogenesis of
Rheumatoid Arthritis (RA) and potentially attractive treatment targets. Upon activation within the joint’s inflammatory milieu, they gain a
transformed phenotype and produce pro-inflammatory cytokines (mainly
IL-6) and tissue destructive enzymes [1]. Materials and methods: Synovial fibroblasts were isolated via enzymatic
processing from synovial tissues obtained from patients with RA or
Osteoarthritis (OA). Synovial fibroblasts (passages 2-4) were stimulated
with TNF-a (10 ng/ml) only on day 1. The expression of TNF-a-target
genes was measured by qPCR in time course experiments (1, 3, 6, 24, 48,
72, 96 and 120 hours after TNF-a stimulation). O35
New mechanisms of action and signaling by TNF-a
Lionel B Ivashkiv1,2
1Arthritis and Tissue Degeneration Program, Hospital for Special Surgery,
New York, NY 10021, USA; 2Department of Medicine and Immunology and
Microbial Pathogenesis Program, Weill Cornell Medical College, New York,
NY 10065, USA
Arthritis Research & Therapy 2012, 14(Suppl 1):O35 Signal transduction inhibitors for the treatment of rheumatoid arthritis
Yoshiya Tanaka*, Kunihiro Yamaoka Rheumatoid arthritis (RA) is a representative autoimmune disease
characterized by chronic and destructive inflammatory synovitis. The
multiple cytokinesand cell surface molecules play a pivotal role in
the pathogenesis of RA and binding of these molecules to their ligands on
the cell surfaceinduce various signal intracellular transduction including
phosphorylation of kinase proteins. The tyrosine kinase is the first
intracellular signals to be phosphorylated and 14 tyrosine kinases are
known to be involved in RA. Among them, members of Janus kinase (Jak)
familyare essential for the signaling pathways of various cytokines and are
implicated in the pathogenesis of RA. An orally available Jak3 inhibitor
tofacitinib is currently in clinical trials for RA with satisfactory effects and
acceptable safety [1,2]. A phase 2 double-blinded study wascarried out to
investigate the efficacy and safety of tofacitinib in Japanese patients with
active RA andinadequate responseto methotrexate (MTX). A total of 140
patients were randomized to tofacitinib 1, 3, 5, 10 mg, or placebotwice
daily and ACR20 response rates at week 12, a primary endpoint, was
significant for all tofacitinib treatment groups [3]. Thus, tofacitinib in
combination with MTX was efficacious and had a manageable safety
profile and tofacitinib 5 and 10 mg twice a day appear suitable for further
evaluation to optimize their potential for the treatment of RA. Although
the mode of action of tofacitinib has remain unclear, we clarified thatthe
inhibitory effects of tofacitinib could be mediated through the suppression
of IL-17 and IFN-g production and proliferation of CD4+ T cells, presumably
Th1 and Th17 cells by in vitro experiments. We next conducted a
treatment study in the SCID-HuRAg mice, an RA animal model utilizing
SCID mice implanted with synovium and cartilage from patients with RA
and tofacitinib was administered via an osmotic mini-pump. Tofacitinib
decreased serum levels of human IL-6 and IL-8 in the mice and reduced
invasion of the synovial tissue into the implanted cartilage as well as
accumulation of immune cells in the synovium.Taken together, orally
available low molecular weight products such as tofacitinibtargeting
intracellular signaling molecules, would provide enormous power and
flexibility in the treatment of RA Conclusions: In contrast to human Mj, synovial fibroblasts display a
sustained inflammatory and tissue destructive response to TNF-a. Our
observations suggest that synovial fibroblasts may lack the homeostatic
mechanisms that control and terminate the effects of TNF-a on human
Mj [2]. y
References 1. Kremer JM, Bloom BJ, Breedveld FC, Coombs JH, Fletcher MP, Gruben D,
et al: The safety and efficacy of a JAK inhibitor in patients with active
rheumatoid arthritis: results of a double-blind, placebo-controlled phase
IIa trial of three dose levels of CP-690,550 versus placebo. Arthritis Rheum
2009, 60:1895-1905. TCZ, even in monotherapy, has been demonstrated to induce DAS28
remission frequently in patients with RA and suppress the radiographic
progression of joint damage. TCZ more significantly reduced radiological
progression in patients with risk factors for rapid progression (i.e. high
urinary C-terminal crosslinking telopeptide, high urinary pyridinoline/
deoxypyridinoline ratio, low body mass index, and presence of joint space
narrowing at baseline) than those without the risk factors. Furthermore,
early decreases in serum type IIA procollagen amino terminal propeptide,
CRP, and/or matrix metalloproteinase 3 (MMP-3) within 12 weeks can
predict for the preventive effects of TCZ on one year progression of joint
destruction in RA. 2. Coombs JH, Bloom BJ, Breedveld FC, Fletcher MP, Gruben D, Kremer JM,
et al: Improved pain, physical functioning and health status in patients
with rheumatoid arthritis treated with Cp-690,550, an orally active Janus
kinase (Jak) inhibitor: results from a randomised, double-blind, placebo-
controlled trial. Ann Rheum Dis 2010, 69:413-416. 2. Coombs JH, Bloom BJ, Breedveld FC, Fletcher MP, Gruben D, Kremer JM,
et al: Improved pain, physical functioning and health status in patients
with rheumatoid arthritis treated with Cp-690,550, an orally active Janus
kinase (Jak) inhibitor: results from a randomised, double-blind, placebo-
controlled trial. Ann Rheum Dis 2010, 69:413-416. 3. Tanaka Y, Suzuki M, Nakamura H, Toyoizumi S, Zwillich SH, Tofacitinib Study
Investigators: Phase II study of tofacitinib (CP-690,550) combined with
methotrexate in patients with rheumatoid arthritis and inadequate
response to methotrexate. Arthritis Care Res (Hoboken) 2011, 63:1150-1158. Although long-term treatment with TCZ is well tolerated, it goes without
saying that it is beneficial not only for the patients but also for medical
economy. To test the possibility of drug free remission introduced by
TCZ, Drug free REmission after cessation of Actemra Monotherapy
(DREAM) study was conducted. A total of 187 patients, who had received
TCZ in the previous clinical trials (the mean treatment duration was 4.3
years), were enrolled, and discontinued TCZ. Remission, defined as DAS28
less than 2.6, was maintained in 10% of the patients without any drug
over 52 weeks. O35
New mechanisms of action and signaling by TNF-a
Lionel B Ivashkiv1,2 1Arthritis and Tissue Degeneration Program, Hospital for Special Surgery,
New York, NY 10021, USA; 2Department of Medicine and Immunology and
Microbial Pathogenesis Program, Weill Cornell Medical College, New York,
NY 10065, USA 1Arthritis and Tissue Degeneration Program, Hospital for Special Surgery,
New York, NY 10021, USA; 2Department of Medicine and Immunology and
Microbial Pathogenesis Program, Weill Cornell Medical College, New York,
NY 10065, USA Human macrophages (Mj) generated in vitro (blood derived CD14+ cells
stimulated for 48 h with M-CSF) were used in similar time course
experiments as controls. ,
Arthritis Research & Therapy 2012, 14(Suppl 1):O35 Arthritis Research & Therapy 2012, 14(Suppl 1):O35 Results: In Mj it was observed a rapid (within 1-3 hours) induction of
TNF-a-target genes (including TNF-a, IL-1b, IL-6 and IL-8) that was
restrained back to the baseline within a few hours (3-24 hours depending
on the gene). In stark contrast, synovial fibroblasts displayed a remarkably
more sustained response to TNF-a. IL-6 mRNA expression was induced
within a few hours by TNF-a, and induction increased continuously for
72-96 h despite the absence of any further exogenous TNF-a stimulation. The levels of IL-6 mRNA induced by TNF-a in synovial fibroblasts were
substantially higher compared to human Mj, suggesting that within the TNF-a is a key pathogenic factor in inflammatory arthritis. Rapid and
transient signaling and functional responses of cells to TNF-a, such as
activation of NF-gB and MAPKs, are well known. These signaling mechanisms
are widely assumed to be functional in cells chronically exposed to TNF-a
and to mediate the pathogenic effects of TNF-a in chronic inflammation. We
investigated the responses of primary macrophages to TNF-a over the
course of several days and compared patterns of signaling and gene Page 15 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Signal transduction inhibitors for the treatment of rheumatoid arthritis
Yoshiya Tanaka*, Kunihiro Yamaoka To support this hypothesis, further investigation is needed at the
level of proximal and distal TNF-a signaling events and at the level of
epigenetic regulation of TNF-a-target genes in synovial fibroblasts. References 1. Bartok B, Firestein GS: Immunol Rev 2010, 233(1):233-55. 2. Ivashkiv LB: Eur J Immunol 2011, 41:2477-81. 1. Bartok B, Firestein GS: Immunol Rev 2010, 233(1):233-55. 2. Ivashkiv LB: Eur J Immunol 2011, 41:2477-81. O37
Interleukin-6 as a therapeutic target in locomotor disorders
Norihiro Nishimoto1*, Miho Murakami1, Takaji Matsutani1, Jun Hashimoto2,
Nobuhiro Takagi3
1 O37
Interleukin-6 as a therapeutic target in locomotor disorders
Norihiro Nishimoto1*, Miho Murakami1, Takaji Matsutani1, Jun Hashimoto2,
Nobuhiro Takagi3
1Laboratory of Immune Regulation, Wakayama Medical University, Ibaraki,
567-0085, Japan; 2Department of Orthopaedic Surgery, NHO Osaka-Minami
Medical Center, Kawachinagano, 586-8521, Japan; 3Chugai Pharmaceutical
Co Ltd, Tokyo, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O37 1Laboratory of Immune Regulation, Wakayama Medical University, Ibaraki,
567-0085, Japan; 2Department of Orthopaedic Surgery, NHO Osaka-Minami
Medical Center, Kawachinagano, 586-8521, Japan; 3Chugai Pharmaceutical
Co Ltd, Tokyo, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O37 1Laboratory of Immune Regulation, Wakayama Medical University, Ibaraki,
567-0085, Japan; 2Department of Orthopaedic Surgery, NHO Osaka-Minami
Medical Center, Kawachinagano, 586-8521, Japan; 3Chugai Pharmaceutical
Co Ltd, Tokyo, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O37 Arthritis Research & Therapy 2012, 14(Suppl 1):O37 Interleukin-6 (IL-6) is a multifunctional cytokine that regulates immune
response, inflammation, and hematopoiesis. Although IL-6 plays several
important physiological roles, deregulated overproduction of IL-6 causes
various clinical symptoms and laboratory abnormalities. In the locomotor
disorders such as rheumatoid arthritis (RA) and juvenile idiopathic arthritis
(JIA), IL-6 overproduction has been shown to be involved in inflammatory
manifestations as well as joint destruction. Thus the blocking IL-6
signaling may be a therapeutic approach in those diseases. Various
therapeutic antibodies targeting IL-6 have been developed, and
tocilizumab (TCZ), an anti-IL-6 receptor antibody, precedes the others in
clinical use. y
References Furthermore, low serum IL-6 (<35 pg/mL) and
normalization of MMP-3 levels at cessation of TCZ were identified as
independent predictive markers for the longer duration of drug free
remission. In addition, retreatment with TCZ in the patients, who
responded to initial TCZ monotherapy, and experienced loss of efficacy
after cessation of TCZ, was well tolerated and showed excellent efficacy
equivalent to that observed at the initial treatment with TCZ. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 joint microenvironment, synovial fibroblasts and not Mj are the main
source of IL-6. By adding the supernatants from 96 h TNF-a-stimulated
fibroblast cultures on unstimulated synovial fibroblasts, a similar robust
induction of IL-6 mRNA was observed, suggesting that there is a TNF-a-
induced soluble factor that mediates the sustained response. A similar
pattern of sustained expression was observed for other TNF-a-target
genes including IL-1b, IL-8 and MMPs. Interestingly, there was no
difference between OA- and RA-derived synovial fibroblasts in their
response to TNF-a. O38 O38
Signal transduction inhibitors for the treatment of rheumatoid arthritis
Yoshiya Tanaka*, Kunihiro Yamaoka
The First Department of Internal Medicine, University of Occupational and
Environmental Health, Japan, Kitakyushu, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O38 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 tissue engineering and regeneration is that ultimately the “regenerate tissue”
needs to be a three-dimensional structure. This may be accomplished
through the use of engineered constructs derived by cell seeding into
natural or synthetic biomaterial scaffolds. While direct cell injection is the
most convenient means of cell delivery, a scaffold-based approach is
capable of producing three-dimensional engineered tissues with mechanical
properties compatible with those of various musculoskeletal tissues. Of the
40-50 million Americans with osteoarthritis (OA), an estimated 10-12% suffer
from post-traumatic OA. We have developed an impact model for the
development of post-traumatic OA. Data on the characteristics of this model
in vitro and in vivo will be presented. Focal lesions developed in vivo
resulting from these traumatic impacts will be repaired using stem cell-laden
hydrogel or nanofiber constructs. Concurrently, cell-hydrogel and cell-
nanofibrous constructs are currently being developed for the engineering of
cartilaginous tissues, and information on the fabrication and biological
attributes of these various tissue-engineered composites will be presented. In conclusion, tissue engineering and regenerative medicine presents an
exciting, emerging inter-disciplinary research field that is a natural platform
for life scientists, engineers, and clinicians working together to develop
therapeutic solutions for diseased or injured tissue and organs. Biological agents targeting a specific molecule provide an effective means
for therapeutic management of rheumatoid arthritis (RA) due to their
specificity and powerful functional capabilities, which has resulted in a
paradigm shift in the treatment strategy of this disease. The dramatic
improvement of the sign and symptoms of a patient with RA first came
from the report with chimeric anti-TNF alpha monoclonal, infliximab in
1993. The observation was confirmed in the double-blind randomized
controlled study comparing this biological agent and placebo in 1994. The first approved biologics for RA was TNF Receptor 1-Ig fusion protein,
etanercept in the United States in 1998. Until now, nine biological agents
are approved in RA worldwide. Revolutionary change of RA management
with biological therapies obtained in western countries and Japan has
been reviewed [1]. Atreatment strategy that uses tightly controlled dosesof administered
biologics, targeting clinical remission or low disease activity, and followed
by discontinuation of the biologics may be advantageous from botha
health and economical point of view. This strategy is now being
examinedin several clinical studies and trials in Japan for several biologics,
including infliximab, etanercept, tocilizumab, and abatacept [1]. Peptide therapy in sepsis and inflammation: a novel strategy to
suppress inflammation Hidechika Okada*, Alan Okada Research Institute for Protein Science, Nakayama-cho, Mizuho-ku, Nagoya, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O40 Antisense homology box (AHB): In 1984, Blalock proposed the possible
role of antisense peptides for molecular interaction among proteins
(BBRC, 121: 203, 1984). Antisense homology box (AHB): In 1984, Blalock proposed the possible
role of antisense peptides for molecular interaction among proteins
(BBRC, 121: 203, 1984). 1. Takeuchi T, Kameda H: The Japanese experiences with biologic therapies
for rheumatoid arthritis. Nat Rev Rheumatol 2010, 6:644-652. 1. Takeuchi T, Kameda H: The Japanese experiences with biologic therapies
for rheumatoid arthritis. Nat Rev Rheumatol 2010, 6:644-652. 2. Takeuchi T, Miyasaka N, Tatsuki Y, Yano T, Yoshinari T, Abe T, Koike T:
Baseline tumor necrosis factor alpha levels predict the necessity for
dose escalation of infliximab therapy in patients with rheumatoid
arthritis. Ann Rheum Dis 2011, 70:1208-15. We speculated that interactions between sense- and antisense-peptides
should play a role in formation of the tertiary structure of proteins. We
developed a novel computer program named ANTIS to find antisense
peptide sequences between proteins to be compared (Nature Med. 1:894,1995). ANTIS revealed the presence of an appreciable number of
sense and antisense peptide pairs within any protein molecule and those
portions were designated as antisense homology boxes (AHB). 3. Okuyama A, Nagasawa H, Suzuki K, Kameda H, Kondo H, Amano K,
Takeuchi T: Fc gamma receptor IIIb polymorphism and usage of
glucocorticoids at baseline are associated with infusion reactions to
infliximab in patients with rheumatoid arthritis. Ann Rheum Dis 2011,
70:299-304. 3. Okuyama A, Nagasawa H, Suzuki K, Kameda H, Kondo H, Amano K,
Takeuchi T: Fc gamma receptor IIIb polymorphism and usage of
glucocorticoids at baseline are associated with infusion reactions to
infliximab in patients with rheumatoid arthritis. Ann Rheum Dis 2011,
70:299-304. Complementary peptide: Each peptide should have specific structure
determined by its amino acid sequence which may react with its antisense
peptide. To generate candidates of complementary peptide (C-pep)
reactive to a target amino acid sequence based upon the sense-antisense
amino acid relationship. We invented an evolutionary computer program
(MIMETIC) that generatesC-pep sequences that have a potential to interact
with a target peptide (Microbiol. Imm.46:211, 2002). Complementary peptide: Each peptide should have specific structure
determined by its amino acid sequence which may react with its antisense
peptide. O39 Adult stem cell-based therapy for degenerative joint diseases
Peter G Alexander*, Rocky S Tuan
Center for Cellular and Molecular Engineering, University of Pittsburgh,
Pittsburgh, PA 15219, USA
Arthritis Research & Therapy 2012, 14(Suppl 1):O39 Cell-based therapy for regenerative medicine is a major field of biomedical
research including its use in the treatment of degenerative joint disease. The
goal of regenerative medicine is to develop methods to repair, replace, and
regenerate diseased, injured, or non-functional tissues. Towards this goal,
stem or progenitor cells have been considered a highly desirable candidate
cell type, because of their expandability and potential to be induced toward
specific cell differentiation lineages. A key requirement in musculoskeletal Page 16 of 54 Page 16 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Peptide therapy in sepsis and inflammation: a novel strategy to
suppress inflammation To generate candidates of complementary peptide (C-pep)
reactive to a target amino acid sequence based upon the sense-antisense
amino acid relationship. We invented an evolutionary computer program
(MIMETIC) that generatesC-pep sequences that have a potential to interact
with a target peptide (Microbiol. Imm.46:211, 2002). 4. Sekiguchi N, Kawauchi S, Furuya T, Matsuda K, Ando S, Ogasawara M,
Inaba N, Abe T, Ito S, Takeuchi T: Monitoring of cDNA microarray profile
in peripheral blood during infliximab treatment of rheumatoid arthritis
patients. Rheumatology 2008, 47:780-88. gy
5. Tanino M, Matoba R, Nakamura S, Kameda H, Amano K, Okayama T,
Nagasawa H, Suzuki K, Matsubara K, Takeuchi T: Prediction of efficacy of
anti-TNF biologic agent, infliximab, for rheumatoid arthritis patients
using a comprehensive transcriptome analysis of white blood cells. Biochem Biophys Res Comm 2009, 387:261-265. C5a inhibitory peptides: C5a anaphylatoxin is considered to be an
effective target for treatment of hyperinflammation since C5a stimulates
generation of tumor necrosis factor alpha (TNFa and other inflammatory
cytokines. Amino acids 37 to 53 of C5a (RAARISLGPRCIKAFTE) is an
antisense peptide to AHBpeptides of the C5a receptor (C5aR), and this has
been designated PL37. This region of C5a is presumed to be a potential
site for C5aR stimulation. Using the computer program MIMETIC, we
generated 19 C-peps to PL37. One of the 7 inhibitory C-peps to PL37
which interfered with C5a function was termed PepA(ASGAPAPGPAGP-
LRPMF). To improve stability, we modified PepA by acetylation of its
N-terminal alanine generating acetylated PepA (AcPepA). Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 It is ideal to personalize medical treatment for individual RA patients by
predicting efficacy and safety of a given biologic. In order to identify
predictive factors, enormous amounts of efforts have put forth. Although
several clinical variables have been associated with efficacy and safety,
they are often unrealistic in clinical practice. We found that the baseline
circulating TNF levels [2] and Fc gamma 3B polymorphism [3] are
important predicting factors for response to infliximab in RA patients, and
discuss the role of these markers in real world. Further clinical studies
using biomarkers and molecular expression pattern [4,5] should provide a
clue to find the appropriate predicting markers or even new therapeutic
targets. In the near future, the information accumulated from these studies
may allow selecting the best biological agents in individual patient. References j
g
Support: Commonwealth of Pennsylvania Department of Health and the
United States Department of Defense. Tetsuzo Tauchi
First Department of Internal Medicine, Tokyo Medical University, Shinjuku-ku,
Tokyo 160-0023, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O43 1. Nakao R, Hirasaka K, Goto J, Ishidoh K, Yamada C, Ohno A, Okumura Y,
Nonaka I, Yasutomo K, Baldwin KM, Kominami E, Higashibata A, Nagano K,
Tanaka K, Yasui N, Mills EM, Takeda S, Nikawa T: Ubiquitin ligase Cbl-b is a
negative regulator for insulin-like growth factor 1 signaling during
muscle atrophy caused by unloading. Mol Cell Biol 2009, 29:4798-4811. Recent studies have demonstrated that hedgehog pathway is activated in
chronic myeloid leukemia (CML) stem cells via up-regulation of
Smoothened (Smo), a seven transmembrane domain receptor protein. LDE225 is a small molecule Smo antagonist which has entered Phase I
clinical evaluation in patients with solid tumors. We performed a
comprehensive drug combination experiment using a broader range of
concentrations for LDE225 and nilotinib. Compared with single agents,
the combination of LDE225 and nilotinib was more effective at reducing
the outgrowth of resistant cell clones. No outgrowth was observed in the
presence of 2 μM nilotinib plus 20 μM LDE225. Also co-treatment with
LDE225 and nilotinib resulted in significantly more inhibition of growth
than treatment with either agent alone in BaF3 cells expressing wt-BCR-
ABL and BCR-ABL mutants (M244V, G250E, Q252H, Y253F, E255K, T315A,
T315I, F317L, F317V, M351T, H396P). The observed data from the
isobologram indicated the synergistic effect of simultaneous exposure to
LDE225 and nilotinib even in BaF3 cells expressing T315I. To assess the in
vivo efficacy of LDE225 and nilotinib, athymic nude mice were injected
s.c. with BaF3 cells expressing random mutagenesis for BCR-ABL
mutation. 7 days after injection (average tumor volume, 100 mm3), the
mice were randomised into four groups (5 mice per group), with each
group receiving either vehicle, LDE225 (20 mg/kg; p.o. once every day),
nilotinib (30 mg/kg; p.o. once every day), LDE225 (20 mg/kg; p.o. once
every day) + nilotinib (30 mg/kg; p.o. once every day). The LDE225 and
nilotinib combination more effectively inhibited tumor growth in mice
compared to either vehicle- or nilotinib- or LDE225-treated mice. Histopathologic analysis of tumor tissue from LDE225 plus nilotinib-
treated mice demonstrated an increased number of apoptotic cells
detected by TUNEL staining. To investigate combined effects of LDE225
and nilotinib on primary Ph-positive acute lymphocytic leukemia (ALL)
cells, NOD/SCID mice were injected i.v. with bone marrow mononuclear
cells from a Ph positive ALL patient. Treatment with LDE225 and nilotinib
demonstrated a marked segregation of apoptotic cells in both the central
bone-marrow cavity and the endosteal surface. O44 O44 O43 O43
Combined effects of the hedgehog pathway inhibitor LDE225 and
nilotinib in a random mutagenesis screen
Tetsuzo Tauchi
First Department of Internal Medicine, Tokyo Medical University, Shinjuku-k
Tokyo 160-0023, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O43 Tetsuzo Tauchi
First Department of Internal Medicine, Tokyo Medical University, Shinjuku-ku,
Tokyo 160-0023, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O43 These results suggest
that the combination with a Smo inhibitor and ABL TKIs may help to
eliminate the Ph positive ALL cells. Taken together, the present study
shows that the combination of LDE225 and nilotinib exhibits a desirable
therapeutic index that can reduce the in vivo growth of mutant forms of
BCR-ABL-expressing cells. 2. Sandri M, Sandri C, Gilbert A, Skurk C, Calabria E, Picard A, Walsh K,
Schiaffino S, Lecker SH, Goldberg AL: Foxo transcription factors induce the
atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle
atrophy. Cell 2004, 117:399-412. 2. Sandri M, Sandri C, Gilbert A, Skurk C, Calabria E, Picard A, Walsh K,
Schiaffino S, Lecker SH, Goldberg AL: Foxo transcription factors induce the
atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle
atrophy. Cell 2004, 117:399-412. y
3. Stitt TN, Drujan D, Clarke BA, Panaro F, Timofeyva Y, Kline WO, Gonzalez M,
Yancopoulos GD, Glass DJ: The IGF-1/PI3K/Akt pathway prevents
expression of muscle atrophy-induced ubiquitin ligases by inhibiting
FOXO transcription factors. Mol Cell 2004, 14:395-403. 3. Stitt TN, Drujan D, Clarke BA, Panaro F, Timofeyva Y, Kline WO, Gonzalez M,
Yancopoulos GD, Glass DJ: The IGF-1/PI3K/Akt pathway prevents
expression of muscle atrophy-induced ubiquitin ligases by inhibiting
FOXO transcription factors. Mol Cell 2004, 14:395-403. O45 O42 In the present study, we found that under both in vitro and in vivo
experimental conditions, Cbl-b ubiquitinated and induced specific
degradation of IRS-1, a key intermediate of skeletal muscle growth
regulated by IGF-1/insulin and growth hormone, resulting in inactivation of
Akt-1. Inactivation of Akt-1 led to upregulation of atrogin-1 through
dephosphorylation (activation) of FOXO3, as well as reduced mitogen
response, in skeletal muscle. Thus, activation of Cbl-b may be an important
mechanism underlying the failure of atrophic muscle to respond to growth
factor-based treatments such as IGF-1 (Figure 1). References In the present study, we found that under both in vitro and in vivo
experimental conditions, Cbl-b ubiquitinated and induced specific
degradation of IRS-1, a key intermediate of skeletal muscle growth
regulated by IGF-1/insulin and growth hormone, resulting in inactivation of
Akt-1. Inactivation of Akt-1 led to upregulation of atrogin-1 through
dephosphorylation (activation) of FOXO3, as well as reduced mitogen
response, in skeletal muscle. Thus, activation of Cbl-b may be an important
mechanism underlying the failure of atrophic muscle to respond to growth
factor-based treatments such as IGF-1 (Figure 1). O42 O42
Anti-TNF antibody therapy induces IL-17 suppressing regulatory T cells
in patients with rheumatoid arthritis
Jenny McGovern*, Clare A Notley, Dao Nguyen, Claudia Mauri,
David A Isenberg, Michael R Ehrenstein
Division of Medicine, University College London, London WC1E 6JF, USA
Arthritis Research & Therapy 2012, 14(Suppl 1):O42 in patients with rheumatoid arthritis
Jenny McGovern*, Clare A Notley, Dao Nguyen, Claudia Mauri,
David A Isenberg, Michael R Ehrenstein
Division of Medicine, University College London, London WC1E 6JF, USA
Arthritis Research & Therapy 2012, 14(Suppl 1):O42 AcPepA rescued Cynomolgusmonkyes at lethal shock induced by bacterial
LPS (4 mg/kg).The excellent therapeutic effect of AcPepA is due to
restriction of high mobility group box 1 (HMGB1) surge induced by the
effect of C5a on C5L2, which is the second C5a receptor, since the
released HMGB1 has the capacity to stimulate TLR4 as an endogeneous
ligand resulting in further activation of inflammatory cells to release
inflammatory cytokines forming positive feedback circuit of inflammation. Biologic therapies not only offer the prospect of improved patient
outcomes in a variety of autoimmune diseases, but also the opportunity to
explore the specific target’s role in the underlying mechanisms of disease. Over recent years we have studied the role of regulatory T cells (Treg) in
patients with rheumatoid arthritis before and after anti-TNF therapy. We
have shown that Treg from patients with rheumatoid arthritis have
defective suppressor function. This Treg defect is linked with abnormalities
in the expression and function of CTLA-4. Anti-TNF antibody therapy did
not reverse CTLA-4 dysfunction but instead induced the differentiation of a
distinct and potent Treg population. These induced Treg were able to
inhibit IL-17 production, in contrast to Treg from healthy individuals,
patients with active RA or RA patients treated with etanercept, a modified O41 Overview of biotherapy in rheumatoid arthritis (RA)
Tsutomu Takeuchi*, Hideto Kameda
Division of Rheumatology, Department of Internal Medicine, School of
Medicine, Keio University, Shinjyuku,Tokyo, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O41 Page 17 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 TNF receptor. These results may provide mechanistic insight into the
therapeutic benefit of switching between different anti-TNF agents and
the differing incidence of tuberculosis between adalimumab and
etanercept. Regulation of immune cell responses by semaphorins and their
receptors Atsushi Kumanogoh1,2*, Hyota Takamatsu1,2
1Department of Respiratory Medicine, Allergy and Rheumatic Diseases,
Graduate School of Medicine Osaka University, 2-2 Yamada-oka, Suita, Osaka
565-0871, Japan; 2World Premier International Immunology Frontier Research
Center, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O45 Atsushi Kumanogoh1,2*, Hyota Takamatsu1,2
1Department of Respiratory Medicine, Allergy and Rheumatic Diseases,
Graduate School of Medicine Osaka University, 2-2 Yamada-oka, Suita, Osaka
565-0871, Japan; 2World Premier International Immunology Frontier Research
Center, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O45 Background: Semaphorins were originally identified as axon guidance
factors involved in the development of the neuronal system. However,
accumulating evidence indicates that several members of semaphorins, so-
called ‘immune semaphorins’, are crucially involved in various phases of
immune responses (Figure 1) [1-3]. In addition, semaphorins and their
receptors have been shown to be crucial for the pathogenesis of
immunological disorders such as atopic dermatitis, multiple sclerosis,
systemic sclerosis, systemic lupus erythematosus and rheumatoid arthritis,
These semaphorins regulate immune cell interactions during physiological
and pathological immune responses. However, conventional static analysis
could not determine definitively whether they regulate immune cell
movement. Materials and methods: Plexin-A1-/- mice were previously established
[4]. Combinational studies, including imaging technique for visualizing
single-cell dynamics and conventional immunological assays were
performed. Results and discussion: We find that plexin-A1-mediated semaphorin
signals are crucially involved in the transmigration of DCs across the
lymphatics to exit the periphery to induce antigen-specific T-cell priming
using plexin-A1-/- mice. In addition, adoptive transfer experiments identify
that Sema3A produced in the lymphatics functions as a ligand for the
plexin-A1/NP-1 receptor complex expressed in DCs. Interestingly, plexin-A1
is localized at the trailing edge but not the leading edge of DCs during
migration. Sema3A induces phosphorylation of the myosin light chain to
promote actomyosin contraction, resulting in increased DC velocity in the
constricted area (Figure 2). Collectively, these findings not only demonstrate
the involvement of semaphorins in immune cell trafficking but also indicate
that semaphorins are therapeutic targets to treat immunological disorders. References 1.
Suzuki K, et al: Semaphorin 7A initiates T-cell-mediated inflammatory
responses through alpha1beta1 integrin. Nature 2007, 446:680-684.
2.
Suzuki K, et al: Semaphorins and their receptors in immune cell
interactions. Nat Immunol 2008, 9:17-23.
3.
Nogi T, et al: Structural basis for semaphorin signalling through the
plexin receptor. Nature 2010, 467:1123-7.
4.
Takegahara N, et al: Plexin-A1 and its interaction with DAP12 in immune
responses and bone homeostasis. Nat Cell Biol 2006, 8:615-622.
5.
Takamatsu H, et al: Semaphorins guide the entry of dendritic cells into
the lymphatics by activating myosin II. Nat Immunol 2010, 11:594-600. References Tetsuzo Tauchi
First Department of Internal Medicine, Tokyo Medical University, Shinjuku-ku,
Tokyo 160-0023, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O43 Molecular mechanism of unloading-mediated muscle atrophy and
development of its countermeasures
k
h
k Takeshi Nikawa
Department of Nutritional Physiology, Institute of Health Biosciences, The
University of Tokushima Graduate School, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O44 The ubiquitin ligase Cbl-b plays a major role in skeletal muscle atrophy
induced by unloading [1]. The mechanism of Cbl-b-induced muscle
atrophy is unique in that it does not appear to involve the degradation of
structural components of the muscle, but rather it impairs muscular
trophic signals in response to unloading conditions. Recent studies on the
molecular mechanisms of muscle atrophy have focused on the role of IGF-
1/PI3K/Akt-1 signaling cascade as a vital pathway in the regulation of the
balance between hypertrophy and atrophy [2,3]. These studies indicate
that under muscle wasting conditions, such as disuse, diabetes and fasting,
decreased IGF-1/PI3K/Akt-1 signaling augments the expression of atrogin-
1, resulting in muscle atrophy. However, these studies did not address the
mechanisms of unloading-induced impairment of growth factor signaling. 1. Suzuki K, et al: Semaphorin 7A initiates T-cell-mediated inflammatory
responses through alpha1beta1 integrin. Nature 2007, 446:680-684. 2. Suzuki K, et al: Semaphorins and their receptors in immune cell
interactions. Nat Immunol 2008, 9:17-23. 3. Nogi T, et al: Structural basis for semaphorin signalling through the
plexin receptor. Nature 2010, 467:1123-7. 4. Takegahara N, et al: Plexin-A1 and its interaction with DAP12 in immune
responses and bone homeostasis. Nat Cell Biol 2006, 8:615-622. 5. Takamatsu H, et al: Semaphorins guide the entry of dendritic cells into
the lymphatics by activating myosin II. Nat Immunol 2010, 11:594-600. 1. Suzuki K, et al: Semaphorin 7A initiates T-cell-mediated inflammatory
responses through alpha1beta1 integrin. Nature 2007, 446:680-684. 2. Suzuki K, et al: Semaphorins and their receptors in immune cell
interactions. Nat Immunol 2008, 9:17-23. 1. Suzuki K, et al: Semaphorin 7A initiates T-cell-mediated inflammatory
responses through alpha1beta1 integrin. Nature 2007, 446:680-684. 2. Suzuki K, et al: Semaphorins and their receptors in immune cell
interactions. Nat Immunol 2008, 9:17-23. 3. Nogi T, et al: Structural basis for semaphorin signalling through the
plexin receptor. Nature 2010, 467:1123-7. 3. Nogi T, et al: Structural basis for semaphorin signalling through the
plexin receptor. Nature 2010, 467:1123-7. 4. Takegahara N, et al: Plexin-A1 and its interaction with DAP12 in immune
responses and bone homeostasis. Nat Cell Biol 2006, 8:615-622. 5. Takamatsu H, et al: Semaphorins guide the entry of dendritic cells into
the lymphatics by activating myosin II. Nat Immunol 2010, 11:594-600. 4. P1
Reconstruction of injured spinal cord by epigenetic regulation of
transplanted neural stem cells In collecting FBB samples, we always keep in mind future biochemical and
molecular analyses and collaborations. The brains are separated into two
hemispheres. One hemisphere is fixed in formalin for neuropathological
analysis and the other is precisely subdivided into coronary sections and
small blocks which are saved in Eppendorf tubes. After samples are
photographed, they are frozen on dry ice (slices) and in liquid nitrogen
(tubes). Finally, all material is stored at -80 degrees in 9 refrigerators for later
use in research. Masahiko Abematsu1,2,3*, Keita Tsujimura3, Mariko Yamano5, Michiko Saito4,
Kenji Kohno4, Takao Setoguchi1,2, Kazunori Yone1, Kinichi Nakashima3,
Setsuro Komiya1,2 1Department of Orthopaedic Surgery, Graduate School of Medical and Dental
Sciences, Kagoshima University, Kagoshima 890-8520, Japan; 2The Near-Future
Locomotor Organ Medicine Creation Course (Kusunoki Kai), Graduate School of
Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8520, Japan;
3Laboratory of Molecular Neuroscience, Graduate School of Biological Sciences,
Nara Institute of Science and Technology, Ikoma 631-0192, Japan; 4Laboratory of
Molecular and Cell Genetics, Graduate School of Biological Sciences, Nara Institute
of Science and Technology, Ikoma 631-0192, Japan; 5Department of Although our bank has gone unrecognized in the past, our farsighted efforts
have been gaining considerable attention in recent years in Japan. We now
have over 20 collaborators and supply more than 30 research institutes with
our samples. In addition, our research institute was approved in 2004 by the
Japanese Ministry of Education, Culture, Sports, Science and Technology, as
one of the non-governmental institutes which is permitted to apply for
governmental grants and we became a member of the Comprehensive
Brain Science Network in 2010. FBB at the Choju Medical Institute,
Fukushimura Hospitalis a unique facility and one of the most active brain
banks in the world. gy
p
p
Comprehensive Rehabilitation, Osaka Prefecture University, Habikino 583-8555,
Japan p
Arthritis Research & Therapy 2012, 14(Suppl 1):P1 Arthritis Research & Therapy 2012, 14(Suppl 1):P1 Background: Neural stem cells (NSCs) possess the ability to self-renew and
to differentiate into the three major cell types found in the central nervous
system (CNS). Recent studies have shown that epigenetic gene regulation
events such as DNA methylation and histone modification play important
roles in regulating NSC fate specification. In this context, we have previously
shown that the histone deacetylase inhibitor valproic acid (VPA) enhances
neuronal differentiation of NSCs. g
References References References 1. Jones J, Wu K, Yang Y, Guerrero C, Nillegoda N, Pan ZQ, Huang L: A 1. Jones J, Wu K, Yang Y, Guerrero C, Nillegoda N, Pan ZQ, Huang L: A
targeted proteomic analysis of the ubiquitin-like modifier NEDDD8 and
associated proteins. J Proteome Res 2008, 7:1274-1287. “Fukushi” is a Japanese word that means welfare and “mura” is a village. We
have several buildings for the aged and disabled, and about 800 elderly
people reside within the complex. targeted proteomic analysis of the ubiquitin-like modifier NEDDD8 and
associated proteins. J Proteome Res 2008, 7:1274-1287. p
2. Ohki Y, Funatsu N, Konishi N, Chiba T: The mechanism of poly-NEDD8 2. Ohki Y, Funatsu N, Konishi N, Chiba T: The mechanism of poly-NEDD8
chain formation in vitro. Biochem Biophys Res Commun 2009, 381:443-447. The Fukushimura Hospital was established in 1982 and is managed by the
Sawarabi MedicalCooperative. It currently has 487 beds. Our patients mainly
have dementia and cerebrovascular problems. The hospital plays a pivotal
role within the village and acts as the central facility. y
3. Alexandra Zhernakova A, et al: Meta-analysis of genome-wide association
studies in celiac disease and rheumatoid arthritis identifies fourteen
non-HLA shared loci. PloS Genet 2011, 7:e10020004. FBB was established in 1990. We have a long history of collecting samples,
not only from patients but also from residents of our care houses and
nursing homes within the Fukushimura complex. This allows us as medical
doctors and researchers to obtain clinical information or blood samples,
sometimes even before the onset of illness. In our institute, all clinical and
pathological dataare held in the office of individual data management. P1
Reconstruction of injured spinal cord by epigenetic regulation of
transplanted neural stem cells Perhaps because these patterns of NSC
differentiation are exquisitely controlled during normal embryonic
development, restoration of damaged neural networks in the injured adult
CNS is severely limited. Here, using a mouse model of spinal cord injury(SCI),
we examined the effectiveness of NSC transplantation and differentiation
control by VPA administration. P2 P2
Fukushimura Brain Bank (FBB) -Based in a private geriatric hospital-
Hiroyasu Akatsu1,2*, Akira Hori1,2, Hidechika Okada1, Yoshio Hashizume1,2,
Takayuki Yamamoto1,2
1Choju Medical Institute, Fukushimura Hospital, Japan; 2Institute of
Neuropathology, Fukushimura Hospital, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P2 PNBP1 interacted with NEDD8, NEDD8-conjugating enzyme Ubc12 and Cul1. PNBP1 strongly associated with wild-type Cul1, but not its NEDDylation
defective Cul1(K720R) mutant, suggesting that the interaction is mediated in
part through NEDD8. Furthermore, PNBP1 promoted NEDDylation of Cul1 in
an in vitro reconstitution assay. These activities were dependent on RING-
finger domain of PNBP1. Finally, knockdown of PNBP1 led to reduction of
the NF-B activation, suggesting that PNBP1 is an important modulator of
the NF-B signaling pathway. Fukushimura Brain Bank (FBB) -Based in a private geriatric hospital-
Hiroyasu Akatsu1,2*, Akira Hori1,2, Hidechika Okada1, Yoshio Hashizume1,2,
Takayuki Yamamoto1,2 y
1Choju Medical Institute, Fukushimura Hospital, Japan; 2Institute of
Neuropathology, Fukushimura Hospital, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P2 Fukushimura Brain Bank (FBB) was established under the auspices of
Fukushimura Hospital, a legally incorporated medical institution. It is
managed completely within the private sector. POSTER PRESENTATIONS P1
Reconstruction of injured spinal cord by epigenetic regulation of
transplanted neural stem cells O46 O46
A novel NEDD8-binding protein modulates NF-B signaling pathway
Osamu Takashima*, Fuminori Tsuruta, Manato Ebina, Yu Kigoshi,
Shingo Nakamura, Tomoki Chiba
Graduate School of Life and Environmental Sciences, University of Tsukuba,
Tennodai, Tsukuba, Ibaraki 305-8577, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):O46 y
Results: We show that transplanting NSCs and administering VPA enhances
the functional recovery of their hindlimbs. Neuronal differentiation of
transplanted NSCs was promoted in VPA-treated mice. Anterograde
corticospinal tract tracing revealed that transplant-derived neurons partially
reconstructed the broken neuronal circuits, most likely in a ‘relay’ manner. Ablation of the transplanted cells abolished the recovery of hindlimb motor
function, indicating that transplanted cells contributed directly to the
improvement of motor function. In canonical NF-B signaling pathway, a ubiquitin ligase called SCF (Skp1,
Cul1, F-box protein) complex is essential for I-B degradation. The activity
of the SCF complex is positively regulated by a post-translational
modification of Cul1 subunit with a ubiquitin-like protein NEDD8. Like
ubiquitin, NEDD8 possesses evolutionary conserved Lys residues on its
surface, and forms poly-NEDD8 chain in vivo and in vitro [1,2]. Despite the
importance of the NEDD8 modification in all eukaryotic cells, little is
known about the function of poly-NEDD8 chain. To elucidate the function
of the poly-NEDD8 chain in vivo, we screened poly-NEDD8 chain binding
proteins (PNBPs) using a yeast two-hybrid system. Of the identified PNBPs,
PNBP1 was identical to a gene present in non-HLA celiac disease and
rheumatoid arthritis risk loci [3]. Conclusions: These data raise the possibility that epigenetic regulation
in transplanted neural stem cells can be exploited to provide treatment
for SCI. Molecular mechanism of unloading-mediated muscle atrophy and
development of its countermeasures
k
h
k Takegahara N, et al: Plexin-A1 and its interaction with DAP12 in immune
responses and bone homeostasis. Nat Cell Biol 2006, 8:615-622. 5. Takamatsu H, et al: Semaphorins guide the entry of dendritic cells into
the lymphatics by activating myosin II. Nat Immunol 2010, 11:594-600. Page 18 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Figure 1(abstract O44) Possible mechanism of unloading-mediated muscle atrophy. Figure 1(abstract O44) Possible mechanism of unloading-mediated muscle atrophy. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Page 19 of 54 Figure 1(abstract O45) Semaphorins are involved in physiological and pathological immune responses. Figure 2(abstract O45) Sema3A produced by the lymphatic induces actomyosin contraction during transmigration. Figure 1(abstract O45) Semaphorins are involved in physiological and pathological immune responses. O45) Semaphorins are involved in physiological and pathological immune responses. Figure 2(abstract O45) Sema3A produced by the lymphatic induces actomyosin contraction during transmigration. Figure 2(abstract O45) Sema3A produced by the lymphatic induces actomyosin contraction during transmigration. Page 20 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 then intraperitoneally administered VPA or saline daily, for 7 days, whereafter
we monitored their hindlimb motor function using the open field locomotor
scale for 6 weeks. We next analyzed the migration, morphology, neuronal
marker expression and viability of these cells after co-administration with
VPA. We examined extensively the roles of the neurons responsible for
reconstruction of broken neuronal networks using two neuronal tracers,
immunoelectron microscopy, and two cell-ablation methods. O46 P6 The compliance of patients with treatment was rated as in 24 good, in 36
fair and in 14 bad. P6
Intermittent cold stress-induced experimental fibromyalgia model in
mice - pharmacology and neurobiology
Kohei Araki*, Michiko Nishiyori, Hiroshi Ueda
Division of Molecular Pharmacology and Neurosciences, Nagasaki University
Graduate School of Biomedical Sciences, Nagasaki 852-8521, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P6 P5 P5
A novel role for monosodium urate monohydrate crystals and gouty
synovial fluids in monocyte migration in gout
M Asif Amin1*, Qiang Shu1, Jonathon W Vargo1, Jeffrey H Ruth1,
Takeo Isozaki1, Solhee Lee1, Alisa E Koch2
1University of Michigan Medical School, Ann Arbor, MI, USA; 2Department of
Veteran’s Affairs and University of Michigan, Ann Arbor, MI, 48109, USA
Arthritis Research & Therapy 2012, 14(Suppl 1):P5 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Recommendations: We need to inform the thalassemic patients about
the risk of osteoporosis and the need for their awareness regard such
complication and the importance of their compliance with therapy. Background:
IL-1
receptor
antagonist
deficient
(Il1rn-/-)
mice
spontaneously develop arthritis. We previously demonstrated that IL-17
plays a crucial role in the development of arthritis in Il1rn-/- mice. Furthermore we showed that IL-1 Ra-deficiency in T cells is important for
the development of arthritis. It is not known, however, which IL-17-
producing cells are involved in the pathogenesis of arthritis in this model. Results: To identify the source of IL-17 in Il1rn-/- mice, we analyzed IL-17-
producing cells. We found that IL-17 production from both CD4+ T cells and
©™T cells was increased in the draining lymph nodes. To clarify the roles of
CD4+ T cells and ©™T cells in the development of arthritis, ©™T cells or CD4+
T cells were depleted in Il1rn-/- mice using antibodies. The development of
disease was suppressed in both cases, suggesting both Th17 cells and IL-17-
producing ©™T cells were involved in the pathogenesis. Then, the
pathogenic role of IL-17-producing ©™T cells in the absence of Th17 cells
was examined. Osteoporosis in Iraqi patients with thalassemia
1*
1 Results: We found a significant two fold increase in in vitro MN migration
in response to MSU crystals, while gouty SFs increased MN migration five
fold compared to negative control (p < 0.05). MSU crystal induced MN
migration was significantly decreased by inhibitors of p38 MAPK, Src, and
NFB, suggesting that crystal induced MN migration occurs via these
pathways. After engrafting SCID mice for 4 weeks, we injected dye-tagged
human PB MNs via tail vein. Simultaneously, we injected MSU crystals or
gouty SFs into ST grafts. After 48 hours, we harvested the STs and found
an increase in MN homing to the grafts injected with MSU crystals or SFs
(p < 0.05), indicating that either of these stimuli could recruit MNs in vivo. Human MNs stimulated with MSU for 24 hours released significantly higher
quantities of the potent leukocyte chemoattractants MIF and ENA-78/
CXCL5. MIF was six fold higher in gouty SFs compared to osteoarthritic
fluids, suggesting the importance of MIF in gouty arthritis. MIF or ENA-78/
CXCL5 secretion depended on the p38 MAPK pathway. Background: Thalassemia is defined as a complete absence of one or more
of the four globins in the red blood cells due to the deletion of or
nonfunctioning of one or more genes. g
g
Osteoporosis is a universal medical problem, affecting both genders. Materials and methods: 74 thalassemic patients 36 male and 38 female
below the age of 25 years. The study was a clinical cross-sectional for both genders with thalassemia
major, Investigation done included a chest × ray, serum iron, total iron
binding capacity (TIBC), transferrin saturation, serum calcium, serum
phosphorus, serum alkaline phosphatase, blood urea, serum creatinine,
and a DXA bone scan. y
Conclusions: This data suggests an intriguing role for MSU crystals and
gouty SFs in MN migration and provides evidence that MNs and their
secreted products may be potential therapeutic targets for treating gout. Statistical analysis:-P-value–S.P.S.S.–chi-square. Results: We found that the bony disorder in thalassemic patients
increased with age (bone pain, carpopedal spasm, osteoporosis), and with
low serum iron and low T.I.B.C. and with increased transferrin saturation. The compliance of patients with treatment was rated as in 24 good, in 36
fair and in 14 bad. Results: We found that the bony disorder in thalassemic patients
increased with age (bone pain, carpopedal spasm, osteoporosis), and with
low serum iron and low T.I.B.C. and with increased transferrin saturation. P5
A novel role for monosodium urate monohydrate crystals and gouty
synovial fluids in monocyte migration in gout
* Takeo Isozaki1, Solhee Lee1, Alisa E Koch2
1University of Michigan Medical School, Ann Arbor, MI, USA; 2Department of
Veteran’s Affairs and University of Michigan, Ann Arbor, MI, 48109, USA
Arthritis Research & Therapy 2012, 14(Suppl 1):P5 Background: Gout is characterized by intra-articular deposition of
monosodium urate monohydrate (MSU) crystals. The role of neutrophil
influx in acute gouty arthritis is well established, while the contribution of
monocytes (MNs) and their secreted inflammatory mediators is not. Here
we demonstrate the role of MSU in MN migration. We generated mice with IL-17 producing ©™T cells, but without Th17 cells,
by adoptively transferring Il17-/-Il1rn-/–T cells into nude mice in which IL-17-
producing ©™T cells are present. We found that these mice still developed
arthritis and that only ©™T cells produced IL-17. Finally, to corroborate that
the development of arthritis in this transfer system is dependent on IL-17,
we adoptively transferred Il17-/-Il1rn-/--T cells into Il17-/-nu/nu mice. The
development of arthritis was significantly suppressed in Il17-/-Il1rn-/--T cell-
transferred Il17-/-nu/nu mice compared with Il-17+/+nu/nu mice transferred
with Il17-/-Il1rn-/--T cells, suggesting that ©™T -cell-derived IL-17 is important
for the develop arthritis. © Materials and methods: To examine the role of MSU crystals in normal
human peripheral blood (PB) MN migration, we performed MN chemotaxis
in a modified Boyden chamber in vitro using either MSU crystals or gouty
synovial fluids (SFs) as stimuli. To examine mechanisms of MN migration, we
performed MN chemotaxis with MSU in the presence or absence of
chemical signaling inhibitors. We determined the in vivo role of MSU crystals
or gouty SFs in homing of dye-tagged MNs using normal human synovial
tissue (ST)-severe combined immunodeficient (SCID) mouse chimeras. To
investigate the contribution of MSU to production of leukocyte
chemoattractants macrophage migration inhibitory factor (MIF) and
epithelial neutrophil activating factor-78 (ENA-78/CXCL5), and the signaling
molecules involved in secretion of these cytokines, we stimulated MNs with
MSU crystals with or without chemical signaling inhibitors, and performed
ELISAs on conditioned medium. We also assayed for MIF in gouty SF by
ELISA. Conclusion: These results indicate that ©™T cell-derived IL-17 plays an
important role in the pathogenesis of arthritis in Il1rn-/- mice. P3 Materials and methods: NSCs were transplanted into the SCI epicenter
7 days after injury. Non-transplanted control and transplanted mice were Page 21 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 P5
A novel role for monosodium urate monohydrate crystals and gouty
synovial fluids in monocyte migration in gout
* P4
Osteoporosis in Iraqi patients with thalassemia
Salim M AL Jadir1*, Mohamed Z Jalal1, Median F AL Ghreer2,
Mozahem S AL Hamdani2, Wamid R AL Omaree3
1Al Salaam General Teaching Hospital in Mosul City, Iraq; 2Ibn Al Atheer
Teaching Hospital, Thalassemia center, Mosul City, Iraq; 3Medical College of
Mosul UNV, Mosul, Iraq
Arthritis Research & Therapy 2012, 14(Suppl 1):P4 P4
Osteoporosis in Iraqi patients with thalassemia
Salim M AL Jadir1*, Mohamed Z Jalal1, Median F AL Ghreer2,
Mozahem S AL Hamdani2, Wamid R AL Omaree3
1Al Salaam General Teaching Hospital in Mosul City, Iraq; 2Ibn Al Atheer
Teaching Hospital, Thalassemia center, Mosul City, Iraq; 3Medical College of
Mosul UNV, Mosul, Iraq
Arthritis Research & Therapy 2012, 14(Suppl 1):P4 P9
PTEN in antigen presenting cells is a master regulator for Th17-
mediated autoimmune pathology
1*
2*
2
1 The high frequency of appearance of ferrous deficit anemia among RA
patients, probably is explained by that in conditions of this disease
changes of pH happen among gastro duodenal area. Besides, wide use of
non steroidal anti inflammatory medicine (NAIM) at RA also may effect to
pH of stomach. And in cases of destroyed reaction of ambience change
of ferrous assimilation. That fact of ferrous deficit anemia may has
independent character at analyzed RA patients is excluded. But on their
history of illness it is impossible to determine this fact. Stephan Blüml1*, Gernot Schabbauer2*, Eva Hainzl2, Birgit Niederreiter1,
Anastasia Hladik1, Tobias Lohmeyer2, Michael Bonelli1, Elisabeth Zinser3,
Marije Koenders4, Wim van den Berg4, Giulio Superti-Furga5, Josef S Smolen1,
Kurt Redlich1 1Division of Rheumatology, Internal Medicine III, Medical University of
Vienna, Austria; 2Institute for Vascular Biology and Thrombosis Research,
Center for Biomolecular Medicine and Pharmacology, Medical University
Vienna, A-1090 Vienna, Austria; 3Department of Dermatology,
Hartmannstasse 14, University Hospital Erlangen, 91052 Erlangen, Germany;
4Rheumatology Research and Advanced Therapeutics, Department of
Rheumatology, Radboud University Nijmegen Medical Center, Nijmegen, The
Netherlands; 5CeMM - Center for Molecular Medicine of the Austrian
Academy of Sciences, Vienna 1090, Austria
Arthritis Research & Therapy 2012, 14(Suppl 1):P9 Study of offenses of appearance of anemia at RA patients depending on age
categories is evidencing on that 83,4% of patients with anemia comes to
patients from 31 to 60 years old, and among patients of 31 to 40 years old
appears 25% patients, from 41 to 50 years old - 26,7% and from 51 to
60 years old - 31,7%, accordingly. Results of these analysis showed that if at patients with debut RA anemia
appears at 1,5% cases, than among RA patients with prolongation of
anamnesis from 1 to 5 years old, from 5 to 10 years old appears in
33,3%, 28,7% and in 34,8% cases accordingly. Therefore as far as
increasing of prolongation of current of RA, specific gravity of patients
with anemia increases. Autoreactive T cells are a central element in many systemic autoimmune
diseases. The generation of these pathogenic T cells is instructed by antigen
presenting cells. However, signalling pathways in APC that drive
autoimmunity are not completely understood. Here we show that that
conditional deletion of PTEN in myeloid cells are almost completely
protected from the development of two prototypic model autoimmune
diseases, collagen induced arthritis (CIA) and experimental autoimmune
encephalomyelitis (EAE). Frequency of appearance of anemias at rheumatoid arthritis - a disease
of autoimmune genesis (on the data of retrospective study)
1*
2
2 p
,
,
1Tashkent Institute of Postgraduate Medical Education, Tashkent, Uzbekistan;
2Tashkent Medical Academy, Tashkent, Uzbekistan
Arthritis Research & Therapy 2012, 14(Suppl 1):P7 The purpose of research is study of offenses of appearance of anemia
among rheumatoid arthritis (RA) patients, revealing of their etiologic
reasons, as well as the estimation of character of used anti anemia means
of medicine on the basis of retrospective analysis of history of disease. Analysing the clinical parameters of RA in hTNFtg mice, we observed a delay
of onset of paw swelling in mice treated with YopM. At histological analysis
of the hind paws, we found reduced bone destruction and decreased
osteoclast formation, as well as less inflammation in YopM treated hTNFtg
mice in comparison to untreated hTNFtg mice. Coming out of above stated histories of illness of RA patients were
analyzed to presence of established as accompanying disease of anemia. Results of this analysis are represented on picture as it seen on the
presented data, 33,3% of patients with RA anemia is verified as
accompanying pathology. Therefore at 1/3 patients with P anemia takes
place. The study of etiologic causes of anemia at these patients shows
that in 76,6% cases anemia bears ferrous deficit character, 20%- anemia
of chronic diseases and only in 3,4% cases - auto immune anemia. Therefore, the majority of patients of RA anemia bears ferrous deficit
character. These results suggest that YopM has the potential to reduce inflammation
and bone destruction in vivo. For this reason YopM may constitute a
novel therapeutic agent for the treatment of RA. P9
PTEN in antigen presenting cells is a master regulator for Th17-
mediated autoimmune pathology
1*
2*
2
1 Intermittent cold stress-induced experimental fibromyalgia model in
mice - pharmacology and neurobiology
* The prevalence of osteoporosis in thalassemic Iraqi patients DXA scans
was found to be 67.5% while osteopenia was found in 9.4% and normal
BMD in 22.9%. Kohei Araki*, Michiko Nishiyori, Hiroshi Ueda
Division of Molecular Pharmacology and Neurosciences, Nagasaki University
Graduate School of Biomedical Sciences, Nagasaki 852-8521, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P6 Discussion: During the last decade, the presence of osteopenia and
osteoporosis in well-treated thalassaemics has been described in different
studies with high prevalence up to 50%. Several factors are implicated in reduction of bone mass in thalassaemia
major. Delayed sexual maturation, growth hormone (GH) and insulin
growth factor-(IGF)-1 deficiency, parathyroid gland dysfunction, diabetes,
hypothyroidism, ineffective haemopoiesis with progressive marrow
expansion, direct iron toxicity on osteoblasts, as well as liver disease have
been indicated as possible etiological factors for thalassaemia-induced
osteoporosis. Furthermore, iron chelating has correlated with growth
failure and bone abnormalities, and high desferrioxamine dosage has
been associated with cartilage alterations. Stress-induced pain, as in Fibromyalgia (FM), is considered to be caused by
intense events involving physical and psychological injury and is reinforced
by successive stress. Previously, we have established a novel mice model of
FM, using intermittent cold stress (ICS) exposure. Mice given ICS caused
abnormal pain, including mechanical allodynia and hyperalgesia to
nociceptive thermal and chemical stimuli, which lasted for more than
2 weeks. In contrast, those given constant cold stress (CCS) did not. The
abnormal pain was generalized, female-predominant and specific for A-delta
and A-beta, but not C-fiber-stimuli in the electrical stimulation-induced
nociceptive test. The mechanical allodynia induced by ICS was effectively
suppressed by intraperitoneal or intracerebroventricular injection of
gabapentin. The potency and duration of anti-allodynia effects were much Conclusions: Osteoporosis in thalassemic Iraqi patient was too high and
even more in those patients with bad compliance regard attendance to
the Thalassemia centre. Page 22 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 protein of Yersinia species that is able to enter host cells by membrane
penetration. In the cell YopM mediates down-regulation of inflammatory
responses. We investigated whether YopM has the potential to act as a
“selfdelivering” immune therapeutic agent by reducing the inflammation
and joint destruction linked to RA. protein of Yersinia species that is able to enter host cells by membrane
penetration. In the cell YopM mediates down-regulation of inflammatory
responses. We investigated whether YopM has the potential to act as a
“selfdelivering” immune therapeutic agent by reducing the inflammation
and joint destruction linked to RA. higher and longer, respectively, than the neuropathic pain induced by sciatic
nerve injury. Taken together, these findings indicate that mice given ICS
manifest most of characteristics observed in fibromyalgia patients in terms
of pharmacology and pain physiology. Acknowledgements: The research described in this article was
supported in part by MEXT KAKENHI (17109015 to Hiroshi Ueda) and
Health Labor Sciences Research Grants from the Ministry of Health, Labor
and Welfare of Japan (to Hiroshi Ueda): “Research on Allergic disease and
Immunology” also supported this work. Using confocal laser scanning we analysed the penetration of recombinant
YopM into bone marrow macrophages (BMMs). Furthermore we studied the
effects of YopM on osteoclastogenesis using in vitro osteoclast formation
assay. To unravel the signaling pathways of YopM, we tested for
phosphorylation of MAP-kinases (ERK, AKT and p-38) and activation of NF-KB
signaling by Western Blot analysis. With respect to a potential in vivo
application of YopM, we injected YopM intra articular and intravenous in
mice and monitored the distribution by fluorescence reflection imaging
(FRI). We treated hTNFtg mice, as animal model for RA, with YopM and
recorded clinical parameters (weight, grip strength and paw swelling). Finally we analysed the destruction of bone and cartilage histologically
compared to untreated hTNFtg mice and wildtype mice. P9
PTEN in antigen presenting cells is a master regulator for Th17-
mediated autoimmune pathology
1*
2*
2
1 Myeloid specific deletion of PTEN lead to a
significant reduction of cytokines pivotal for the induction of systemic
autoimmunity such as IL-23 and IL-6 in vitro and in vivo. In addition, PTEN
deficient dendritic cells showed reduced activation of p38 MAP-kinase and
increased inhibitory phosphorylation of GSK3b in vitro. Dendritic cell and
macrophage phenotypic maturation and migration to lymph nodes as well
as collagen specific T and B cell activation was comparable in wt and
myeloid specific PTEN-/-. However, analysing the impact of myeloid specific
PTEN deficiency on T cell polarization, we found a significant reduction of a
Th17 type of immune response characterized by reduced production of P8 References 1. Nishiyori M, Ueda H: Prolonged gabapentin analgesia in an experimental
mouse model of fibromyalgia. Mol Pain 2008, 4:52. 1. Nishiyori M, Ueda H: Prolonged gabapentin analgesia in an experimental
mouse model of fibromyalgia. Mol Pain 2008, 4:52. 1. Nishiyori M, Ueda H: Prolonged gabapentin analgesia in an experimental
mouse model of fibromyalgia. Mol Pain 2008, 4:52. 2. Nishiyori M, Nagai J, Nakazawa T, Ueda H: Absence of morphine analgesia
and its underlying descending serotonergic activation in an experimental
mouse model of fibromyalgia. Neurosci Lett 2010, 472:184-187. 2. Nishiyori M, Nagai J, Nakazawa T, Ueda H: Absence of morphine analgesia
and its underlying descending serotonergic activation in an experimental
mouse model of fibromyalgia. Neurosci Lett 2010, 472:184-187. As seen in confocal scanning microscopy, YopM penetrated the cell
membrane of BMMs and accumulated near the nucleus. Studying the
signaling pathways affected by YopM, we found that YopM reduced the
TNFa induced activation of NF-kB via reducing the phosphorylation of IkBa. TNFa mediated phosphorylation of MAP kinases were not altered by YopM. Most interestingly, we found a strong reduction of osteoclast formation by
YopM. Incubation of BMMs with YopM led to a 90% reduction in osteoclasts
precursors and osteoclasts. YopM-Cy5 injected into the hind paws of hTNFtg
mice was detectable in the joint without a systemic distribution for 48 hours
and elimination mediated through renal clearance. P7
Frequency of appearance of anemias at rheumatoid arthritis - a disease
of autoimmune genesis (on the data of retrospective study)
Abdumalik N Aripov1*, Marif SH Karimov2, Aida A Eshmurzaeva2
1Tashkent Institute of Postgraduate Medical Education, Tashkent, Uzbekistan;
2Tashkent Medical Academy, Tashkent, Uzbekistan
Arthritis Research & Therapy 2012, 14(Suppl 1):P7 Egr-1 mediates the suppressive effect of IL-1 on PPARg expression in
human OA chondrocytes Egr-1 mediates the suppressive effect of IL-1 on PPARg expression in
human OA chondrocytes
Sarah S Nebbaki*, Fatima Ezzahra El Mansouri, Mohamed Benderdour,
Johanne Martel-Pelletier, Jean-Pierre Pelletier, Hassan Fahmi
Osteoarthritis Research Unit, Montreal, H2L 4M1, Canada
Arthritis Research & Therapy 2012, 14(Suppl 1):P12 y
Sarah S Nebbaki*, Fatima Ezzahra El Mansouri, Mohamed Benderdour,
Johanne Martel-Pelletier, Jean-Pierre Pelletier, Hassan Fahmi
Osteoarthritis Research Unit, Montreal, H2L 4M1, Canada
Arthritis Research & Therapy 2012, 14(Suppl 1):P12 Results: Notch1 and its ligands DLL-4 and HRT-1 were expressed in RAST
both in the lining layer and perivascular regions. Additionally avb3, b1-
integrin and F-actin predominantly localised to vascular endothelium and
lining cells in RAST, compared with osteoarthritis and normal control
synovial tissue. A-SAA significantly upregulated levels of Notch1 mRNA
and protein in ECs. Differential effects were observed on Notch ligands
HRT-1 and Jagged 1 mRNA in response to A-SAA stimulation. In contrast,
A-SAA inhibited DLL-4 mRNA (p < 0.05), consistent with a negative
feedback loop controlling interactions between NOTCH1 IC and DLL-4 in
the regulation of EC tip vs. stalk cells development. A-SAA induced
disassembly of endothelial cell F-actin cytoskeleton and loss of focal
adhesions as demonstrated by a reduction in vinculin staining. Finally,
A-SAA-induced angiogenesis, cell migration and invasion were inhibited
in the presence of NOTCH 1 siRNA (p < 0.05). Background: Peroxisome proliferator-activated receptor gamma (PPARg)
is a ligand activated transcription factor and member the nuclear
hormone receptor superfamily. Several lines of evidence indicate that
PPARg have protective effects in osteoarthritis (OA). Indeed, PPARg has
been shown to down-regulate several inflammatory and catabolic
responses in articular joint cells and to be protective in animal models of
OA. We have previously shown that IL-1 down-regulated PPARg
expression in OA chondrocytes. In the present study we will investigate
the mechanisms underlying this effect of IL-1. Materials and methods: Chondrocytes were stimulated with IL-1, and
the level of PPARg and Egr-1 protein and mRNA were evaluated using
Western blotting and real-time reverse-transcription polymerase chain
reaction, respectively. The PPARg promoter activity was analyzed in
transient transfection experiments. Egr-1 recruitment to the PPARg
promoter was evaluated using chromatin immunoprecipitation (ChIP)
assays. Conclusion: A-SAA induces the NOTCH signalling pathway and
cytoskeletal
rearrangement
which
allows temporal
and
spatial
reorganization of cells during cell migratory events and EC morphology. Together these results suggest a critical role for A-SAA in driving cell
shape, migration and invasion in the inflamed joint. The bacterial effector protein YopM reduces rheumatoid arthritis (RA)
outcome by inhibiting inflammation and bone destruction
d1*
2
3
h
2
h
d 2
3 Osteoclasts mediate the degradation of bone during RA and are derived
from macrophages. The yersinia outer protein M (YopM) is an effector Page 23 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Background: Cigarette smoking has been shown as major environmental
risk factor for rheumatoid arthritis (RA). Epidemiological studies indicate an
association of cigarette smoking with development of RA [1,2], although
molecular mechanisms remain unknown. The aim of this study is to
analyze the influence of cigarette smoke on the gene expression regulated
by histone deacetylases (HDACs) in RA synovial fibroblasts (RASF). IL-17 and IL-22. Moreover, there was an increase in IL-4 production and
higher numbers of regulatory T cells myeloid specific PTEN-/-. In contrast,
myeloid specific PTEN deficiency did not affect serum transfer arthritis,
which is independent of the adaptive immune system and solely depends
on innate effector functions. These data demonstrate that the presence of
PTEN in myeloid cells is required for the development of systemic
autoimmunity. Deletion of PTEN in myeloid cells inhibits the development of
CIA and EAE by preventing the generation of a pathogenic Th17 type of
immune response. y
y
y
Methods: RASF obtained from patients undergoing joint replacement
surgery were stimulated with freshly prepared cigarette smoke extract
(CSE) for 24 hours. Expression of HDACs was measured at the mRNA level
by Real-time TaqMan and SYBR green PCR and at the protein level by
immunoblot analysis. Global histone 3 (H3) acetylation was analyzed by
immunoblot. P12 Egr-1 mediates the suppressive effect of IL-1 on PPARg expression in
human OA chondrocytes Acute Serum Amyloid A induces cell migration cytoskeletal
rearrangement and Notch signalling in rheumatoid arthritis
* Ann Rheum Dis
2001, 60:223-227. 2. Hutchinson D, Shepstone L, Moots R, Lear JT, Lynch MP: Heavy cigarette
smoking is strongly associated with rheumatoid arthritis (RA),
particularly in patients without a family history of RA. Ann Rheum Dis
2001, 60:223-227. 2. Hutchinson D, Shepstone L, Moots R, Lear JT, Lynch MP: Heavy cigarette
smoking is strongly associated with rheumatoid arthritis (RA),
partic larl in patients
itho t a famil histor of RA A
Rh
Di P11
Cigarette smoke downregulates HDAC2 in rheumatoid arthritis synovial
fibroblasts
Anna Engler1*, Astrid Jüngel1, Christoph Kolling2, Beat A Michel1, Renate Gay1,
Steffen Gay1, Caroline Ospelt1
1Center of Experimental Rheumatology, University Hospital Zurich and Zurich
Center of Integrative Human Physiology (ZIHP), Zurich, Switzerland;
2Schulthess Clinic, Zurich, Switzerland
Arthritis Research & Therapy 2012, 14(Suppl 1):P11 Egr-1 mediates the suppressive effect of IL-1 on PPARg expression in
human OA chondrocytes y
Results: We demonstrated that the suppressive effect of IL-1 on PPARg
expression requires de novo protein synthesis and was concomitant with
the induction of the transcription factor Egr-1. ChIP analyses revealed that
IL-1 induced Egr-1 recruitment at the PPARg promoter. IL-1 inhibited
the activity of PPARg promoter and overexpression of Egr-1 potentiated
the inhibitory effect of IL-1, suggesting that Egr-1 may mediate the
suppressive effect of IL-1. P10 Results: Stimulation of RASF (n = 8-10) with CSE significantly enhanced
the expression of HDAC1 (x-fold: 2.0 ± 0.4; p = 0.04), HDAC2 (1.9 ± 0.3;
p = 0.02) and HDAC3 (2.4 ± 0.4; p = 0.01) at the mRNA level while the
expression of HDAC 4-11 remained unchanged. On the protein level,
expression of HDAC1 and HDAC3 were not altered, whereas the
expression of HDAC2 protein was decreased in CSE stimulated RASF. No
measurable changes in global acetylation of H3 were induced by CSE in
RASF (n = 6). Acute Serum Amyloid A induces cell migration cytoskeletal
rearrangement and Notch signalling in rheumatoid arthritis
Mary Connolly*, Peadar Rooney, Wei Gao, Douglas Veale, Ursula Fearon
Translational Research Group, Dublin Academic Medical Centre, St. Vincent’s
University Hospital, Dublin, Ireland
Arthritis Research & Therapy 2012, 14(Suppl 1):P10 Acute Serum Amyloid A induces cell migration cytoskeletal
rearrangement and Notch signalling in rheumatoid arthritis
* Acute Serum Amyloid A induces cell migration cytoskeletal
rearrangement and Notch signalling in rheumatoid arthritis
* Mary Connolly*, Peadar Rooney, Wei Gao, Douglas Veale, Ursula Fearon
Translational Research Group, Dublin Academic Medical Centre, St. Vincent’s
University Hospital, Dublin, Ireland
Arthritis Research & Therapy 2012, 14(Suppl 1):P10 Background: Acute Serum Amyloid A (A-SAA) is an acute phase protein
strongly expressed in rheumatoid arthritis (RA) synovial tissue (ST) critically
involved in regulating cell migration and angiogenesis. These processes are
dependent on downstream interactions between extracellular matrix and
cytoskeletal components. Additionally the Notch signalling pathway has
been show to regulate endothelial cell (EC) morphogenesis and is critically
involved in vessel formation, branching and morphogenesis. The aim of this
study was to examine if A-SAA-induced angiogenesis, cell migration and
invasion are mediated by the NOTCH signalling pathways. Conclusion: CSE specifically downregulates the expression of HDAC2 in
RASF. Differential regulation of HDAC2 at the mRNA and protein level
points to post-transcriptional degradation mechanisms induced by
smoking. Even though global H3 acetylation was not changed by CSE,
decreased HDAC2 levels might be associated with hyper-acetylation and
thus increased expression of specific HDAC2 regulated genes. References 1. Bergstrom U, Jacobsson LT, Nilsson JA, Berglund G, Turesson C: Pulmonary
dysfunction, smoking, socioeconomic status and the risk of developing
rheumatoid arthritis. Rheumatology (Oxford) 2011, 50:2005-2013. 1. Bergstrom U, Jacobsson LT, Nilsson JA, Berglund G, Turesson C: Pulmonary
dysfunction, smoking, socioeconomic status and the risk of developing
rheumatoid arthritis. Rheumatology (Oxford) 2011, 50:2005-2013. y
g
g p
y
Materials and methods: Immunohistology was used to examine Notch1,
DLL-4 and HRT-1 in RA synovial tissue (RAST). avb3 and b1-integrins,
filamentous actin (F-actin) and focal adhesion expression in RAST and
rheumatoid arthritis synovial fibroblast cells (RASFC) was assessed by
immunofluorescence. NOTCH1 IC, its ligands DLL-4, JAGGED 1 and
downstream signaling components HRT1, HRT2 were quantified by Real-
time PCR. NOTCH1 IC protein was assessed by western blot. A-SAA-induced
angiogenesis cell migration and invasion were assessed by Matrigel tube
formation, scratch and invasion assay. A-SAA modulation of filamentous
actin (F-actin) and focal adhesions (vinculin) was examined by dual
immunofluorescence. Finally, A-SAA-induced angiogenesis, invasion, altered
cell shape and migration were performed in the presence or absence of
siRNA against NOTCH 1. 2. Hutchinson D, Shepstone L, Moots R, Lear JT, Lynch MP: Heavy cigarette
smoking is strongly associated with rheumatoid arthritis (RA),
particularly in patients without a family history of RA. P11 Conclusions: These results indicate that Egr-1 contributes to IL-1-
mediated down-regulation of PPARg expression in OA chondrocytes and
suggest that this pathway could be a potential target for pharmacologic
intervention in the treatment of OA and possibly other arthritic diseases. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Page 24 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 diagnosis of SSc), were collected from Sulaimani internal Medicine
teaching hospital from July 2009 to July 2010. Immune cell - derived microparticles contribute to the resistance of
rheumatoid arthritis synovial fibroblasts to death receptor-mediated
apoptosis
1*
1
1
2 Our aim was to analyze miRs as potential systemic markers in early stages
of the disease and to find new miRs locally at the site of inflammation
that play a role in the pathogenesis of RA. Mojca Frank1*, Meike Dahlhaus1, Maria Filkova1, Christoph Kolling2,
Beat A Michel1, Diego Kyburz1, Blaž Rozman3, Renate E Gay1, David Pisetsky4,
Steffen Gay1, Astrid Jüngel1 Mojca Frank1*, Meike Dahlhaus1, Maria Filkova1, Christoph Kolling2,
Beat A Michel1, Diego Kyburz1, Blaž Rozman3, Renate E Gay1, David Pisetsky4,
Steffen Gay1, Astrid Jüngel1 Methods: MiRs from sera of patients with treatment naïve early RA (ERA),
with treated established RA and HC were isolated by phenol-chloroform
extraction. TaqMan Low Density Array was used to analyze the expression
of 260 miRs in RASF and OASF. MiR-196a expression was further analyzed
in additional RASF and OASF, RA and OA synovial tissues. TaqMan
RealTime-PCR was used for quantification of miRs and functional
experiments (MTT, scratch assay, AnnexinV FACS) were performed
following transfection with pre-miR or miR-196a inhibitor. 1Center of Experimental Rheumatology, University Hospital Zürich, Zürich,
Switzerland; 2Schultess Clinic, Zürich, Switzerland; 3Department of
Rheumatology, University Medical Centre Ljubljana, Ljubljana, Slovenia;
4Medical Research Service, Durham Veterans Administration Medical Center,
Durham, NC, USA 1Center of Experimental Rheumatology, University Hospital Zürich, Zürich,
Switzerland; 2Schultess Clinic, Zürich, Switzerland; 3Department of
Rheumatology, University Medical Centre Ljubljana, Ljubljana, Slovenia;
4Medical Research Service, Durham Veterans Administration Medical Center,
Durham, NC, USA Arthritis Research & Therapy 2012, 14(Suppl 1):P15 Background: Immune cell-derived microparticles (MPs) are present at
increased amounts in synovial fluid of rheumatoid arthritis (RA) patients
[1] and can activate disease-relevant signalling pathways in RA synovial
fibroblasts (SF) [2,3]. Increased resistance to apoptosis is one of the main
characteristics of aggressive phenotype of RASF [4,5] and MPs have been
shown to mediate both pro- and anti- apoptotic effects in different
target cells [6,7]. The aim of the present study was to investigate the
functional role of immune cell-derived MPs in modulating the apoptosis
of SF in RA. g
p
Results: In sera of patients with ERA, the expression of miR-146a was lower
than in both HC (p < 0.05) and established RA sera (p < 0.001) while miR-
155, 132, -203 and -223 showed no differences. In RASF, the expression of miR-196a is significantly lower than in OASF
(p < 0.0001) as well as in RA synovial tissues compared with OA (p =
0.01). References References
1. Stanczyk J, Ospelt C, Karouzakis E, Filer A, Raza K, Kolling C, Gay R,
Buckley CD, Tak PP, Gay S, Kyburz D: Altered expression of microRNA-203
in rheumatoid arthritis synovial fibroblasts and its role in fibroblast
activation. Arthritis Rheum 2011, 63:373-81. P14
MiRs in RA: possible biomarkers and therapeutic targets
Maria Filkova1*, Caroline Ospelt1, Joanna Stanczyk1, Serena Vettori1,
Ladislav Senolt2, Mojca Frank1, Christoph Kolling3, Beat A Michel1,
Renate E Gay1, Steffen Gay1, Astrid Jüngel1
1Center of Experimental Rheumatology, University Hospital Zurich, Zurich,
Switzerland; 2Institute of Rheumatology, Department of Experimental
Rheumatology of the 1st Faculty of Medicine, Charles University in Prague,
Prague, Czech Republic; 3Schultess Clinic, Zurich, Switzerland
Arthritis Research & Therapy 2012, 14(Suppl 1):P14 2. Stanczyk J, Pedrioli DM, Brentano F, Sanchez-Pernaute O, Kolling C, Gay RE,
Detmar M, Gay S, Kyburz D: Altered expression of MicroRNA in synovial
fibroblasts and synovial tissue in rheumatoid arthritis. Arthritis Rheum
2008, 58:1001-9. 3. Pauley KM, Satoh M, Chan AL, Bubb MR, Reeves WH, Chan EK: Upregulated
miR-146a expression in peripheral blood mononuclear cells from
rheumatoid arthritis patients. Arthritis Res Ther 2008, 10:R101. Background and objective: New concepts of therapy highlight an early
use of effective treatment to prevent further joint damage in RA. Altered
expression of epigenetic marks like miRs offers us the possibility to
develop new diagnostic tools and novel therapeutic targets. 4. Fulci V, Scappucci G, Sebastiani GD, Giannitti C, Franceschini D, Meloni F,
Colombo T, Citarella F, Barnaba V, Minisola G, Galeazzi M, Macino G: miR-
223 is overexpressed in T-lymphocytes of patients affected by
rheumatoid arthritis. Hum Immunol 2010, 71:206-11. We found miR-146, -155 and -203 to be upregulated in rheumatoid
arthritis (RA) synovial fibroblasts (SF) compared to osteoarthritis (OA) SF
[1,2]. Based on the comprehensive analysis of the expression of 260 miRs
we found miR-196a to be one of the most downregulated miRs in RASF. In peripheral blood mononuclear cells, miR-132 and -223 are upregulated
in established RA compared with healthy controls (HC) [3,4]. Prevalence of interstitial lung disease among patients with systemic
sclerosis in Iraqi Kurdistan
1*
2
1 Patients and methods: A sample of thirty patients with SSc (whom
fulfilled the American Rheumatism Association preliminary criteria for the Table 2(abstract P13) Distribution of HRCT scans abnormalities
Variables
Frequency
Percent
CT chest
Normal
14
46.7
Abnormal
16
53.3
Fibrosis
No
19
63.3
Yes
11
36.7
Traction bronchiactetic changes
No
19
63.3
Yes
11
36.7
Ground glass
No
22
73.3
Yes
8
26.7
Honey comb
No
26
86.7
Yes
4
13.3
Table 1(abstract P13) Results of pulse oximetry both during rest and exertion, chest x-ray finding,
pulmonary function test
Frequency
Percent
O2 Saturation (rest)
Above 92
20
66.7
Below 92
10
33.3
O2 Saturation (exertion)
Above 92
13
43.3
Below 92
17
56.7
CXR
Normal
19
63.3
Basal reticular shadowing
11
36.7
Pulmonary function test
Normal
15
50.0
Restrictive
15
50.0
Obstructive
0
00.0 ble 1(abstract P13) Results of pulse oximetry both during rest and exertion, chest x-ray finding,
ulmonary function test Table 2(abstract P13) Distribution of HRCT scans abnormalities
Variables
Frequency
Percent
CT chest
Normal
14
46.7
Abnormal
16
53.3
Fibrosis
No
19
63.3
Yes
11
36.7
Traction bronchiactetic changes
No
19
63.3
Yes
11
36.7
Ground glass
No
22
73.3
Yes
8
26.7
Honey comb
No
26
86.7
Yes
4
13.3 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Page 25 of 54 Figure 1(abstract P13) Subsets of Systemic sclerosis. Figure 1(abstract P13) Subsets of Systemic sclerosis. igure 1(abstract P13) Subsets of Systemic sclerosis. Figure 1(abstract P13) Subsets of Systemic sclerosis. References
1. Stanczyk J, Ospelt C, Karouzakis E, Filer A, Raza K, Kolling C, Gay R,
Buckley CD, Tak PP, Gay S, Kyburz D: Altered expression of microRNA-203
in rheumatoid arthritis synovial fibroblasts and its role in fibroblast
activation. Arthritis Rheum 2011, 63:373-81. 2. Stanczyk J, Pedrioli DM, Brentano F, Sanchez-Pernaute O, Kolling C, Gay RE,
Detmar M, Gay S, Kyburz D: Altered expression of MicroRNA in synovial
fibroblasts and synovial tissue in rheumatoid arthritis. Arthritis Rheum
2008, 58:1001-9. 3. Pauley KM, Satoh M, Chan AL, Bubb MR, Reeves WH, Chan EK: Upregulated
miR-146a expression in peripheral blood mononuclear cells from
rheumatoid arthritis patients. Arthritis Res Ther 2008, 10:R101. 4. Prevalence of interstitial lung disease among patients with systemic
sclerosis in Iraqi Kurdistan
1*
2
1 All patients were evaluated in a cross sectional study for the evidence
of ILD, almost all patients were submitted to chest radiographs (CXR),
pulmonary function tests (PFT) and oxygen saturation by pulse
oximetry (Spo2) and high-resolution computed tomography (HRCT)
scan. Taha Ahmad Qaradakhy1*, Kosar Mohamed Ali2, Omer Hama Karim1
1Department of Rheumatology, Sulaimani Internal Medicine Teaching
Hospital, Sulaimani, Iraq; 2Respiratory/General Medical Department, College
of Medicine, Sulaimani, Iraq
Arthritis Research & Therapy 2012, 14(Suppl 1):P13 Results: Patients ages ranged from 23-68 years with mean (45.57)
years, with female predominance 27(90%) compare to 3(10%) male. Majority of patients had limited type of systemic sclerosis 21(70%), and 15
(50%) cases had restirictive ventilatory defect. Out of the thirty patients in
the study 16(53.3%) patients had evidence of ILD on HRCT. Background: Systemic sclerosis (SSc) associated interstitial lung disease
(ILD) is the leading cause of morbidity and mortality in SSc patients. Aim of the study: To detect and determine the prevalence of ILD in
patients with SSc in Sulaimani Governorate. Conclusion: 1. ILD is common among patients with SSc (dcSSc type). 2. PFT & HRCT are sensitive tools for diagnosis ILD among patients
with SSc. Prevalence of interstitial lung disease among patients with systemic
sclerosis in Iraqi Kurdistan
1*
2
1 Fulci V, Scappucci G, Sebastiani GD, Giannitti C, Franceschini D, Meloni F,
Colombo T, Citarella F, Barnaba V, Minisola G, Galeazzi M, Macino G: miR-
223 is overexpressed in T-lymphocytes of patients affected by
rheumatoid arthritis. Hum Immunol 2010, 71:206-11. Results: Poly(I:C)-induced MPs but not MPs from unstimulated U937
cells increased the production of IL-6 in RASF (mean ± SE: 1873 ± 325 References 1. Berckmans RJ, Nieuwland R, Kraan MC, Schaap MC, Pots D, Smeets TJ,
Sturk A, Tak PP: Synovial microparticles from arthritic patients modulate
chemokine and cytokine release by synoviocytes. Arthritis Res Ther 2005,
7(3):R536-544. Figure 1(abstract P16) sLAG3 concentrations in SLE and other
autoimmune diseases measured by ELISA. 2. Beyer C, Pisetsky DS: The role of microparticles in the pathogenesis of
h
ti di
N t R
Rh
t l 2010 6(1) 21 29 2. Beyer C, Pisetsky DS: The role of microparticles in the pathogenesis of
rheumatic diseases. Nat Rev Rheumatol 2010, 6(1):21-29. 3. Distler JH, Jüngel A, Huber LC, Seemayer CA, Reich CF, Gay RE, Michel BA,
Fontana A, Gay S, Pisetsky DS, Distler O: The induction of matrix
metalloproteinase and cytokine expression in synovial fibroblasts
stimulated with immune cell microparticles. Proc Natl Acad Sci USA 2005,
102(8):2892-2897. 0.14. In addition, sLAG3 concentrations showed a significant correlation
with SLEDAI. Interestingly, elevation of sLAG3 was observed even in
patients with SLEDAI = 0. These results suggested that sLAG3 could be a
specific and novel marker for SLE. 4. Pundt N, Peters MA, Wunrau C, Strietholt S, Fehrmann C, Neugebauer K,
Seyfert C, van Valen F, Pap T, Meinecke I: Susceptibility of rheumatoid
arthritis synovial fibroblasts to FasL- and TRAIL-induced apoptosis is cell
cycle-dependent. Arthritis Res Ther 2009, 11(1):R16. 4. Pundt N, Peters MA, Wunrau C, Strietholt S, Fehrmann C, Neugebauer K,
Seyfert C, van Valen F, Pap T, Meinecke I: Susceptibility of rheumatoid
arthritis synovial fibroblasts to FasL- and TRAIL-induced apoptosis is cell
cycle-dependent. Arthritis Res Ther 2009, 11(1):R16. Conclusions: sLAG3 can be a novel marker for SLE. sLAG3 in sera of SLE
patient may reflect the activation of pDCs. Because sLAG3 shows
adjuvant effect when combined with active immunization [3], sLAG3 may
contribute to the exacerbation of lupus. The association between
elevated sLAG3, type I interferon signature and activation of pDCs should
be investigated further. y
5. Korb A, Pavenstädt H, Pap T: Cell death in rheumatoid arthritis. Apoptosis
2009, 14(4):447-454. 5. Korb A, Pavenstädt H, Pap T: Cell death in rheumatoid arthritis. Apoptosis
2009, 14(4):447-454. 6. Distler JH, Akhmetshina A, Dees C, Jüngel A, Stürzl M, Gay S, Pisetsky DS,
Schett G, Distler O: Induction of apoptosis in circulating angiogenic cells
by microparticles. Arthritis Rheum 2011, 63(7):2067-2077. Immune cell - derived microparticles contribute to the resistance of
rheumatoid arthritis synovial fibroblasts to death receptor-mediated
apoptosis
1*
1
1
2 RASF transfection with pre-miR/miR-196a inhibitor resulted in
down/upregulation of predicted targets HOXC8 and ANXA1. Pre-miR-
196a suppressed cell proliferation (27.5%) and migration (41.5%) and
induced apoptosis (54.1%) while miR-196a inhibitor enhanced both
proliferation (81.9%) and migration (231%) and reduced apoptosis
(52.3%) in RASF. Methods: MPs were isolated by the differential centrifugation from cell
culture supernatants of U937 cells, untreated or stimulated with TNFa or
poly(I:C) for 16 h. Flow cytometry was used to measure the counts and
surface expression of CD4 and Fas on MP. Proinflammatory response of
RASF induced by MPs was determined by measuring IL-6 protein levels
by ELISA. Proliferation of OASF (n = 3) and RASF (n = 4) stimulated with
MPs for 24 h was investigated by MTT Cell Proliferation Assay. Functional
role of MPs (after 24 h treatment) in spontaneous apoptosis and
apoptosis mediated by Fas Ligand (FasL) or TNFa-Related Apoptosis
Inducing Ligand (TRAIL) was measured by flow cytometry using Annexin
V/propidium iodide staining of RASF and OASF. Conclusion: In contrast to established RA synovial fibroblasts where an
increased expression of miR-146a was reported, our data showed that in
early arthritis sera miR-146a is significantly downregulated and might
characterize an early clinical stage of the disease. The low expression of
miR-196a in both RA synovial tissue and in isolated SF contributes to the
aggressive and invasive phenotype of RASF by modifying proliferation,
migration and apoptosis with an impact on the pathogenesis of RA. Acknowledgements: This work was supported by IAR-EPALINGES, FP7
Masterswitch, MH CR- grant project No.10065-4 and ARTICULUM
fellowship. Conclusion: In contrast to established RA synovial fibroblasts where an
increased expression of miR-146a was reported, our data showed that in
early arthritis sera miR-146a is significantly downregulated and might
characterize an early clinical stage of the disease. The low expression of
miR-196a in both RA synovial tissue and in isolated SF contributes to the
aggressive and invasive phenotype of RASF by modifying proliferation,
migration and apoptosis with an impact on the pathogenesis of RA. Acknowledgements: This work was supported by IAR-EPALINGES, FP7
Masterswitch, MH CR- grant project No.10065-4 and ARTICULUM
fellowship. Immune cell - derived microparticles contribute to the resistance of
rheumatoid arthritis synovial fibroblasts to death receptor-mediated
apoptosis
1*
1
1
2 Results: Poly(I:C)-induced MPs but not MPs from unstimulated U937
cells increased the production of IL-6 in RASF (mean ± SE: 1873 ± 325 Page 26 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 pg/mL, p = 0.002, n = 9 and 476 ± 182 pg/mL, n = 6, respectively)
when compared to unstimulated RASF (304 ± 61 pg/mL, n = 9). No changes in proliferation or spontaneous rate of apoptosis were
observed in RASF or OASF stimulated with MPs. Treatment of RASF
(n = 5) and OASF (n = 5) with FasL or treatment of RASF (n = 7) with
TRAIL for 24 h significantly increased apoptosis of SF (p = 0.010; p =
0.036 and p = 0.016, respectively). Poly(I:C)-induced MPs inhibit FasL-
induced apoptosis of RASF (% decrease ± SE: 40.2 ± 7.0%; p = 0.001; n
= 5) and OASF (41.1 ± 9.5%; p = 0.036, n = 5) and decreased TRAIL-
induced apoptosis of RASF (29.9 ± 6.8%, p = 0.093). In contrast, TNFa-
induced MPs had no effect on Fas-induced apoptosis in SF (n = 3). MPs
from untreated U937 cells did not influence FasL- or TRAIL-induced
apoptosis of RASF (n = 5) and OASF (n = 4). Fas was not expressed on
the surface of MPs, indicating that Poly(I:C)-induced MP did not act as a
decoy to decrease the effective concentration of FasL in cell culture
supernatants. Figure 1(abstract P16) sLAG3 concentrations in SLE and other
autoimmune diseases measured by ELISA. Conclusions: Immune cells and SF can communicate via MPs. The
impairment of the death receptor-induced apoptosis pathway mediated
by immune cell-derived MPs may contribute to synovial hyperplasia and
joint destruction in RA. Conclusions: Immune cells and SF can communicate via MPs. The
impairment of the death receptor-induced apoptosis pathway mediated by immune cell-derived MPs may contribute to synovial hyperplasia and
joint destruction in RA. Acknowledgements: This work was supported by IAR-EPALINGES, FP7
Masterswitch, and ARTICULUM Fellowship. P17 7. Castellana D, Zobairi F, Martinez MC, Panaro MA, Mitolo V, Freyssinet JM,
Kunzelmann C: Membrane microvesicles as actors in the establishment
of a favorable prostatic tumoral niche: a role for activated fibroblasts
and CX3CL1-CX3CR1 axis. Cancer Res 2009, 69(3):785-793. P17
GCIP, Id like HLH protein, negatively regulates cell proliferation of
rheumatoid synovial cells via interaction with CBP
Hidetoshi Fujita1,2*, Minako Nakazawa1, Satoko Aratani1,3, Kusuki Nishioka3,
Akiyoshi Fukamizu4, Toshihiro Nakajima1,3
1Institute of Medical Science, St Marianna University School of Medicine,
Kanagawa 216-8512, Japan; 2Advanced Radiation Biology Research Program,
and Hospital, Research Center for Charged Particle Therapy, National Institute
of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba, 263-8555, Japan;
3Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjyuku,
Shinjyuku-ku, Tokyo, 160-8402, Japan; 4Graduate School of Life and
Environmental Sciences, University of Tsukuba, Ibaraki 305-8572, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P17 P17
GCIP, Id like HLH protein, negatively regulates cell proliferation of
rheumatoid synovial cells via interaction with CBP
Hidetoshi Fujita1,2*, Minako Nakazawa1, Satoko Aratani1,3, Kusuki Nishioka3,
Akiyoshi Fukamizu4, Toshihiro Nakajima1,3
1Institute of Medical Science, St Marianna University School of Medicine,
Kanagawa 216-8512, Japan; 2Advanced Radiation Biology Research Program,
and Hospital, Research Center for Charged Particle Therapy, National Institute
of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba, 263-8555, Japan;
3Institute of Medical Science, Tokyo Medical University, 6-1-1 Shinjyuku,
Shinjyuku-ku, Tokyo, 160-8402, Japan; 4Graduate School of Life and
Environmental Sciences, University of Tsukuba, Ibaraki 305-8572, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P17 References Nucleotide sensing-TLRs (Toll-like receptors) recognize pathogen derived-
nucleic acids and trigger immune response [1]. Because of the highly
conserved structure of nucleic acids, these TLRs have risk to recognize
host derived-nucleic acids and induce autoimmune disease, therefore it is
important to clarify the mechanisms and control the response. 1. Harris ED: Rheumatoid arthritis. Pathophysiology and implications for
therapy. N Engl J Med 1990, 322:1277-1289. 1. Harris ED: Rheumatoid arthritis. Pathophysiology and implications for
therapy. N Engl J Med 1990, 322:1277-1289. 2. Feldmann M, Brennan FM, Maini RN: Rheumatoid arthritis. Cell 1996,
85:307-310. 2. Feldmann M, Brennan FM, Maini RN: Rheumatoid arthritis. Cell 1996,
85:307-310. 3. Aarvak T, Natvig JB: Cell-cell interactions in synovitis: antigen presenting
cells and T cell interaction in rheumatoid arthritis. Arthritis Res 2001,
3:13-17. 3. Aarvak T, Natvig JB: Cell-cell interactions in synovitis: antigen presenting
cells and T cell interaction in rheumatoid arthritis. Arthritis Res 2001,
3:13-17. We found that the responses of TLR7 and TLR9 are balanced reciprocally,
and Unc93 homolog B1 (Unc93B1) is a key molecule for this balancing
system [2]. Unc93B1 is known as an essential molecule for TLR3, TLR7,
and TLR9 responses, and the function depends on its C-terminal region
[3]. The balancing function of Unc93B1 is located on 34th aspartic acids
from N-terminal, and alanine mutant (D34A) Unc93B1 up-regulates TLR7
response and down-regulates TLR9 response (Figure 1) [2]. 4. Schett G, Tohidast-Akra M, Steiner G, Smolen J: The stressed synovium. Arthritis Res 2001, 3:80-86. 4. Schett G, Tohidast-Akra M, Steiner G, Smolen J: The stressed synovium. Arthritis Res 2001, 3:80-86. 5. Amano T, Yamasaki S, Yagishita N, Tsuchimochi K, Shin H, Kawahara K,
Aratani S, Fujita H, Zhang L, Ikeda R, Fujii R, Miura N, Komiya S, Nishioka K,
Maruyama I, Fukamizu A, Nakajima T: Synoviolin/Hrd1, an E3 ubiquitin
ligase, as a novel pathogenic factor for arthropathy. Genes Dev 2003,
17:2436-2449. g
g
It is reported that TLR7 or TLR9 response contributes to some kinds of
autoimmune disease and TLR7 overexpressed mice develop SLE like
autoimmune disease [4-8]. To investigate the significance of reciprocal
TLR7/TLR9 balance in vivo, we generated Unc93b1D34A/D34A mice and
observed the phenotypes. 6. Workman CJ, Wang Y, El Kasmi KC, Pardoll DM, Murray PJ, Drake CG,
Vignali DA: LAG-3 regulates plasmacytoid dendritic cell homeostasis. J Immunol 2009, 182:1885-1891. P16
Increased concentration of serum soluble LAG3 in systemic lupus
erythematosus Seri Yu*, Keishi Fujio, Kazuyoshi Ishigaki, Hirofumi Shoda, Tomohisa Okamura,
Tanita Noor, Shuji Sumitomo, Kazuhiko Yamamoto Department of Allergy and Rheumatology, Graduate School of Medicine, The
University of Tokyo, 113-0033, Japan Department of Allergy and Rheumatology, Graduate School of Medicine, The
University of Tokyo, 113-0033, Japan Arthritis Research & Therapy 2012, 14(Suppl 1):P16 Arthritis Research & Therapy 2012, 14(Suppl 1):P16 Arthritis Research & Therapy 2012, 14(Suppl 1):P16 Background: Rheumatoid arthritis (RA) is one of the most common
articular diseases with a prevalence of 1% worldwide [1,2]. The clinical
features of RA include chronic inflammation of systemic joints associated
with synovial hyperplasia followed by impairment of quality of life [3,4]. Recently, we have shown that Synoviolin/Hrd1, an E3 ubiquitin ligase, is a
novel causative factor for arthropathy [5]. However, the mechanism that
regulates synovial cell outgrowth is not fully understood. Background: In systemic lupus erythematosus (SLE), type I interferon
and plasmacytoid DCs (pDCs) are supposed to play important roles. However, there are few evidences for pDCs activation in SLE. Murine
pDCs are reported to produce soluble LAG3 (sLAG3) upon activation and
pDCs are responsible for most of sLAG3 in mice serum [1]. Therefore,
serum sLAG3 concentration was examined in SLE and other autoimmune
diseases. Materials and methods: Human embryonic kidney (HEK)-293 cells, HEK-
293T cells, NIH3T3 cells and synovial cells were cultured in DMEM medium. Transient transfection assays were performed in HEK-293 cells and HEK-
293T cells. HEK-293 cells transfected with NF-B-Luc were treated with 100
ng/ml of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), or 10
ng/ml of TNF-a for 24 h, and luciferase activities were measured. siRNAs
with 21 nucleotides for human GCIP were chemically synthesized. Transfection with siRNAs and cell survival assay were carried out. Materials and methods: This study enrolled 45 SLE patients who met
ACR criteiria. Disease activity was rated using a SLE disease activity index
(SLEDAI). sLAG3 concentrations were measured by a quantitative
sandwich enzyme immunoassay [2]. Results: The ratio of sLAG3 concentration in SLE to control was 3.10+/-
1.05, PM/DM to control was 1.04+/-0.08, and RA to control was 0.77+/- Page 27 of 54 Page 27 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Shirokanedai, Minatoku, Tokyo 108-8639, Japan; 2Laboratory of Innate
Immunity, The Institute of Medical Science, The University of Tokyo, 4-6-1
Shirokanedai, Minatoku, Tokyo 108-8639, Japan; 3Laboratory of
Developmental Genetics, Center for Experimental Medicine and Systems
Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1
Shirokanedai, Minatoku, Tokyo 108-8639, Japan; 4Department of Pathology,
Faculty of Medicine, Kinki University, Osaka 589-8511, Japan; 5Department of
Gastroenterology and Metabology, Ehime University Graduate School of
Medicine, Ehime 791-0295, Japan; 6Division of Molecular Immunology,
Institute for Enzyme Research, University of Tokushima, Tokushima 770-8504,
Japan; 7Laboratory of Host Defense, World Premier International
Immunology Frontier Research Center, Osaka 565-0871, Japan; 8Department
of Host Defense, Research Institute for Microbial Diseases, Osaka University,
Osaka 565-0871, Japan
h
h
h
l Shirokanedai, Minatoku, Tokyo 108-8639, Japan; 2Laboratory of Innate
Immunity, The Institute of Medical Science, The University of Tokyo, 4-6-1
Shirokanedai, Minatoku, Tokyo 108-8639, Japan; 3Laboratory of
Developmental Genetics, Center for Experimental Medicine and Systems
Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1
Shirokanedai, Minatoku, Tokyo 108-8639, Japan; 4Department of Pathology,
Faculty of Medicine, Kinki University, Osaka 589-8511, Japan; 5Department of
Gastroenterology and Metabology, Ehime University Graduate School of
Medicine, Ehime 791-0295, Japan; 6Division of Molecular Immunology,
Institute for Enzyme Research, University of Tokushima, Tokushima 770-8504,
Japan; 7Laboratory of Host Defense, World Premier International
Immunology Frontier Research Center, Osaka 565-0871, Japan; 8Department
of Host Defense, Research Institute for Microbial Diseases, Osaka University,
Osaka 565-0871, Japan
h
h
h
l Results: Grap2 cyclin D interacting protein (GCIP), Id like HLH protein,
was down-regulated in the rheumatoid synovial cells. Introduction of
GCIP into mouse fibroblast NIH3T3 cells resulted in growth suppression,
whereas knockdown with siRNAs in synovial cells enhanced cell growth. GCIP associated with CBP and repressed transcription of CREB-target
genes such as cyclin D1 by inhibition of interaction between CBP and
RNA polymerase II complexes. Binding assays revealed that GCIP bound
to CBP via acidic region, not HLH domain, and this interaction was
regulated by phosphorylation of GCIP in a cell cycle-dependent manner. Therefore, GCIP has inhibitory effect on cell proliferation via interference
with CBP-mediated transcription. Conclusions: We propose the novel inhibitory mechanisms of Id protein
family; the coactivator CBP is a functional target. Furthermore, down-
regulation of GCIP may be a key factor in rheumatoid synovial cell
outgrowth. Arthritis Research & Therapy 2012, 14(Suppl 1):P18 References p
yp
As results, Unc93b1D34A/D34A mice were born according to Mendelian rule
but started to die spontaneously at 10 weeks old and over half of
Unc93b1D34A/D34A mice died within 1 year (Figure 2A) [9]. Unc93b1D34A/
D34A 7. Treibel F, Hacene K, Pichon MF: A soluble lymphocyte activation gene-3
(sLAG-3) protein as a prognostic factor in human breast cancer
expressing estrogen or progesterone receptors. Cancer Letters 2006,
235:147-153. y
g
D34A mice developed various phenotypes, for example, splenomegaly,
hepatitis, glomerulonephritis, thrombocytopenia, myeloproliferative
disorder (Figure 2B-2E). Especially, lethal acute hepatitis was observed in
moribund mice and infiltrated myeloid cells in liver were expanded in
spleen. These phenotypes are vanished by TLR7 deficient Unc93B1D34A/
D34A mice, thus TLR7 hyper-response caused by TLR7/TLR9 balance
disruption is factor of phenotypes in Unc93b1D34A/D34A mice (Figure 2). Not only innate immune system, acquired immune system is also affected
by D34A mutation. Expanded memory T cells, up-regulation of ICOS and
CD69 on T cells were observed by TLR7 dependent manner and some
classes of serum immunoglobulin level is increased in Unc93b1D34A/D34A
mice. In addition, Th1 and Th17 cells were expanded and activated in
Unc93b1D34A/D34A mice. The activation of T cells were TLR7 dependent, and D34A mice developed various phenotypes, for example, splenomegaly,
hepatitis, glomerulonephritis, thrombocytopenia, myeloproliferative
disorder (Figure 2B-2E). Especially, lethal acute hepatitis was observed in
moribund mice and infiltrated myeloid cells in liver were expanded in
spleen. These phenotypes are vanished by TLR7 deficient Unc93B1D34A/
D34A mice, thus TLR7 hyper-response caused by TLR7/TLR9 balance
disruption is factor of phenotypes in Unc93b1D34A/D34A mice (Figure 2). 8. Triebel F: LAG-3: a regulator of T-cell and DC responses and its use in
therapeutic vaccination. Trends Immunol 2003, 24:619-622. 8. Triebel F: LAG-3: a regulator of T-cell and DC responses and its use in
therapeutic vaccination. Trends Immunol 2003, 24:619-622. P18
Unc93 homolog B1 restricts systemic lethal inflammation by
orchestrating TLR7 and TLR9 response
Ryutaro Fukui1*, Shin-Ichiroh Saitoh1, Atsuo Kanno1, Masahiro Onji1,
Takuma Shibata1,2, Akihiko Ito4, Morikazu Onji5, Mitsuru Matsumoto6,
Shizuo Akira7,8, Nobuaki Yoshida3, Kensuke Miyake1,2
1Division of Infectious Genetics, Department of Microbiology and
Immunology, The Institute of Medical Science, The University of Tokyo, 4-6-1 P18 Unc93 homolog B1 restricts systemic lethal inflammation by
orchestrating TLR7 and TLR9 response
* p
p
yp
( g
)
Not only innate immune system, acquired immune system is also affected
by D34A mutation. Expanded memory T cells, up-regulation of ICOS and
CD69 on T cells were observed by TLR7 dependent manner and some
classes of serum immunoglobulin level is increased in Unc93b1D34A/D34A mice. In addition, Th1 and Th17 cells were expanded and activated in
Unc93b1D34A/D34A mice. The activation of T cells were TLR7 dependent, and Figure 1(abstract P18) The D34A mutation of Unc93B1 up-regulates TLR7 response and down-regulates TLR9 response. (A and B). Empty vector
was transfected to bone marrow derived stem cells (BMSCs) from wild tipe mice (gray bars). Empty vector (yellow bars), wild type Unc93B1 expressing
vector (blue bars), or D34A Unc93B1 expressing vector (red bars) were transfected to BMSCs from 3d mice. Transfected BMSCs were cultured with
puromycin and GM-CSF to differentiate to dendritic cells (DCs). After differentiation, DCs were harvested and stimulated by TLR7 ligands (A, loxoribine,
μg/ml) or TLR9 ligands (B, CpG-B, nM). Culture supernatant was corrected and subjected to ELISA for measurement of IL-12p40 (ng/ml). Figure 1(abstract P18) The D34A mutation of Unc93B1 up-regulates TLR7 response and down-regulates TLR9 response. (A and B). Empty vector
was transfected to bone marrow derived stem cells (BMSCs) from wild tipe mice (gray bars). Empty vector (yellow bars), wild type Unc93B1 expressing
vector (blue bars), or D34A Unc93B1 expressing vector (red bars) were transfected to BMSCs from 3d mice. Transfected BMSCs were cultured with
puromycin and GM-CSF to differentiate to dendritic cells (DCs). After differentiation, DCs were harvested and stimulated by TLR7 ligands (A, loxoribine,
μg/ml) or TLR9 ligands (B, CpG-B, nM). Culture supernatant was corrected and subjected to ELISA for measurement of IL-12p40 (ng/ml). Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Page 28 of 54 Figure 2(abstract P18) Unc93b1D34A/D34A mice develop systemic lethal inflammation spontaneously. (A) Survival curves of Unc93b1WT/WT,
Unc93b1D34A/D34A, or Unc93b1D34A/D34ATlr7-/- mice (blue, red, or green line, respectively). (B and C) Macroscopic images of spleen (B) and liver (C). (D) Microscopic analyses of liver. Histological samples were stained by Hematoxyline and Eosine (H&E) or silver impregnation. (E) Platelet counts in
peripheral blood from indicated genotypes of mice. Bars in the graph indicate averages. Figure 2(abstract P18) Unc93b1D34A/D34A mice develop systemic lethal inflammation spontaneously. 6.
Asagiri M, Hirai T, Kunigami T, Kamano S, Gober HJ, Okamoto K,
Nishikawa K, Latz E, Golenbock DT, Aoki K, et al: Cathepsin K-dependent
toll-like receptor 9 signaling revealed in experimental arthritis. Science
2008, 319:624-627. 4.
Pisitkun P, Deane JA, Difilippantonio MJ, Tarasenko T, Satterthwaite AB,
Bolland S: Autoreactive B cell responses to RNA-related antigens due to
TLR7 gene duplication. Science 2006, 312:1669-1672.
5.
Santiago-Raber ML, Kikuchi S, Borel P, Uematsu S, Akira S, Kotzin BL, Izui S:
Evidence for genes in addition to Tlr7 in the Yaa translocation linked with
acceleration of systemic lupus erythematosus. J Immunol 2008,
181:1556-1562.
6.
Asagiri M, Hirai T, Kunigami T, Kamano S, Gober HJ, Okamoto K,
Nishikawa K, Latz E, Golenbock DT, Aoki K, et al: Cathepsin K-dependent
toll-like receptor 9 signaling revealed in experimental arthritis. Science
2008, 319:624-627.
7.
Deane JA, Pisitkun P, Barrett RS, Feigenbaum L, Town T, Ward JM,
Flavell RA, Bolland S: Control of toll-like receptor 7 expression is essential
to restrict autoimmunity and dendritic cell proliferation. Immunity 2007,
27:801-810.
8.
Lande R, Gregorio J, Facchinetti V, Chatterjee B, Wang YH, Homey B, Cao W,
Su B, Nestle FO, Zal T, et al: Plasmacytoid dendritic cells sense self-DNA
coupled with antimicrobial peptide. Nature 2007, 449:564-569. 7.
Deane JA, Pisitkun P, Barrett RS, Feigenbaum L, Town T, Ward JM,
Flavell RA, Bolland S: Control of toll-like receptor 7 expression is essential
to restrict autoimmunity and dendritic cell proliferation. Immunity 2007,
27:801-810. P18 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Page 29 of 54 Figure 3(abstract P18) T cells and B cells are activated in Unc93b1D34A/D34A mice. (A or D) Flow cytometry analysis for memory T cells (A upper, or
D) or Th1/Th17 cells (A lower). (B) Expression of ICOS was measured by cell surface staining of CD4+ T cells. Mean fluorescent intensity (MFI) was
calculated and indicated by dots. Black bars in the graph indicate average of MFI. (C) Flow cytometry analysis for matrginal zone B cells. (E) Survival
curves of Unc93b1D34A/D34A (Red) or Unc93b1D34A/D34AIghm-/- (Green) mice. Fi
3( b t
t P18) T
ll
d B
ll
ti
t d i
U
93b1D34A/D34A
i
(A
D) Fl
t
t
l
i f
T
ll (A Figure 3(abstract P18) T cells and B cells are activated in Unc93b1D34A/D34A mice. (A or D) Flow cytometry analysis for memory T cells (A upper, or
D) or Th1/Th17 cells (A lower). (B) Expression of ICOS was measured by cell surface staining of CD4+ T cells. Mean fluorescent intensity (MFI) was
calculated and indicated by dots. Black bars in the graph indicate average of MFI. (C) Flow cytometry analysis for matrginal zone B cells. (E) Survival
curves of Unc93b1D34A/D34A (Red) or Unc93b1D34A/D34AIghm-/- (Green) mice. 9. Fukui R, Saitoh SI, Kanno A, Onji M, Shibata T, Ito A, Matsumoto M, Akira S,
Yoshida N, Miyake K: Unc93B1 restricts systemic lethal inflammation by
orchestrating toll-like receptor 7 and 9 trafficking. Immunity 2011. 9. Fukui R, Saitoh SI, Kanno A, Onji M, Shibata T, Ito A, Matsumoto M, Akira S,
Yoshida N, Miyake K: Unc93B1 restricts systemic lethal inflammation by
orchestrating toll-like receptor 7 and 9 trafficking. Immunity 2011. Fas is a member of the TNF receptor family and crucial for induction of
apoptosis. MRL- lpr/lpr mice, which carry a mutation of Fas, spontaneously
develop systemic autoimmune disease including arthropathy, indicating that
Fas plays an important role in elimination of self-reactive immunocytes by
apoptosis. In addition to autoimmune diseases, we found a novel
phenotype of FasKO mice exclusively in Balb/c genetic background that is
allergic blepharitis. Allergic blepharitis is revealed in Balb/c FasKO mice from
15 week-old and about 85% of the mice suffered from allergic blepharitis at
35 week-old. P18 (A) Survival curves of Unc93b1WT/WT
Unc93b1D34A/D34A, or Unc93b1D34A/D34ATlr7-/- mice (blue, red, or green line, respectively). (B and C) Macroscopic images of spleen (B) and liver (C). (D) Microscopic analyses of liver. Histological samples were stained by Hematoxyline and Eosine (H&E) or silver impregnation. (E) Platelet counts in
peripheral blood from indicated genotypes of mice. Bars in the graph indicate averages. mature B cell depleted Ighm-/-Unc93b1D34A/D34A mice did not induce T cell
activation and moderated phenotypes (Figure 3D and 3E). It suggests that B
cells are activated by TLR7 hyper-response, and the B cells activate T cells to
generate phenotypes of Unc93b1D34A/D34A mice.However, thrombocytopenia
was not completely recovered in Ighm-/-Unc93b1D34A/D34A mice but
completely recovered in Rag2-/-Unc93b1D34A/D34A mice. Interaction between
cell types and phenotypes should be confirmed as a future plan. References 4. Pisitkun P, Deane JA, Difilippantonio MJ, Tarasenko T, Satterthwaite AB,
Bolland S: Autoreactive B cell responses to RNA-related antigens due to
TLR7 gene duplication. Science 2006, 312:1669-1672. 5. Santiago-Raber ML, Kikuchi S, Borel P, Uematsu S, Akira S, Kotzin BL, Izui S:
Evidence for genes in addition to Tlr7 in the Yaa translocation linked with
acceleration of systemic lupus erythematosus. J Immunol 2008,
181:1556-1562. 6. Asagiri M, Hirai T, Kunigami T, Kamano S, Gober HJ, Okamoto K,
Nishikawa K, Latz E, Golenbock DT, Aoki K, et al: Cathepsin K-dependent
toll-like receptor 9 signaling revealed in experimental arthritis. Science
2008, 319:624-627. 1. Blasius AL, Beutler B: Intracellular toll-like receptors. Immunity 2010,
32:305-315. 2. Fukui R, Saitoh S, Matsumoto F, Kozuka-Hata H, Oyama M, Tabeta K,
Beutler B, Miyake K: Unc93B1 biases Toll-like receptor responses to
nucleic acid in dendritic cells toward DNA- but against RNA-sensing. J Exp Med 2009, 206:1339-1350. 7. Deane JA, Pisitkun P, Barrett RS, Feigenbaum L, Town T, Ward JM,
Flavell RA, Bolland S: Control of toll-like receptor 7 expression is essential
to restrict autoimmunity and dendritic cell proliferation. Immunity 2007,
27:801-810. 3. Tabeta K, Hoebe K, Janssen EM, Du X, Georgel P, Crozat K, Mudd S,
Mann N, Sovath S, Goode J, et al: The Unc93b1 mutation 3d disrupts
exogenous antigen presentation and signaling via Toll-like receptors 3,
7 and 9. Nat Immunol 2006, 7:156-164. 8. Lande R, Gregorio J, Facchinetti V, Chatterjee B, Wang YH, Homey B, Cao W,
Su B, Nestle FO, Zal T, et al: Plasmacytoid dendritic cells sense self-DNA
coupled with antimicrobial peptide. Nature 2007, 449:564-569. Stimulation of bone formation in cortical bone of the mice treated with a
novel bone anabolic peptide with osteoclastogenesis inhibitory activity
Yuriko Furuya1*, Kohji Uchida1, Hisataka Yasuda2 y
j
1Nagahama Institute for Biochemical Science, Oriental Yeast Co Ltd, Shiga,
Japan; 2Planning & Development Group Bioindustry Division, Oriental Yeast
Co Ltd, Tokyo, Japan y
j
1Nagahama Institute for Biochemical Science, Oriental Yeast Co Ltd, Shiga,
Japan; 2Planning & Development Group Bioindustry Division, Oriental Yeast
Co Ltd, Tokyo, Japan T-regs and Th17 cells are the new generation of CD4+T-cells which play
crucial role in autoimmunity. Both of subsets can influence each other
and probably have common precursor. A key question for understanding
the mechanism of autoimmunity is to recognize how T-regs and Th17
cells turn from self-protection to autoreactivity. Based on literature data
and own observations, we have constructed a conception of age-
dependent thymic T-cells maturation “peripherialisation” as cause of
errors in Th17-T-reg cells interrelations. The connection of T-regs with
thymus is determined currently. Connection of Th17 cells with thymus
remains to be determined properly. Main, there may be naturally
occurring Tregs of thymic origin that are resistant to cell death and serve
as “reserve pool” for autoimmunity protective suppressors. This
mechanism could be affected by external factors producing profound
lymphopenia [1]. Previously we found that RA patients with numerous
rheumatoid nodules (RN) and lymphopenia had statistically reliable
decrease of CD3+T-cells level. We found definite negative correlation
between CD3+PBL amount and RN number (p = 0,029). In all RA patients
with and without RN we didn’t found the decrease of CD4 receptor. Hereby we expected to find unusual CD3-4+ and CD3-8+ cells in RA. Otherwise the percentage of CD3+4+ and CD3+8+ cells was normal in
general. But in 4 RA patients after magnetic separation of CD3+T-cells we
detected reliable amount of CD3-4+ lymphocytes (25-28%) These cells
were not detected before separation. One of possible explanation of this
phenomenon is CD3 molecule modulation after the contact with anti-CD3
antibodies conjugated with magnetic particles. So the presence of T-cells
with unusual phenotype in peripheral blood of RA patients doesn’t give
absolute evidence of T-cells maturation disorders. According to our
viewpoint “recent thymic emigrants” (RTE) fraction presence among Arthritis Research & Therapy 2012, 14(Suppl 1):P20 Arthritis Research & Therapy 2012, 14(Suppl 1):P20 Arthritis Research & Therapy 2012, 14(Suppl 1):P20 Background: Receptor activator of nuclear factor-B ligand (RANKL), a
member of tumor necrosis factor (TNF)-a, is produced by osteoblasts (Obs)
and stimulates its receptor RANK on osteoclast (Oc) progenitors to
differentiate them to osteoclasts. P18 Serum concentrations of both IgG1 and IgE Abs were about
100-times higher in 20-week old FasKO mice than in WT mice; however,
there was no significant difference between WT and FasKO mice in the
ability of B cells to produce IgG1 and IgE Abs in the presence of IL-4 and
anti-CD40 Ab inducing co-stimulatory signals. Additionally, the production of
IL-4 by T cells was same. These results suggested that other type of cells Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Page 30 of 54 Figure 1(abstract P19) Balb/c FasKO mice develope allergic blepharitis. Figure 1(abstract P19) Balb/c FasKO mice develope allergic blepharitis. enhanced IgG1 and IgE Abs production from B cells in Balb/c FasKO mice. To
identify the cells enhancing IgG1 and IgE Abs production, we cultured B cells
in vitro in the presence of IL-4 and anti-CD40 Ab together with various types
of cells from Balb/c FasKO mice. In the result, we found FasKO non-T non-B
cells upregulated the production of both IgG1 and IgE from B cells. Moreover, the number of these cells was specifically increased in Balb/c
FasKO mice.All the results indicate that these cells enhance production of
IgG1 and IgE from B cells in the presence of IL-4 and anti-CD40 Ab, and
excessive accumulation of these cells may cause allergy via hyper-
production of IgE. References cell cultures. The anabolic effect of WP9QY peptide was enhanced
markedly by addition of BMP2. Increases in mRNA expression of IGF1, collagen type I, and osteocalcin
were observed in E1 cells treated with the peptide for 12 and 96 h in
GeneChip analysis. Addition of p38 MAP kinase inhibitor reduced ALP
activity in E1 cells treated with the peptide, suggesting a signal through
p38 was involved in the mechanisms. Conclusions: Taken together, the peptide abrogated osteoclastogenesis
by blocking RANKL-RANK signaling and stimulated Ob differentiation/
mineralization with unknown mechanism in vitro. However, in our
experimental conditions the peptide exhibited bone anabolic effect
dominantly in vivo. Since the peptide is known to bind RANKL, we
hypothesize that the peptide shows the bone anabolic activity with
reverse signaling through RANKL on Obs. 1. Moro K, Yamada T, Tanabe M, Takeuchi T, Ikawa T, Kawamoto H,
Furusawa J, Ohtani M, Fujii H, Koyasu S: Innate production of T(H)2
cytokines by adipose tissue-associated c-Kit(+)Sca-1(+) lymphoid cells. Nature 2010, 463:540-544. 1. P20 P20
Stimulation of bone formation in cortical bone of the mice treated with a
novel bone anabolic peptide with osteoclastogenesis inhibitory activity
Yuriko Furuya1*, Kohji Uchida1, Hisataka Yasuda2 Stimulation of bone formation in cortical bone of the mice treated with a
novel bone anabolic peptide with osteoclastogenesis inhibitory activity
Yuriko Furuya1*, Kohji Uchida1, Hisataka Yasuda2 WP9QY peptide designed to mimics TNF
receptor’s contact site to TNF-a was known to abrogate osteoclastogenesis
in vitro by blocking RANKL-RANK signaling. WP9QY ameliorated collagen-
induced arthritis and osteoporosis in mouse models. Here we report that
the peptide surprisingly exhibited bone anabolic effect in vitro and in vivo. Materials and methods: WP9QY was administered subcutaneously to mice
three times per day for 5 days at a dose of 10 mg/kg in normal mice,
followed by peripheral quantitative computed tomography (pQCT) and
histomorphometrical analyses. To clarify the mechanism by which the
peptide exerted the bone anabolic effect, we examined the effects of the
peptide on osteoblast (Ob) differentiation/mineralization with mouse MC3T3-
E1 (E1) cells and human mesenchymal stem (MSC) cells, and those on
osteoclast (Oc) differentiation with RAW264 cells in the presence of sRANKL. Background: Receptor activator of nuclear factor-B ligand (RANKL), a
member of tumor necrosis factor (TNF)-a, is produced by osteoblasts (Obs)
and stimulates its receptor RANK on osteoclast (Oc) progenitors to
differentiate them to osteoclasts. WP9QY peptide designed to mimics TNF
receptor’s contact site to TNF-a was known to abrogate osteoclastogenesis
in vitro by blocking RANKL-RANK signaling. WP9QY ameliorated collagen-
induced arthritis and osteoporosis in mouse models. Here we report that
the peptide surprisingly exhibited bone anabolic effect in vitro and in vivo. Materials and methods: WP9QY was administered subcutaneously to mice
three times per day for 5 days at a dose of 10 mg/kg in normal mice,
followed by peripheral quantitative computed tomography (pQCT) and
histomorphometrical analyses. To clarify the mechanism by which the
peptide exerted the bone anabolic effect, we examined the effects of the
peptide on osteoblast (Ob) differentiation/mineralization with mouse MC3T3-
E1 (E1) cells and human mesenchymal stem (MSC) cells, and those on
osteoclast (Oc) differentiation with RAW264 cells in the presence of sRANKL. Results: WP9QY augmented bone mineral density (BMD) significantly in
cortical bone not in trabecular bone. Histomorphometrical analysis showed
that the peptide had little effect on osteoclasts in distal femoral
metaphysis, but markedly increased bone formation rate in femoral
diaphysis. The peptide markedly increased alkaline phosphatase (ALP, a
marker for Ob) activity in E1 and MSC cell cultures and decreased tartrate-
resistant acid phosphatase (TRAP, a marker for Oc) activity in RAW264 cell
culture in a dose-dependent manner, respectively. P18 Moro K, Yamada T, Tanabe M, Takeuchi T, Ikawa T, Kawamoto H,
Furusawa J, Ohtani M, Fujii H, Koyasu S: Innate production of T(H)2
cytokines by adipose tissue-associated c-Kit(+)Sca-1(+) lymphoid cells. Nature 2010, 463:540-544. 2. Neill DR, Wong SH, Bellosi A, Flynn RJ, Daly M, Langford TK, Bucks C,
Kane CM, Fallon PG, Pannell R, Jolin HE, McKenzie AN: Nuocytes represent
a new innate effector leukocyte that mediates type-2 immunity. Nature
2010, 464:1367-1370. P21
T-regs/Th17 function defect in systemic autoimmunity as a result of
“recent thymic emigrants” maturation defect
Mark Goloviznin1*, Natalia Lakhonina1, Alexander Yarilin2, Yulia Buldakova1,
Vitaly Timofeev3, Tatiana Kremenchugskaya1, Marina Struchkova1
1Department of Internal Diseases of Dental Faculty, Moscow State University
of Medicine and Dentistry, Russia; 2Laboratory of Cell Immunology, Research
Center Institute of Immunology, Moscow, Russia; 3Department of Faculty
Therapy of Russian State Medical University, Moscow, Russia
Arthritis Research & Therapy 2012, 14(Suppl 1):P21 P21
T-regs/Th17 function defect in systemic autoimmunity as a result of
“recent thymic emigrants” maturation defect
1*
1
2
1 Mark Goloviznin1*, Natalia Lakhonina1, Alexander Yarilin2, Yulia Buldakova1,
Vitaly Timofeev3, Tatiana Kremenchugskaya1, Marina Struchkova1
1Department of Internal Diseases of Dental Faculty, Moscow State University
of Medicine and Dentistry, Russia; 2Laboratory of Cell Immunology, Research
Center Institute of Immunology, Moscow, Russia; 3Department of Faculty
Therapy of Russian State Medical University, Moscow, Russia
Arthritis Research & Therapy 2012, 14(Suppl 1):P21 P22 )
y
g p
Result: The mean of serum 25(OH)D3 level was 22.80 ± 16,23 ng/mL.14
patients (25.5%) had vitamin D deficiency (<10 ng/mL), 34 patients
(61.8%) had vitamin D insufficiency (10-30 ng/mL), and 7 patients (14.7%)
had normal vitamin D levels. There were significant difference level of
anti-dsDNA antibodies (112.46 vs 267.13 U/ml; p < 0.05) and IgM ACA
(16.40 vs 29.7 IU/ml; p < 0,05) in patients with vitamin D insufficiency
and vitamin D defisiency. Serum level of 25(OH)D3 were negatively
related with level of anti-dsDNA and IgM ACA (r: - 0, and r: - 0,72
respectively). The mean of SLEDAI was 15,0 + 10.46. Serum vitamin D
levels were inversely correlated with SLEDAI (R=-0.319, p<0.05). Normal
BMD at lumbal spine found in 21 (38.2%) patients. 26 patients (47.3%)
were osteopenia, and 8 (14.5%) patients were osteoporosis. At femoral
neck, 25 (45.5%) patients had normal BMD, 23(41.8%) patients were
osteopenia, 7 (12.7%) patients were osteoporosis. There were no
significant correlation between vitamin D level and BMD at lumbal spine
(p = 0,531) and at femoral neck (p = 0,175). Dendritic cell immunoreceptor (DCIR) is associated with anti-cyclic
citrullinated peptides (anti-CCP) antibody - negative rheumatoid
arthritis in Chinese Han population Stimulation of bone formation in cortical bone of the mice treated with a
novel bone anabolic peptide with osteoclastogenesis inhibitory activity
Yuriko Furuya1*, Kohji Uchida1, Hisataka Yasuda2 In addition, the peptide
stimulated mineralization evaluated by alizarin red staining in E1 and MSC p
Results: WP9QY augmented bone mineral density (BMD) significantly in
cortical bone not in trabecular bone. Histomorphometrical analysis showed
that the peptide had little effect on osteoclasts in distal femoral
metaphysis, but markedly increased bone formation rate in femoral
diaphysis. The peptide markedly increased alkaline phosphatase (ALP, a
marker for Ob) activity in E1 and MSC cell cultures and decreased tartrate-
resistant acid phosphatase (TRAP, a marker for Oc) activity in RAW264 cell
culture in a dose-dependent manner, respectively. In addition, the peptide
stimulated mineralization evaluated by alizarin red staining in E1 and MSC Page 31 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 References 1. Toloza SM, Cle DE, Gladman DD, Ibanez D, Urowitz MB: Vitamin D
insufficiency in large female cohort. Lupus 2010, 19:13-19. 2. Ruiz-Irastorza G, Egurbide MV, Olivares N, Martinez-Berriotxoa A, Aguirre C:
Vitamin D deficiency in sustemic lupus erythematosus: prevalence,
predictor and clinical consequences. Rheumatology 2008, 47:920-923. 3. Arnson Y, Amital H, Shoenfeld Y: Vitamin D and autoimmunity: new
aetiological and therapeutic considerations. Ann Rheum 2007,
66:1137-1142. 1. Toloza SM, Cle DE, Gladman DD, Ibanez D, Urowitz MB: Vitamin D
insufficiency in large female cohort. Lupus 2010, 19:13-19. 2. Ruiz-Irastorza G, Egurbide MV, Olivares N, Martinez-Berriotxoa A, Aguirre C:
Vitamin D deficiency in sustemic lupus erythematosus: prevalence,
predictor and clinical consequences. Rheumatology 2008, 47:920-923. 3. Arnson Y, Amital H, Shoenfeld Y: Vitamin D and autoimmunity: new
aetiological and therapeutic considerations. Ann Rheum 2007,
66:1137-1142. p
Results: The DCIR rs2377422 was found significantly associated with RA
(allele analysis: OR 1.17, 95%CI 1.04-1.31, p=3.67 × 10-3; genotype analysis
(recessive model C/C vs. T/T + T/C): OR 1.37, 95%CI 1.08-1.73, p=9.04 ×
10-3). Following stratification for anti-CCP status, a suggestive association
of rs2377422 with anti-CCP-positive RA was observed (p = 0.058, OR 1.34,
95%CI 0.99-1.82). In contrast, the CC genotype of rs2377422 was found
specifically to confer susceptible risk for anti-CCP-negative RA (OR 1.92,
95%CI 1.27-2.90, p=1.99 × 10-3), despite loss of power in the analysis. The
relative risk of RA was 3.0 (95%CI 1.33-6.91, p=6.48 × 10-3) in individuals
carrying rs2377422 TT genotype with SE alleles, and 9.06 (95%CI 3.33-
25.61, p=2.08 × 10-6) in individuals carrying rs2377422 CC genotype with
SE genes. The interaction between rs2377422 and SE alleles was
significant, as measured by the attributable proportion (AP) due to
interaction (0.60). DCIR gene transcription quantification analysis further
proved the dominant effect of rs2480256 CC genotype on DCIR
expression levels in RA patients (C/C vs. T/T + T/C: 0.55 ± 0.09 vs. 0.24 ±
0.02, p=1.67 × 10-3). 4. Amital H, Szekanecz Z, Szucs G, et al: Serum concentration of 25-OH
vitamin D in patients with systemic lupus erythematosus (SLE) are
inversely related to disease activity: is it time to routinely supplement
patients with SLE with vitamin D? Ann Rheum Dis 2010, 69:1155-1157. Uncoupling protein 3 attenuates generation of reactive oxygen
species by interacting with thioredoxin 2 in the mitochondrial
intermembrane space Katsuya Hirasaka1*, Edward M Mills2, Shohei Kohno1, Tomoki Abe1,
Chika Ikeda1, Tasuku Maeda1, Shigetada Kondo1, Ayako Maita1,
Yuushi Okumura1, Takeshi Nikawa1
1Department of Nutritional Physiology, Institute of Health Biosciences,
University of Tokushima, Tokushima, 770-8503, Japan; 2Division of
Pharmacology/Toxicology, University of Texas at Austin, Austin, TX 78712, USA
Arthritis Research & Therapy 2012, 14(Suppl 1):P24 Conclusions: Our study provides evidence for association between DCIR
rs2377422 and RA, particularly with anti-CCP-negative RA in non-
Caucasian populations. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Backround: Vitamin D defficiency has been reported to have negative
association with clinical manifestation and disease activity of SLE [1,2]. Vit
D has an important role in the pathogenesis of SLE [3] and it is necessary
to give vit D supplementation to the patients [4]. The objective of our
study was to determine the association between serum vitamin D level
with auto antibodies expression, disease activity and bone mineral
density in SLE patients. T-regs and hypothetically among Th17-cells is the sign of normal Th17/T-
regs function. Otherwise the absence of RTE among them leads to
immunopathology. CD31 receptor and T cell receptor rearrangement
excision circles (TREC) are now markers of RTE. We investigated the
number of CD4+CD31+T-cells in RA patients. The preliminary results
permit us to suggest the diminution of RTE in RA (less then 1%/ml) We
also found the diminution of TREC amount in PBL of 22 rheumatoid
arthritis patients, (Median 0,035539 units). FOXP3, RORg, RORa and CD31
expression in RA will permit to establish role of RTE in autoimmunity. y
Patients and methods: 55 female patients with SLE were recruited from
Clinic of Rheumato-Immunology, Saiful Anwar Hospital, Malang, Indonesia. Mean age of the patients 31.12 years (12-64 yo) with duration of illness
18,4 months (2-54 mo). Serum vitamin (25 (OH)D3 level was assayed using
ELISA method (Cusabio, normal value>30 ng/mL). Anti ds-DNA and Anti
Cardiolipin antibodies were assayed using ELISA method (Diagnostic
Automation, Inc, USA). Disease activity assessed by SLE disease activity
index (SLEDAI) and BMD was assessed by bone densitometry using DEXA. Association between variables (serum vitamin D and autoantibodies level,
BMD and SLEDAI) were analyzed using Spearman correlation. Acknowledgements: The work is done in framework of project 11-04-
01670 sponsored by Russian Foundation of Basic Research. Reference 1. Littman D, Rudensky A: Th17 and regulatory cells in mediating and
restraining inflammation. Cell 2010, 140:845-858. P23
Association between serum level of Vitamin D with autoantibodies
expression, disease activity (SLEDAI) and bone mineral density (BMD) in
patients with Systemic Lupus Erythematosus (SLE)
Handono Kalim1*, Singgih Wahono1, Putra Suryana BP1, Lenny Puspitasari1,
Fajar Hadi Wijayanto1, Kusworini Handono2
1Rheumato-Immunology Division, Department of Internal Medicine,
Brawijaya University, Malang, Indonesia; 2Department of Clinical Pathology
Faculty of Medicine, Brawijaya University, Malang, Indonesia
Arthritis Research & Therapy 2012, 14(Suppl 1):P23 Dendritic cell immunoreceptor (DCIR) is associated with anti-cyclic
citrullinated peptides (anti-CCP) antibody - negative rheumatoid
arthritis in Chinese Han population Jianping Guo, Xinyu Wu, Xiaolan Lu, Yin Su, Ru Li, Jing He, Xu Liu,
Zhanguo Li* g
Department of Rheumatology and Immunology, Peking University People’s
Hospital, Beijing, 100044, China Arthritis Research & Therapy 2012, 14(Suppl 1):P22 Background: The dendritic cell immunoreceptor (DCIR) is an important
member of C-type lectin superfamily, which has been shown evidence for
susceptibility to arthritis in multiple animal models. The human DCIR
polymorphisms have been shown a nominal association with rheumatoid
arthritis (RA) susceptibility, mainly with anti-cyclic citrullinated peptides
(anti-CCP) antibody -negative RA in Swedish population. We aimed to
investigate the possible association of DCIR with RA susceptibility in
Chinese Han population. Conclusion: A large proportion ofSLE patients had low vitamin D levels. There were positive association between vit D level and autoantibodies
expression in SLE and negative association between serum vitamin D
levels with SLEDAI. No association was found between serum vit D level
and BMD. p p
Methods: A total of 1193 patients with RA and 1278 healthy controls
were genotyped for single-nucleotide polymorphism (SNP) rs2377422 and
rs10840759. Association analyses were performed on the whole data set
and on RA subsets based on the status of anti-CCP antibody in RA
patients. The interaction between rs2377422 and HLA-DRB1 shared
epitope (SE) was also analyzed for RA susceptibility. Finally, we carried
out association analysis of rs2377422 with DCIR mRNA expression in RA
patients. P26 P26
Community-based epidemiological study on hyperuricemia and gout
over 5 years in Huang-pu district, Shanghai
Hui Du1*, Shun-Le Chen1, Chun-De Bao1, Xiao-Dong Wang1, Yuan Wang1,
Yue-Ying Gu1, Kusuki Nishioka2
1Department of Rheumatology, Ren Ji Hospital, Shanghai Jiaotong University
School of Medicine, Shanghai 200001, China; 2Institute of Medical Science,
Tokyo Medical University, Tokyo 160-8402, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P26 Adrenal function in rheumatoid arthritis: acorrelation with disease
activity Richard Imrich1,2*, Miroslav Vlcek1,2, Jana Kerlik1, Michael Vogeser3,
Fabian Kirchhoff3, Adela Penesova1,2, Zofia Radikova1, Jozef Lukac4,
Jozef Rovensky4 1Laboratory of Human Endocrinology, Institute of Experimental
Endocrinology, Slovak Academy of Sciences, Bratislava, 833 06, Slovakia;
2Center for Molecular Medicine, Slovak Academy of Sciences, Bratislava, 831
01, Slovakia; 3Institute of Clinical Chemistry, Hospital of the University of
Munich, Munich, 81377, Germany; 4National Institute of Rheumatic Diseases,
Piestany, 921 12, Slovakia
Arthritis Research & Therapy 2012, 14(Suppl 1):P27 P27 Results/conclusions: We found that temporal disruption of TACE under
the control of Mx1 transgene prevented lethality from endotoxin shock. Furthermore, inactivation of TACE in macrophage/monocyte lineage cells
also rendered significant protection against LPS-induced septic shock. Consistent with these findings, serum TNFa levels in the TACE mutant
mice were much lower than those in control mice. The present study thus
shows that 1) TACE is indeed a principal enzyme responsible for the
release of soluble TNFa in vivo, and that 2) inactivation of TACE in
macrophage/monocyte lineage cells is sufficient to yield strong protection
against LPS-induced endotoxin shock. Taken together, the present data
indicate inhibition of TACE activity as a potential therapeutic target for
TNFa-related disorders. P25 P25
Conditional inactivation of the ectodomain shedding of pro-TNFa in
monocytes prevents lethality from LPS-induced septic shock
Keisuke Horiuchi1*, Tokuhiro Kimura2, Yasunori Okada2, Kazuhiro Chiba1,
Carl P Blobel3, Yoshiaki Toyama1
1Department of Orthopedic Surgery, School of Medicine, Keio Univ. Tokyo,
Japan; 2Department of Pathology, School of Medicine, Keio Univ. Tokyo,
Japan; 3Arthritis and Tissue Degeneration Program, Hospital for Special
Surgery, New York, USA
Arthritis Research & Therapy 2012, 14(Suppl 1):P25 yp
Results: The prevalence of hyperuricemia was 15.1% (51 cases/338,
SUA>7 mg/dl) in men, 8.7% in women (41 cases/469, SUA>6 mg/dl) and
seven gout male patients were found in 1996. The prevalence of
hyperuricemia was 19.5% (52 cases/266, SUA>7 mg/dl) in men, 12.6% (71
cases/564, SUA>6 mg/dl) in women in 2001.The prevalence of gout in
2037 dwellers in Huangpu District was 0.77% in men and 0.34% in both
sexes in 1996 [1]. Background: TNFa is synthesized as a membrane-bound precursor and
proteolytically released from cells. Soluble TNFa is the primary mediator of
pathologies such as rheumatoid arthritis, Crohn’s disease, and endotoxin
shock. Although several different enzymes have been implicated in this
proteolytic activity, recent studies lean toward the TNFa converting
enzyme (TACE/ADAM17) as the most relevant TNFasheddasein vivo.In the
present study, we asked whether the inactivation TACE could yield a
protection from lipopolysaccharide(LPS)-induced septic shockin mice. Conclusions: The mean SUA level in each age group in 2001 was higher
than that of in 1996 (see Table 1). The prevalence of hyperuricemia was
increased rapidly (Male: 15.1% in 1996 to 19.5% in 2001; Female: 8.7% in
1996 to 12.6% in 2001 p < 0.05). Azotemia (≥23 vs. <23 mg/dl),
hypertriglyceridemia (≥200 vs. <150 mg/dl, 150–200 vs. <150 mg/dl) were
the associated risk factors by multiple logistic regression analyzing the
independent effect of each variable on hyperuricemia. Materials and methods: To abrogate TNFa shedding activity in vivo, we
generated conditional TACE-deficient mice using Cre-loxP system [1]. We
mated these mice with Mx1-Cretg mice and LysM-Cretg mice to inactivate
TACE in BM cells and macrophage/monocyte lineage cells, respectively. Endotoxin shock was induced by i.p. injection of 5 μg of LPS and 20 mg of
D-galactosamine. All injected mice were closely monitored every hour for
the first 16 h and every 3-6 h thereafter. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Table 1(abstract P26) Comparison of SUA levels in
different age group over 5 years
Year/Age
40-49
50-59
60+
Male 2001
**5.85 ± 1.02 (56)
*6.04 ± 1.14 (74)
6.20 ± 1.32 (136)
1996
5.38 ± 1.06 (100)
5.53 ± 1.30 (50)
5.90 ± 1.45 (188)
Female 2001
4.19 ± 0.88 (164)
4.72 ± 1.07 (146)
**5.14 ± 1.17 (254)
1996
4.13 ± 0.94 (118)
4.49 ± 1.05 (84)
4.74 ± 1.07 (267) Table 1(abstract P26) Comparison of SUA levels in
different age group over 5 years Trx2. In addition, Trx2 directly associated with UCP3 through a
mitochondrial targeting signaling sequence, was processed in the
intermembrane space, and thereby allowing redox reactions. A bimolecular
fluorescence complementation analysis demonstrated that the interaction
of these proteins occurs in the mitochondrial intermembrane space. Furthermore, increased UCP3 expression significantly attenuated ROS
production in isolated mitochondrial without effects on membrane
potential, however this effect is lost by Trx2 knock down. These results
suggest that UCP3 binds to Trx2 in the mitochondrial intermembrane
space and attenuates ROS production. with the uricase-peroxidase enzymatic method. In 2001, 830 residents
≥40 years of age were taken for SUA levels measured with the same
enzymatic method. Cholesterol, triglyceride, blood urea nitrogen,
glycosylated hemoglobin, ESR, rheumatoid factor etc were measured as
possible risk factors to enter the multiple logistic regression analysis on
hyperuricemia. P23 P23
Association between serum level of Vitamin D with autoantibodies
expression, disease activity (SLEDAI) and bone mineral density (BMD) in
patients with Systemic Lupus Erythematosus (SLE)
Handono Kalim1*, Singgih Wahono1, Putra Suryana BP1, Lenny Puspitasari1,
Fajar Hadi Wijayanto1, Kusworini Handono2
1Rheumato-Immunology Division, Department of Internal Medicine,
Brawijaya University, Malang, Indonesia; 2Department of Clinical Pathology
Faculty of Medicine, Brawijaya University, Malang, Indonesia
Arthritis Research & Therapy 2012, 14(Suppl 1):P23 Uncoupling protein 3 (UCP3) is primarily expressed in the inner membrane
of skeletal muscle mitochondria. It has been proposed that UCP3 reduces
production of reactive oxygen species (ROS) and oxidative damage. However, the mechanisms by which UCP3 attenuates ROS production are
not well understood. Here we report that UCP3 interacts with the non-
processed form of thioredoxin 2 (Trx2), a redox protein that is localized in
mitochondria, but not processed Trx2, which is involved in cellular
responses to ROS. The hydrophilic sequences within the N-terminal tail of
UCP3, which faces the intermembrane space, are necessary for binding to Association between serum level of Vitamin D with autoantibodies
expression, disease activity (SLEDAI) and bone mineral density (BMD) in
patients with Systemic Lupus Erythematosus (SLE)
1*
1
1
1 Page 32 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Reference 1. Chen S, Du H, Wang Y, Xu L: The epidemiology study of hyperuricemia
and gout in a community population of Huangpu District in Shanghai. Chin Med J (Engl) 1998, 111(3):228-30. Reference 1. Horiuchi K, Kimura T, Miyamoto T, et al: TNF-a-converting enzyme (TACE/
ADAM17) inactivation in mouse myeloid cells prevents lethality from
endotoxin shock. J Immunol 2007, 179:2686-2689. 1. Horiuchi K, Kimura T, Miyamoto T, et al: TNF-a-converting enzyme (TACE/
ADAM17) inactivation in mouse myeloid cells prevents lethality from
endotoxin shock. J Immunol 2007, 179:2686-2689. Background: Hypothalamic-pituitary-adrenocortical dysfunction contributes
to a complex pathogenesis of rheumatoid arthritis (RA). Decreased
production of adrenal androgens and subtle changes in cortisol production
has been observed in RA, particularly in femalepatients with premenopausal
onset of the disease. Our study was aimed to investigate (1) adrenocortical
function in relation to disease and inflammatory activity and to analyze
cortisol bioavailability in RA females. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 exposure in osteoblast still speculative [3,4]. The aim of our study is to assess
the role of specific reactive oxygen species signalling on the effect of MG as
an AGE on increased caspase-3 expression in pre-osteoblast. plasma cortisol, 17-hydroxyprogesterone, dehydroepiandrosterone and
androstenedione responses in the ACTH test compared to healthy controls. Patients with DAS28>3.2 had lower (p < 0.05) dehydroepiandrosterone
response in the ACTH test compared to patients with DAS28≤3.2. C-reactive
protein (CRP), DAS28, and interleukin (IL)-6 negatively correlated with
androstenedione response to Synacthen. Responses of all measured adrenal
steroids were lower (p < 0.05) in patients on low-dose glucocorticoids
compared to healthy controls. RA patients not treated with glucocorticoids
had lower total cortisol response (p = 0.038) compared to controls, however,
these patients did not differ in free plasma cortisol in the ACTH test. p
p
p
Materials and methods: Pre-osteoblast MC3T3E1 cell line was obtained
from American Type Culture Cell. Caspase-3 expression in the cells were
assayed in basal condition and after the cells exposed with methyl glyoxal
on dose 5 μM for 6 hours incubation. Diethylthiocarbamoic acid,
mercaptosuccinate, or deferoxamine was added in the culture media to
block specific reactive oxygen species signalling for the development of
osteoblast apoptosis. The caspase 3 expression were assesses from each
different groups of preosteoblast culture: preosteoblast exposed to nothing,
preosteoblast exposed to methyl glyoxal, preosteoblast exposed to
diethylthiocarbamoic (SOD blocker), exposed to mercaptosuccinate
(glutathion peroxidase blocker) and exposed to deferoxamine (Fe++ blocker);
and osteoblast exposed to methyl glyoxal and diethylthiocarbamoic, or
mercaptosuccinate, or deferoxamine. The result were analyzed using
Kruskall Wallis test with p < 00.5 significant. Conclusions: The present data indicate an association of increased
disease activity with a decrease in adrenal androgen-producing
zonareticularisin RA. A modest suppression of stimulated cortisol in
glucocorticoid-untreated RA patients is not associated with decreased
cortisol bioavailability. g
Results: Our study showed that MG significantly increased caspase3
expression (apoptosis) of osteoblast. Expression of caspase3 in osteoblast
were significantly highest when the cells exposed to SOD blocker compare
with when the cells exposed to GSH and Fe++ blocker whether the cells
exposed to MG. Hydroxyl radical increase caspase-3 expression higher than
another reactive oxygen species (•OH > H2O2 >•O2
-) in pre-osteoblast
MC3T3E1 without exposed methyl glyoxal. The result showed that
superoxide radical more dominant in increasing caspase-3 expression than
another reactive oxygen species (•O2
- >•OH > H2O2) in pre-osteoblast
MC3T3E1 with MG exposure. Community-based epidemiological study on hyperuricemia and gout
over 5 years in Huang-pu district, Shanghai y
Materials and methods: Adrenal steroids including free plasma cortisol
responses to the low-dose ACTH stimulation test (1 µg Synactheni.v.) were
investigated in 23 premenopausal RA and in 15 age- and BMI-matched
healthy females. Twelve (N = 12) out of 23 RA patients were on low-dose
glucocorticoids (<8.5 mg/day of prednisone or equivalent). When patients
were divided into low (disease activity score 28; DAS28≤3.2) and moderate
to high disease activity (DAS28>3.2) subgroups, glucocorticoid-treated
patients comprised 53% and 50% of patients in each of the subgroups. Plasma C-reactive protein, interleukin (IL)-1b, IL-4, IL-6, IL-8, IL-10, IL-17,
interferon gamma and tumor necrosis factor alpha concentrations were
measured at the baseline. Hui Du1*, Shun-Le Chen1, Chun-De Bao1, Xiao-Dong Wang1, Yuan Wang1,
Yue-Ying Gu1, Kusuki Nishioka2 1Department of Rheumatology, Ren Ji Hospital, Shanghai Jiaotong University
School of Medicine, Shanghai 200001, China; 2Institute of Medical Science,
Tokyo Medical University, Tokyo 160-8402, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P26 1Department of Rheumatology, Ren Ji Hospital, Shanghai Jiaotong University
School of Medicine, Shanghai 200001, China; 2Institute of Medical Science,
Tokyo Medical University, Tokyo 160-8402, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P26 Background: A community-based survey on the prevalence of
hyperuricemia and associated factors was carried out in 1996 and 2001. Materials and methods: In the target community in1996, 2037 dwellers
(age≥15 years old) were interviewed with relevant questionnaires from
house to house. According to even house number, 807 blood samples
(age≥40 years old) were taken for serum uric acid (SUA) levels measured Results: RA patients had high C-reactive protein, IL-6, IL-8 and tumor
necrosis factor alpha. Patients with DAS28>3.2 had lower (p < 0.05) total Page 33 of 54 Page 33 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 There is no significant differences regarding
the effecfts of GSH and Fe++block on osteoblast caspase3 expression. Rheumatoid arthritis fibroblast-like synoviocytes show the upregulation
of myeloid cell specific transcription factor PU.1 and B cell specific
transcriptional co-activator OBF-1, and express functional BCMA
Kenji Itoh1*, Katsuya Nagatani2 j
,
y
g
1Department of Rheumatology, National Defense Medical College,
Tokorozawa, Saitama, Japan; 2Department of Rheumatology & Clinical
Immunology, Jichi Medical University, Shimotsuke, Tochigi, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P28 Objective: Fibroblast-like synoviocytes (FLS) are among the principal
effector cells in the pathogenesis of rheumatoid arthritis (RA).This study
shows the variety of stimulating effects of a proliferation-inducing ligand
(APRIL), and its specific effect on the FLS in the affected RA synovium
(RA-FLS). Conclusion: The increased osteoblast apoptosis caused by AGE (MG) is
mediated by specific reactive oxygen signalling, SOD activation. References 1. Hein G, Wiegand R, Lehmann G, Stein G, Franke S: Advanced glycation
end-products pentosidine and Nεcarboxymethyllysine are elevated in
serum of patients with osteoporosis. Rheumatology (Oxford) 2003,
42:1242-1246. 1. Hein G, Wiegand R, Lehmann G, Stein G, Franke S: Advanced glycation
end-products pentosidine and Nεcarboxymethyllysine are elevated in
serum of patients with osteoporosis. Rheumatology (Oxford) 2003,
42:1242-1246. Results: A significantly higher level of soluble APRIL was detected in RA
serum compared with in normal serum. Among the three receptors of APRIL
tested, RA-FLS expressed only the B cell maturation antigen (BCMA),
whereas the FLS in the affected osteoarthritis synovium (OA-FLS) expressed
none of the receptors. Moreover, RA-FLS expressed transcription factor PU.1
and B cell specific-transcriptional co-activator OBF.1, which were normally
expressed during myeloid and B-lymphoid cell development. The expression
levels of PU.1 and OBF-1 were correlated with those of BCMA in RA-FLS. APRIL stimulated RA-FLS but not OA-FLS to produce interleukin (IL)-6, tumor
necrosis factor (TNF)-a, IL-1b and APRIL itself. APRIL also enhanced the
receptor activator of nuclear factor kappa B ligand (RANKL) expression in
RA-FLS. Moreover, APRIL enhanced the cell-cycle progression of RA-FLS. Neutralization of APRIL by BCMA-Fc fusion protein attenuated all these
stimulating effects of APRIL on RA-FLS. 2. Franke S, Siggkelkow H, Wolf G, Hein G: Advanced glycation end products
influence the mRNA expression of RAGE, RANKL and various
osteoblastic genes in human osteoblasts. Arch Physiol Biochem 2007,
113(3):154-161. 3. Chan W, Wu H, Shiao N: Apoptotic signaling in methylglyoxal-treated
human osteoblasts involves oxidative stress, c-Jun N-terminal kinase,
caspase-3, and p21-activated kinase 2. J Cell Biochem 2007, 100:1056-1069. 3. Chan W, Wu H, Shiao N: Apoptotic signaling in methylglyoxal-treated
human osteoblasts involves oxidative stress, c-Jun N-terminal kinase,
caspase-3, and p21-activated kinase 2. J Cell Biochem 2007, 100:1056-1069. 3. Rheumatoid arthritis fibroblast-like synoviocytes show the upregulation
of myeloid cell specific transcription factor PU.1 and B cell specific
transcriptional co-activator OBF-1, and express functional BCMA
Kenji Itoh1*, Katsuya Nagatani2 Chan W, Wu H, Shiao N: Apoptotic signaling in methylglyoxal-treated
human osteoblasts involves oxidative stress, c-Jun N-terminal kinase,
caspase-3, and p21-activated kinase 2. J Cell Biochem 2007, 100:1056-1069. 4. Natalia M, Ahmed H, Etcheverry SB: Regulation of advanced glycation and
product (AGE) receptors and apoptosis by AGEs in osteoblast-like cells. Mol Cell Biochem 2007, 306:87-94. 4. Natalia M, Ahmed H, Etcheverry SB: Regulation of advanced glycation and
product (AGE) receptors and apoptosis by AGEs in osteoblast-like cells. Mol Cell Biochem 2007, 306:87-94. g
Conclusions: RA-FLS express BCMA, and are stimulated by APRIL. These
results provide evidence that APRIL is one of the main regulators in the
pathogenesis of RA.Epigenetic regulation of BCMA transcription in RA-FLS
might contribute to the underlying mechanisms of this condition. Discrepancy between patient and physician in assessment of global
severity in early rheumatoid arthritis Yuko Kaneko*, Masataka Kuwana, Tsutomu Takeuchi
Division of Rheumatology, Department of Internal Medicine, Keio University
School of Medicine, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P30 P28 P28
Rheumatoid arthritis fibroblast-like synoviocytes show the upregulation
of myeloid cell specific transcription factor PU.1 and B cell specific
transcriptional co-activator OBF-1, and express functional BCMA
Kenji Itoh1*, Katsuya Nagatani2
1Department of Rheumatology, National Defense Medical College,
Tokorozawa, Saitama, Japan; 2Department of Rheumatology & Clinical
Immunology, Jichi Medical University, Shimotsuke, Tochigi, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P28 Rheumatoid arthritis fibroblast-like synoviocytes show the upregulation
of myeloid cell specific transcription factor PU.1 and B cell specific
transcriptional co-activator OBF-1, and express functional BCMA
Kenji Itoh1*, Katsuya Nagatani2 Cartilage-specific deletion of prar-gamma in mice results in early
endochondral ossification defects and accelerated aging-dependent
development of osteoarthritis
1*
1
1
1 Along with a huge amount of works about the importance of a metabolic
syndrome in development of cardiovascular diseases, within last decade in
the literature there was a series of reports on a pathogenetic role of this
syndrome in formation and more serious current of some other diseases of
an internal. In process of doctrine development about a metabolic
syndrome [1], there was new data about existence at gout of various signs
insulin resistance [2]. At the same time, there are insufficiently studied
questions on a role of various categories of a hyperglycemia (a
hyperglycemia on an empty stomach and a postloading hyperglycemia) in
a pathogenesis and gout and hyperuricemia clinic. Roxana Monemdjou1*, Faezeh Vasheghani1, Hassan Fahmi1, Gemma Perez1,
Meryem Blati1, Noboru Taniguchi2, Martin Lotz2, René St-Arnaud3,
Jean-Pierre Pelletier1, Johanne Martel-Pelletier1, Frank Beier4, Mohit Kapoor1
1Osteoarthritis Research Unit, University of Montreal Hospital Research Centre
(CRCHUM) and Department of Medicine, University of Montreal, Montreal,
Quebec, Canada, H2L 4M1; 2Department of Molecular and Experimental
Medicine, The Scripps Research Institute, La Jolla, California 92037, USA; 3Genetics
Unit, Shriners Hospital for Children, Montreal, Quebec, Canada, H3G 1A6;
4Department of Physiology and Pharmacology, Schulich School of Medicine and
Dentistry, University of Western Ontario, London, Ontario, Canada, N6A 5C1
Arthritis Research & Therapy 2012, 14(Suppl 1):P31 Method of the inquiry: 120 males with gout at age 30-69 were
examined to investigate the connection between different categories of
hyperglycemia and level of uric acid in patients with gout. Gout was
revealed on the basis of criteria of American Rheumatic Association. Glucose tolerance condition was revealed by carrying out standard test of
glucose tolerance (TGT) with revealing of glycemia on an empty stomach,
and also in one and two hours after taking 75 gr glucose by the
examined patients. Background: Long bones develop through a strict coordinated process of
endochondral ossification within the growth plate resulting in the
replacement of cartilage by bone and defect in this coordinated process
may result in skeletal abnormalities such as dwarfism, kyposis and also
age-related defects such as osteoarthritis (OA). PPARg, a transcription
factor, plays a key role in lipid homeostasis but its in vivo role in cartilage/
bone development is unknown. Therefore, we determined the specific in
vivo role of PPARg in endochondral bone ossification, cartilage/bone
development and in OA using cartilage-specific PPARg knockout (KO) mice. Materials and methods: Cartilage-specific PPARg KO mice were generated
using LoxP/Cre system. Cartilage-specific deletion of prar-gamma in mice results in early
endochondral ossification defects and accelerated aging-dependent
development of osteoarthritis
1*
1
1
1 Histomorphometric/immunohistochemical analysis
was performed to account for ossification patterns, chondrocyte
proliferation, differentiation, hypertrophy, skeletal organization, bone
density, calcium deposition and mouse OA phenotypic changes during
aging using OARSI scoring. Real-Time PCR and western blotting was
performed to determine the expression of key markers involved in
endochondral ossification and cartilage degradation. The results: According to the revealed findings average levels of
uric acid in patients with gout with normal glucose tolerance had
531,56 ± 0,38 mcmol/l. With damaged glucose tolerance on an empty
stomach and in two hours after glucose loading, levels of uric acid were
more higher(658,18 ± 0,27 mcmol/l and 656,22 ± 0,34 mcmol/l
correspondingly). At the same time on damaged glucose tolerance in an
hour after glucose loading average level of uric acid was 501,16 ± 0,33
mcmol/l. We should draw attention that the difference of average levels
of uric acid among people with disorders glucose tolerance on an
empty stomach and in two hours after glucose loading was more differ
from level of uric acid among people with glucose tolerance disorder in
an hour after glucose loading (p*<0,05). Results: Histomorphometric analyses of embryonic and adult mutant mice
demonstrate reduced long bone growth, calcium deposition, bone density,
vascularity as well as delayed primary and secondary ossification. Mutant
growth plates are disorganized with reduced cellularity, proliferation,
differentiation, hypertrophy and loss of columnar organization. Isolated
chondrocytes and cartilage explants from E16.5 and 3 weeks old mutant
mice further show decreased expression of ECM production products,
aggrecan and collagen II, and increased expression of catabolic enzyme,
MMP-13. Furthermore, aged mutant mice exhibit accelerated OA-like
phenotypes associated with enhanced cartilage degradation, synovial
inflammation, and increased expression of MMP-13, and MMP-generated
aggrecan and collagen II neoepitopes. Subsequently, we show that loss of
PPARg and subsequent downstream alterations in phosphatase and tensin
homolog on chromosome ten (PTEN)/Akt pathway contribute towards
increased expression of OA catabolic and inflammatory markers, thus
enabling the articular cartilage of PPARg-deficient mice to be more
susceptible to degradation during aging. Conclusion: According to these results we can come to the conclusion
that the level of hyperglycemia has connection with existence in
patients with hyperglycemia on an empty stomach and two hours after
glucose loading. At the same time the problem about connection of
uric acid level with hyperglycemia in an hour after glucose loading
should be examined farther. P31 Cartilage-specific deletion of prar-gamma in mice results in early
endochondral ossification defects and accelerated aging-dependent
development of osteoarthritis
1*
1
1
1 P33
Effect of B cell depletion using peptide tetramers in
collagen-induced arthritis
* Conclusions: For the first time, we demonstrate that loss of PPARg in the
cartilage results in endochondral bone defects and subsequently
accelerated OA in mice. PPARg is essential for normal development of
cartilage and bone. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Table 1(abstract P32) Average findings of uric acid in
blood in patients with different categories of
hyperglycemia
Categories of hyperglycemia
n
M
± m
Normal tolerance
13
531,56
0,38
Hyperglycemia on an empty stomach
11
658,18 *
0,27
Hyperglycemia after 1 hour
20
501,16
0,33
Hyperglycemia after 2 hours
76
656,22 *
0,34
* Note: in the table is shown the reliability of differences concerning an
indicator in hyperglycemia group in 1 hour after loading a glucose. Table 1(abstract P32) Average findings of uric acid in
blood in patients with different categories of
hyperglycemia physicians. Tender joint count, DAS28 3-variables, CRP andHAQ were
significantly higher in patients with discordance group where patients
rated themselves worse than physicians than in patients with
concordance (p < 0.05). HAQ score was correlated with the degree of the
difference (R = 0.49, p < 0.05). Conclusions: Higher disease activity and higher HAQ score was
associated the discordance between patient’s and physician’s VAS in early
RA patients, indicating the possibility of physicians underestimating the
patient’s global disease severity at 1-year since diagnosis. References 1. McNeill AM, Rosamond WD, Girman CJ, et al: The metabolic syndrome
and 11-year risk of incident cardiovascular disease in the atherosclerosis
risk in Communities Study. Diabetes Care 2005, 28(2):385-390. 1. McNeill AM, Rosamond WD, Girman CJ, et al: The metabolic syndrome
and 11-year risk of incident cardiovascular disease in the atherosclerosis
risk in Communities Study. Diabetes Care 2005, 28(2):385-390. 2. Vázquez-Mellado J, et al: Metabolic syndrome and ischemic heart disease
in gout. J Clin Reumatol 2004, 10(3):105-109. 2. Vázquez-Mellado J, et al: Metabolic syndrome and ischemic heart disease
in gout. J Clin Reumatol 2004, 10(3):105-109. P33
Effect of B cell depletion using peptide tetramers in
collagen-induced arthritis
Kazuya Michishita*, Kimito Kawahata, Takeyuki Kanzaki, Lisa Akahira, Toshiki Eri,
Kazuhiko Yamamoto
Department of Allergy and Rheumatology, Graduate School of Medicine, the
University of Tokyo, Tokyo, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P33 Cartilage-specific deletion of prar-gamma in mice results in early
endochondral ossification defects and accelerated aging-dependent
development of osteoarthritis
1*
1
1
1 Perhaps, that rising of glycemia level in an
hour after glucose loading is a compensator mechanism in patients
with gout. P29
Methyl glyoxal increase apoptosis (CASPASE-3 expression) in
pre-osteoblast MC3T3E1 cell line via SOD activity
1*
2 Objective: To evaluate the discrepancy between patient and physician in
assessment of global severity in early rheumatoid arthritis (RA) and to
explore factors affecting the discrepancy at 1-year since the diagnosis of RA. Methods: One hundred nine patients with RA with median disease duration
of 4 months were enrolled in this study. The global assessment was
performed using 100 mm-visual analog scale (VAS). The difference between
patient’s and physician’s assessment wascalculated by subtracting
physician’s VAS from patient’s VAS, and the difference more than 20 mm
was defined as discordant.RA patients were stratified by concordance and
discordance of VAS scoring at 1-year after the diagnosis. To clarify the
factors affecting the discrepancy, clinical characteristics, disease activity
using Disease Activity Score (DAS28) 3-variables, functional status by Health
Assessment Questionnaire (HAQ) were compared between patients with
concordance and discordance. Izaak Zoelkarnain Akbar1*, Handono Kalim2, Djoko Wahono Soeatmadji2,
Mohammad Hidayat3 Izaak Zoelkarnain Akbar1*, Handono Kalim2, Djoko Wahono Soeatmadji2,
Mohammad Hidayat3
1 1Department of Orthopaedic, Ulin General Hospital, Faculty of Medicine,
Lambung Mangkurat University, Banjarmasin, Indonesia; 2Department of
Internal Medicine, Saiful Anwar General Hospital, Faculty of Medicine,
Brawijaya University, Malang, Indonesia; 3Department of Orthopaedic, Saiful
Anwar General Hospital, Faculty of Medicine, Brawijaya University, Malang,
Indonesia Arthritis Research & Therapy 2012, 14(Suppl 1):P29 Background: Increased advanced glycation end (AGE) products have been
reported to be an important cause of increased osteoblast apoptosis in
osteoporosis [1,2]. Methylglyoxal (MG) is a reactive dicarbonyl compound
endogenously produced mainly from glycolytic intermediates. The
involvement of specific reactive oxygen spesies (•OH/ H2O2 / •O2
-) in
increased apoptosis (caspase-3 expression) caused by methyl glyoxal Results: The discordance between patient’s and physician’s VAS at 1-year
was found in 41 patients (37%), consisting of 5 patients whose VAS was
better than physicians and 36 patients whose VAS was worse than Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Page 34 of 54 P33 Kazuya Michishita*, Kimito Kawahata, Takeyuki Kanzaki, Lisa Akahira, Toshiki Eri,
Kazuhiko Yamamoto
Department of Allergy and Rheumatology, Graduate School of Medicine, the
University of Tokyo, Tokyo, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P33 P34 P34
Peripheral tolerance induced by apoptotic cells and PD-1+ CD8 T cells
Hirotaka Kazama1*, Tomonori Iyoda1, Satoko Yokoyama1, Kayo Inaba1,
Thomas A Ferguson2, Shin Yonehara1
1Department of Biostudies, Kyoto University Graduate School, Kyoto 606-
8501 Japan; 2Department of Ophthalmology and Visual Science, Washington
University School of Medicine, MO 63110 USA
Arthritis Research & Therapy 2012, 14(Suppl 1):P34 We aimed to explore frequency, plating efficiency and osteoblastogenic
potential of synovial mesenchymal progenitors and correlate them with
intensity of local and systemic inflammation in patients with JIA. Materials and methods: Synovial fluid cells were collected from 19
patients with oligoarticular JIA (oJIA) and 8 patients with poliarticular JIA
(pJIA), plated in density 1.5 × 106/mL in 24-well plates, and cultured in
aMEM + 10% FCS. Osteoblastogenesis was stimulated by the addition of
50 μg/ml ascorbic acid and 5 mmol b-glycerophosphate. To exclude
inflammatory and hematopoietic cells, adherent cells were passaged
three times, and osteoblastogenesis again induced in fourth passage (P4). Osteoblastogenesis was assessed by intensity of alkaline phospatase (AP)
histochemical staining. In addition, osteoblast (Runx2, AP, OPG, RANKL)
and cytokine/chemokine (IL-1, IL-4, CCL2, CCL4 and MIP1a) gene
expression were assessed in P4 osteoblastogenic cultures. Self tolerization in peripheral is critical to prevent autoimmune diseases
including arthritis and here we focus on the role of PD-1 in tolerance
induction against the antigen associated with apoptotic cellsdelivered
intravenously(i.v.). We accessed delayed type hypersensitivity (DTH)
reaction against hapten (TNP) as antigen specific immune response, in
which the injection of TNP-apoptotic cells i.v.suppressedDTH in wild type
mice but we found not in PD-1 KO mice (Figure 1). Adaptive transfer of
CD8 T cells into PD-1 KO mouse from wild type mice tolerated with TNP-
apoptotic cells suppresses DTH. This result shows PD-1 functions on CD8
T cells for immune suppression.Additionally we neutralized the PD-1 with
antibody to determine the phase when PD-1 functions for immune
tolerance by apoptotic cells, and identified PD-1functionsparticularly at
the initial phase of antigen specific immune response. We are further
studying the mechanism of suppressive role of PD-1+ CD8 T cells that
should be activated with apoptotic cells. Results: Plating efficiency of synovial mesenchymal progenitors was
decreased in patients with pJIA in comparison to patients with oJIA. Passage was successful only in 3 (37.5%) pJIA patients, and 18 (94.7%)
oJIA patients. Plated at equal density, P4 synovial adherent cells from
pJIA patients formed less fibroblastic colonies. P32 P32
Average findings of uric acid in blood in patients with gout with
different categories of hyperglycemia
1*
2
1 Background: B cell depletion therapy is effective in the treatment of
various autoimmune diseases. However, this therapy is shown to be
associated with increased risk of adverse effects such as opportunistic
infections. Therefore, in this study, we developed and analyzed the g
yp
g y
Ulugbek K Kayumov1*, Marif Sh Karimov2, Nargiza A Abdukhakimova1
1Tashkent Institute of Postgraduate Medical Education, Tashkent, Uzbekistan;
2Tashkent Medical Academy, Tashkent, Uzbekistan
Arthritis Research & Therapy 2012, 14(Suppl 1):P32 Page 35 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 P35 Decreased plating efficiency, proliferation and osteogenic
differentiation of synovial fluid mesenchymal progenitors as a
marker of severity of juvenile idiopathic arthritis
Elvira Lazic Mosler1*, Marija Jelusic-Drazic2, Danka Grcevic1,3, Ana Marusic4,
Natasa Kovacic1,5 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 selective depletion therapy of pathogenic B cells using peptide tetramers
in collagen-induced arthritis model. selective depletion therapy of pathogenic B cells using peptide tetramers
in collagen-induced arthritis model. P34 Osteoblastogenesis was
higher in children with oJIA than in children with pJIA, both from
primary synovial cells (median 1119.08; IQR 476.57-1470.26 vs. 141.58;
IQR 14.47-237.50, arbitrary units, p < 0.005, Mann-Whitney test), and P4
cells (median 1162.00; IQR 102.00 to 5484.50 vs. 12.00; IQR 6.00-307.37,
arbitrary units, p < 0.05). Osteoblastogenesis from primary synoviocytes
negatively correlated with erythrocyte sedimentation rate (r= -0.4139,
p = 0.03), and synovial concentration of IL-17 (r= -0.4174, p = 0.04). Expression of osteoprotegerin and CCL2 was decreased in P4
osteoblastogenic cultures from pJIA in comparison with oJIA patients
(p < 0.05). Acknowledgements: We were kindly provided the neutralizing
antibodies to PD-1 and PD-L2 by Dr. Hideo Yagita (Juntendo University)
and hybridoma to PD-L1 from Dr. Miyuki Azuma (Tokyo Medical and
Dental University). Conclusions: Severe forms of JIA are characterized by decreased
proliferation, osteogenic differentiation and immunoregulatory potential
of synovial mesenchymal cells, correlating with inflammatory activity. References Figure 1(abstract P34) PD-1 is essential for tolerance induced by
apoptotic cells. TNP-apoptotic cells were injected intravenously into
PD-1 hetero- or homo- deficient mice. The mice were immunized with
TNP (Filled bar) or preconditioned with apoptotic cells before
immunization with TNP (open bar). 1. Barr T, Carmichael NM, Sándor GK: Juvenile idiopathic arthritis: a chronic
pediatric musculoskeletal condition with significant orofacial
manifestations. J Can Dent Assoc 2008, 74:813-821. 1. Barr T, Carmichael NM, Sándor GK: Juvenile idiopathic arthritis: a chronic
pediatric musculoskeletal condition with significant orofacial
manifestations. J Can Dent Assoc 2008, 74:813-821. 2. Li X, Makarov SS: An essential role of NF-kappaB in the “tumor-like”
phenotype of arthritic synoviocytes. Proc Natl Acad Sci USA 2006,
103:17432-17437. 2. Li X, Makarov SS: An essential role of NF-kappaB in the “tumor-like”
phenotype of arthritic synoviocytes. Proc Natl Acad Sci USA 2006,
103:17432-17437. 1Department of Systems BioMedicine, National Research Institute for Child
Health and Development, Setagaya-ku, Tokyo 157-8535, Japan; 2Department
of Molecular Life Sciences, Basic Medical Science and Molecular Medicine, Decreased plating efficiency, proliferation and osteogenic
differentiation of synovial fluid mesenchymal progenitors as a
marker of severity of juvenile idiopathic arthritis Methods: Since the antigenic targets of pathogenic antibodies are
identified in collagen-induced arthritis (CIA) model, we developed toxin-
conjugated peptide tetramers, which contained pathogenic epitope of
mouse type II Collagen (CII). The male DBA/1J mice were immunized with
bovine CII and injected with toxin-conjugated peptide tetramers on day
10 and day 20 after CIIimmunization.We analyzed the effect of toxin-
conjugated peptide tetramers on the production of autoantibodies and
clinical course of arthritis. Decreased plating efficiency, proliferation and osteogenic
differentiation of synovial fluid mesenchymal progenitors as a
marker of severity of juvenile idiopathic arthritis y
j
p
Elvira Lazic Mosler1*, Marija Jelusic-Drazic2, Danka Grcevic1,3, Ana Marusic4,
Natasa Kovacic1,5 1Laboratory for Molecular Immunology, University of Zagreb School of
Medicine, Zagreb HR-10000, Croatia; 2Department of Pediatrics, Division of
Pediatric Rheumatology and Immunology, University Hospital Centre Zagreb,
Zagreb HR-10000, Croatia; 3Department of Physiology and Immunology,
University of Zagreb School of Medicine, Zagreb HR-10000, Croatia;
4Department of Research in Biomedicine and Health, University of Split
School of Medicine, Split HR-21000, Croatia; 5Department of Anatomy,
University of Zagreb School of Medicine, Zagreb HR-10000, Croatia
Arthritis Research & Therapy 2012, 14(Suppl 1):P35 Results: The incidence of arthritis was significantly lower (P < 0.05) in the
tetramer-treated group than in the control group. The mean serum
antibody levels for CII did not differ significantly, but there were
significant differences in the anti-peptide antibodies over time. Conclusions: Peptide tetramer is effective in the selective depletion of
antigen-specific B cells and decreased the incidence of arthritis in CIA
model. Therefore, depletion of antigen-specific B cells using this
strategy might be a new therapeutic intervention of autoimmune
diseases. Background: Juvenile idiopathic arthritis (JIA) is a rheumatic pediatric
disease characterized by synovial inflammation in one or more joints [1]. Inflammation results in hyperplastic changes of the synovium, destruction
of articular cartilage and subchondral osteoresorption. Murine models of
arthritis revealed impaired osteogenic/chondrogenic differentiation of
synovial mesenchymal progenitors via inflammation-induced activation of
NF-B [2]. P37
T cell receptor rearrangement excision circles (TREC) study as an
approach to “in vivo” thymus gland function investigation
N t li L kh
i
1* M k G l
i
i 1 Al i
D
t k
2 T cell receptor rearrangement excision circles (TREC) study as an
approach to “in vivo” thymus gland function investigation
N t li L kh
i
1* M k G l
i
i 1 Al i
D
t k
2 4Department of Systems BioMedicine, Tokyo Medical and Dental University,
Bunkyo-ku, Tokyo 113-8510, Japan; 5Core Research for Evolutional Science
and Technology, Japan Science and Technology Corporation, Saitama
332-0012, Japan Natalia Lakhonina , Mark Goloviznin , Almira Donetskova ,
Margarita Nikonova2, Alexander Yarilin2, Yulia Buldakova1, Anna Tektova1
1Department of Internal Diseases of Dental faculty, Moscow State University
of Medicine and Dentistry, Russia; 2Laboratory of cell immunology, Research
center Institute of Immunology, Moscow, Russia
Arthritis Research & Therapy 2012, 14(Suppl 1):P37 E-mail: riho@hope.tokai-u.jp E-mail: riho@hope.tokai-u.jp Arthritis Research & Therapy 2012, 14(Suppl 1):P36 microRNAs (miRNAs), which are class of post-transcriptional regulators
such as short 19 to 23-nucleotide non-coding RNAs, complementarily
bind seed sequences in the 3’-untranslational region of multiple target
mRNAs, resulting in their suppression of translation or degradation [1]. In
the former case, since the mRNA expression of the targets does not any
change, transcriptomics approach, such as expression array, cannot
identify the targets. Age-dependent changes in immune system such as thymus gland
involution, T-cells amount decrease, are typical both for autoimmune
diseases (Rheumatoid arthritis - RA), and progressive atherosclerosis
characterized as “accelerated ageing”. But till now processes of T-cell
maturation were studied only by indirect methods. The introduction of
T-cell receptor excision circle (TREC) PCR-assay seemed to enable direct
detection of recent thymic emigrants in peripheral blood and therefore
the quantification of thymic output [1]. High TREC levels were detected
during childhood, and were decreasing with age, but TREC-expressing
cells are not completely lost in the elderly. At the first stage of our
investigation we studied TREC level in 3 groups of healthy donors: 16
people. 16 - 30 years old (group 1, TREC Median 0,156299 Units),
8 persons 30 - 45 years old (group 2, TREC Median 0,08782 Units) and 9
people over 45 years (group 3 TREC Median 0,051858 Units). Thereby we
confirmed age-related decline of thymic output in healthy donors. y
Recent studies shed light on the fine-tuning mechanism of miRNAs in
myriad biological processes including development [2], tumorigenesis [3]
and inflammation [4]. We have identified enhancement of mir-146a
expression in rheumatoid arthritis synoviocyte and macrophages [5],
whilst suppression of them in osteoarthritis [6]. LC-MS/MS-based shotgun proteomics identified the targets of arthritis-
related microRNA Figure 1(abstract P34) PD-1 is essential for tolerance induced by
apoptotic cells. TNP-apoptotic cells were injected intravenously into
PD-1 hetero- or homo- deficient mice. The mice were immunized with
TNP (Filled bar) or preconditioned with apoptotic cells before
immunization with TNP (open bar). Riho Kurata1,2*, Tomo Yonezawa1, Hideki Nakajima3, Shyuji Takada1,
Hiroshi Asahara1,4,5 1Department of Systems BioMedicine, National Research Institute for Child
Health and Development, Setagaya-ku, Tokyo 157-8535, Japan; 2Department
of Molecular Life Sciences, Basic Medical Science and Molecular Medicine, Page 36 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 P37
T cell receptor rearrangement excision circles (TREC) study as an
approach to “in vivo” thymus gland function investigation
N t li L kh
i
1* M k G l
i
i 1 Al i
D
t k
2 Another group also have
identified the enhancement of mir-146a and mir-155 in response to
bacterial pathogen such as lipopolysaccaride [7]. Recently, mice lacking of
mir-155 are resistant to collagen-induced arthritis (CIA) [8], whilst
administration of mir-146a complexed with aterocollagen into joint
attenuates pathological condition of CIA [9]. These results indicate that
mir-146a and mir-155 plays an important role for developing arthritis and
inflammation. However, the targets of both two miRNAs and their
molecular mechanisms are not still fully identified. y
y
In RA patients we found age-dependent statistical definite difference of
TREC expression. In the 1-st group (n-12, age range 40,4+2,8 y) TREC
amount was following: Median 0,00766 I/U lower level 0,00045, upper level
0,01961. In the 2-nd group (n = 16, age range 57,5+1,32) TREC were
diminished (Median 0,00065, lower level 0,000002, upper level 0,00095). Detected high TREC amount in some young RA patients is not entirely
consistent with the data of literature.TREC level in patients with chronic
forms of coronary heart disease (age 55 - 70 years old) was lower but
comparable with donors group 3 (TREC Median 0,0200 Units). Unexpectedly high level of TREC comparable with donors group 2 we
detected in patients with Acute Myocardial Infarction (AMI) (10 patients,
age range 48 - 71 y) (TREC Median 0,089845 Units). According to our
viewpoint, the content of TREC in peripheral blood lymphocytes depends
both on thymic output and “peripheral” factors, such as survival time of
“naive” T cells in periphery. Recent data give evidence that the up-
regulation of Th1 cell-functions and interferon-g hyperproduction existed
in patients with AMI after the onset of symptoms. This may participate in
the immune-mediated ventricular remodeling after AMI. The slowing of
“naive"-T-cells turnover and Th1/Th2 imbalance could be the reason of
TREC increase in AMI patients. In this study, in order to identify the targets of them in translational level, we
established gain of function models using adenovirus- and CMV promoter-
mediated overexpression in several culture models and performed liquid
chromatography-tandem mass spectrometry-based shotgun proteomics in
these models. Acknowledgements: The authors sincerely thank Dr. Yanagiya R for
helpful advice on preparation of adenovirus, and Dr. Inoue A for the gift
of CMV vector for mir-146a. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Tokai University School of Medicine, Isehara, Kanagawa, Japan; 3Department
of Pediatric Hematology and Oncology Research, National Research Institute
for Child Health and Development, Setagaya-ku, Tokyo 157-8535, Japan;
4Department of Systems BioMedicine, Tokyo Medical and Dental University,
Bunkyo-ku, Tokyo 113-8510, Japan; 5Core Research for Evolutional Science
and Technology, Japan Science and Technology Corporation, Saitama
332-0012, Japan
E-mail: riho@hope.tokai-u.jp
Arthritis Research & Therapy 2012 14(Suppl 1):P36 Tokai University School of Medicine, Isehara, Kanagawa, Japan; 3Department
of Pediatric Hematology and Oncology Research, National Research Institute
for Child Health and Development, Setagaya-ku, Tokyo 157-8535, Japan; Reference 1. Hazenberg MD, Verschuren MC, Hamann D, Miedema F, van Dongen JJ: T
cell receptor excision circles as markers for recent thymic emigrants:
basic aspects, technical approach, and guidelines for interpretation. J Mol Med 2001, 79(11):631-40. 1. Hazenberg MD, Verschuren MC, Hamann D, Miedema F, van Dongen JJ: T
cell receptor excision circles as markers for recent thymic emigrants:
basic aspects, technical approach, and guidelines for interpretation. J Mol Med 2001, 79(11):631-40. 4. Lu LF, Boldin MP, Chaudhry A, Lin LL, Taganov KD, Hanada T,
Yoshimura A, Baltimore D, Rudensky AY: Function of miR-146a in
controlling Treg cell-mediated regulation of Th1 responses. Cell 2010,
142:914-929. 5. Nakasa T, Miyaki S, Okubo A, Hashimoto M, Nishida K, Ochi M, Asahara H:
Expression of microRNA-146 in rheumatoid arthritis synovial tissue. Arthritis Rheum 2008, 58:1284-1292. 5. Nakasa T, Miyaki S, Okubo A, Hashimoto M, Nishida K, Ochi M, Asahara H:
Expression of microRNA-146 in rheumatoid arthritis synovial tissue. Arthritis Rheum 2008, 58:1284-1292. References 1. Lee RC, Feinbaum RL, Ambros V: The C. elegans heterochronic gene lin-4
encodes small RNAs with antisense complementarity to lin-14. Cell 1993,
75:843-854. 1. Lee RC, Feinbaum RL, Ambros V: The C. elegans heterochronic gene lin-4
encodes small RNAs with antisense complementarity to lin-14. Cell 1993,
75:843-854. 2. Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE,
Horvitz HR, Ruvkun G: The 21-nucleotide let-7 RNA regulates
developmental timing in Caenorhabditis elegans. Nature 2005,
403:901-906. 2. Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE,
Horvitz HR, Ruvkun G: The 21-nucleotide let-7 RNA regulates
developmental timing in Caenorhabditis elegans. Nature 2005,
403:901-906. The work is done in framework of project 11-04-01670 sponsored by
Russian Foundation of Basic Research. Project director Dr. Goloviznin M.V. Reference 3. Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A,
Labourier E, Reinert KL, Brown D, Slack FJ: RAS is regulated by the let-7
microRNA family. Cell 2005, 120:635-647. 3. Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A,
Labourier E, Reinert KL, Brown D, Slack FJ: RAS is regulated by the let-7
microRNA family. Cell 2005, 120:635-647. P37 P37
T cell receptor rearrangement excision circles (TREC) study as an
approach to “in vivo” thymus gland function investigation
Natalia Lakhonina1*, Mark Goloviznin1, Almira Donetskova2,
Margarita Nikonova2, Alexander Yarilin2, Yulia Buldakova1, Anna Tektova1
1Department of Internal Diseases of Dental faculty, Moscow State University
of Medicine and Dentistry, Russia; 2Laboratory of cell immunology, Research
center Institute of Immunology, Moscow, Russia
Arthritis Research & Therapy 2012, 14(Suppl 1):P37 P37
T cell receptor rearrangement excision circles (TREC) study as an
approach to “in vivo” thymus gland function investigation
Natalia Lakhonina1*, Mark Goloviznin1, Almira Donetskova2,
Margarita Nikonova2, Alexander Yarilin2, Yulia Buldakova1, Anna Tektova1
1Department of Internal Diseases of Dental faculty, Moscow State University
of Medicine and Dentistry, Russia; 2Laboratory of cell immunology, Research
center Institute of Immunology, Moscow, Russia
Arthritis Research & Therapy 2012, 14(Suppl 1):P37 P39
Abnormal expressions of immune response-related genes in RA bone
marrow cells Methods: A multicenter, prospective, and observational study was started
in April, 2011 for patients with RA in Taiwan University Hospital, Taipei
Veterans General Hospital, and Chang Gung Memorial Hospital in Keelung. Patients who take anti-TNFa regiments or not (defined as naïve or never
take agent) were both enrolled in the study. The clinical history, DAS-28
score, chest film finding, sputum survey for active TB, and QFT screening
results were collected. Hooi-Ming Lee1*, Chieko Aoki2, Yasunori Shimaoka3, Kensuke Ochi4,
Takahiro Ochi5, Norihiro Nishimoto1,2 Hooi-Ming Lee1*, Chieko Aoki2, Yasunori Shimaoka3, Kensuke Ochi4,
Takahiro Ochi5, Norihiro Nishimoto1,2 1Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-Oka,
Suita, Osaka 565-0871, Japan; 2Laboratory of Immune Regulation, Wakayama
Medical University, 105 Saito Bio Innovation Center, 7-7-20 Saito-Asagi,
Ibaraki, Osaka 567-0085, Japan; 3Yukioka Hospital, 2-2-3 Ukita, Kita-ku, Osaka
530-0021, Japan; 4Kawasaki Municipal Kawasaki Hospital, 12-1 Shinkawa-dori,
Kawasaki-ku, Kawasaki, Kanagawa 210-0013, Japan; 5Osaka Police Hospital,
10-31 Kitayama-chou, Tennoji-ku, Osaka 543-0035, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P39 Results: A total of 147 patients were enrolled in the study, in which five of
them (3.4%) had history of anti-TB treatment and none had active TB at
the beginning of the investigation. There were 75 patients undergoing
anti-TNFa treatment before the study (42 patients (56%) took etanercepts
and the other 33 (46%) ones took adalimumabs) and 72 patients had not
(Table 1). Background: Rheumatoid arthritis (RA) is a systemic autoimmune disease
characterized by chronic synovitis that progresses to destruction of cartilage
and bone. Bone marrow (BM) cells have been shown to contribute to this
pathogenesis. In this study, we compared differentially expressed molecules
in BM cells from RA and osteoarthritis (OA) patients and analyzed abnormal
regulatory networks to identify the role of BM cells in RA. Based on QFT test, the frequency of latent TB infection (LTBI) were 12.5%
(9/72) for naïve patients, and 10.7% (8/75) for biologics users (p > 0.05). Risk analysis showed no difference between different QFT results in study
patients (Table 2). The interval between starting etanercepts or adalimumabs treatment and
screening for QFT test were 22.5 and 14.4 months (p > 0.05), respectively. Subgroup analysis showed possible risk factors for LTBI in patients who had
history of adalimumabs or etanercept treatment were the history of anti-TB
treatment and negative for BCG scar, respectively (p < 0.05). Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Results: The BM mononuclear cells showed 764 up-regulated and 1,910
down-regulated genes in RA patients relative to the OA group. EASE
revealed that the gene category response to external stimulus, which
included the gene category immune response, was overrepresented by the
up-regulated genes. So too were the gene categories signal transduction
and phosphate metabolism. Down-regulated genes were dominantly
classified in three gene categories: cell proliferation, which included mitotic
cell cycle, DNA replication and chromosome cycle, and DNA metabolism. Most genes in these categories overlapped with each other. IPA analysis
showed that the up-regulated genes in immune response were highly
relevant to the antigen presentation pathway and to interferon signaling. The major histocompatibility complex (MHC) class I molecules, HLA-E, HLA-F,
and HLA-G, tapasin (TAP) and TAP binding protein, both of which are
involved in peptide antigen binding and presentation via MHC class I
molecules, are depicted in the immune response molecule networks. Interferon gamma and interleukin 8 were overexpressed and found to play
central roles in these networks. synoviocytes (FLS) with reduced ability to differentiate into osteoblasts,
chondroblasts or adipocytes [2]. Since Fas is shown to inhibit osteoblast
differentiation [3], we were interested whether such inhibitory effect may
contribute to the pathogenesis of AIA. Materials and methods: AIA was induced in mice with a Fas gene
knockout (Fas -/-). Three weeks after pre-immunization with mBSA in
complete Freund’s adjuvant, wild-type (C57BL/6, wt) and Fas -/- mice
were injected with mBSA into each knee, whereas controls were injected
with equal volume of phosphate buffered saline (PBS). Three weeks after
injection we assessed joint diameters, histology, μCT scans, and
differentiation of bone marrow- and synovia-derived osteoblasts. Results: Knee diameters were increased in mBSA-injected wt mice
compared to PBS-injected controls (3.21 ± 0.2 vs. 2.98 ± 0.1, p < 0.05,
t-test), and this increase was not significant in Fas -/- mice (2.97 ± 0.2 vs. 2.87 ± 0.1). Histology revealed presence of synovial hyperplasia in both
mBSA-injected groups, but mBSA-injected wt mice had decreased
trabecular bone volume in distal femoral metaphyses (BV/TV) compared
to controls (1.08 ± 0.57 vs. 2.55 ± 0.43; p < 0.05, t-test). There was no
significant difference between mBSA-injected and control group in Fas -/-
mice (2.34 ± 0.62 vs. 2.61 ± 0.65). μCT analysis showed that mBSA-
injected wt mice had decreased BV/TV (2.99 ± 0.19 v. P39 P39 Fas deficiency attenuates bone loss during antigen induced arthritis in
mice 6. Yamasaki K, Nakasa T, Miyaki S, Ishikawa M, Deie M, Adachi N, Yasunaga Y,
Asahara H, Ochi M: Expression of MicroRNA-146a in osteoarthritis
cartilage. Arthritis Rheum 2009, 60:1035-1041. 6. Yamasaki K, Nakasa T, Miyaki S, Ishikawa M, Deie M, Adachi N, Yasunaga Y,
Asahara H, Ochi M: Expression of MicroRNA-146a in osteoarthritis
cartilage. Arthritis Rheum 2009, 60:1035-1041. Elvira Lazic Mosler1*, Sania Kuzmac1,2, Sanja Ivcevic1,3, Danka Grcevic1,3,
Ana Marusic4, Natasa Kovacic1,2
1Laboratory for Molecular Immunology, University of Zagreb School of
Medicine, Zagreb HR-10000, Croatia; 2Department of Anatomy, University of
Zagreb School of Medicine, Zagreb HR-10000, Croatia; 3Department of
Physiology and Immunology, University of Zagreb School of Medicine,
Zagreb HR-10000, Croatia; 4Department of Research in Biomedicine and
Health, University of Split School of Medicine, Split HR-21000, Croatia
Arthritis Research & Therapy 2012, 14(Suppl 1):P38 7. Taganov KD, Boldin MP, Chang KJ, Baltimore D: NF-kappaB-dependent
induction of microRNA miR-146, an inhibitor targeted to signaling
proteins of innate immune responses. Proc Natl Acad Sci USA 2006,
103:12481-12486. 8. Kurowska-Stolarska M, Alivernini S, Ballantine LE, Asquith DL, Millar NL,
Gilchrist DS, Reilly J, Ierna M, Fraser AR, Stolarski B, McSharry C, Hueber AJ,
Baxter D, Hunter J, Gay S, Liew FY, McInnes IB: MicroRNA-155 as a
proinflammatory regulator in clinical and experimental arthritis. Proc Natl
Acad Sci USA 2011, 108:11193-11198. Background: Antigen induced arthritis (AIA) is an experimental model of
rheumatoid arthritis induced by methylated bovine serum albumin
(mBSA) [1]. Hyperplastic synovia in AIA contains fibroblast-like 9. Nakasa T, Shibuya H, Nagata Y, Niimoto T, Ochi M: The inhibitory effect of
microRNA-146a expression on bone destruction in collagen-induced
arthritis. Arthritis Rheum 2011, 63:1582-1590. Page 37 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 P40 P40
Risk factors for latent tuberculosis infection in RA patients treated with
anti-tumor necrosis factor
1*
1
2
2 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 1.96 ± 0.19; p <
0.001, t-test) and trabecular number (TbN) (1.03 ± 0.03 vs. 0.64 ± 0.02), as
well as increased trabecular separation (TbSep) (256,89 ± 1395,12 vs. 312.40 ± 1323.91), compared to controls. mBSA injected Fas -/- mice had
decreased TbN compared to controls (0.815 ± 0.01 vs. 0.64 ± 0.04; p <
0.05, t-test), with no significant difference in other trabecular parameters. Osteoblast differentiation was increased in both wt and Fas -/- mBSA-
injected mice. Conclusions: Abnormal regulatory networks in the immune response and
cell cycle categories were identified in BM mononuclear cells from RA
patients, indicating that the BM is pathologically involved in RA. Risk factors for latent tuberculosis infection in RA patients treated with
anti-tumor necrosis factor
1*
1
2
2 Shiang-Fen Huang1 , Wei-Juin Su1, Sheng-Yuan Ruan2, Chong-Jen Yu2,
Song-Chou Hsieh3, Yu-Chih Liu4, Yeong-Jian Jan Wu5, Hsiao-Yi Lin6
1Department of Chest Medicine, Taipei Veterans General Hospital, School of
Medicine, National Yang-Ming University, Taipei, Taiwan; 2Division of
Pulmonary and Critical Care Medicine, Department of Internal Medicine,
National Taiwan University Hospital, Taipei, Taiwan; 3Division of Allergy,
Immunology and Rheumatology, Department of Internal Medicine, National
Taiwan University Hospital, Taipei, Taiwan; 4Department of Medicine, Division
of Pulmonary Medicine, Chang Gung Memorial Hospital, Keelung, Taiwan;
5Department of Medicine, Division of Allergy, Immunology and
Rheumatology, Chang Gung Memorial Hospital, Keelung, Taiwan; 6Division of
Allergy, Immunology and Rheumatology, Department of Medicine, Taipei
Veterans General Hospital, Taipei, Taiwan Conclusions: Our study demonstrated that Fas deficiency attenuated the
development of clinical signs and bone loss in AIA. The mechanisms of
this phenomenon need to be clarified. References 1. van den Berg WB, et al: Murine antigen-induced arthritis. Methods in
Molecular Medicine, Volume 136: Arthritis Research Cope AP Totowa (NJ):
Humana Press Inc 2007, 2:243-253. 1. van den Berg WB, et al: Murine antigen-induced arthritis. Methods in
Molecular Medicine, Volume 136: Arthritis Research Cope AP Totowa (NJ):
Humana Press Inc 2007, 2:243-253. Molecular Medicine, Volume 136: Arthritis Research Cope AP Totowa (NJ):
Humana Press Inc 2007, 2:243-253. 2. Li X, Makarov SS: An essential role of NF-kappaB in the “tumor-like”
phenotype of arthritic synoviocytes. Proc Natl Acad Sci USA 2006,
103:17432-7. 2. Li X, Makarov SS: An essential role of NF-kappaB in the “tumor-like”
phenotype of arthritic synoviocytes. Proc Natl Acad Sci USA 2006,
103:17432-7. 3. Kovacic N, Lukic IK, Grcevic D, Katavic V, Croucher P, Marusic A: The E-mail: syruan@ntu.edu.tw E-mail: syruan@ntu.edu.tw 3. Kovacic N, Lukic IK, Grcevic D, Katavic V, Croucher P, Marusic A: The
Fas/Fas-ligand system inhibits differentiation of murine osteoblasts but
has a limited role in osteoblast and osteoclast apoptosis. J Immunol
2007, 178:3379-89. Arthritis Research & Therapy 2012, 14(Suppl 1):P40 Arthritis Research & Therapy 2012, 14(Suppl 1):P40 Background: To estimate the prevalence of latent tuberculosis (TB)
infection according to the interferon-gamma release assay (IGRA,
QuantiFERON®-TB Gold In-Tube, QFT) in patients with rheumatoid arthritis
(RA), and assess the risk factors for incidence of active TB after TNF alpha
blocking agents treatment. P39
Abnormal expressions of immune response-related genes in RA bone
marrow cells Other factors
including DAS-28 score, presence of rheumatoid factor, white cell count, and
previous immunosuppressant dosage (ie, prednisolone and methotrexate)
were not related to the LTBI status (Table 3). Materials and methods: Gene expression profiles (GEPs) in BM-derived
mononuclear cells from 9 RA and 10 OA patients were obtained by DNA
microarray. Up- and down-regulated genes were identified by comparing
the GEPs from the two patient groups. Bioinformatics was performed by
Expression Analysis Systemic Explorer (EASE) 2.0 based on gene ontology,
followed by network pathway analysis with Ingenuity Pathways Analysis
(IPA) 7.5. Page 38 of 54 Page 38 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 P41
TGF-b signaling induces SnoN to suppress BMP-induced hypertrophic
maturation of chondrocytes
1*
1 2
1
1 2 TGF-b signaling induces SnoN to suppress BMP-induced hypertrophic
maturation of chondrocytes
1*
1 2
1
1 2 Shingo Maeda1*, Ichiro Kawamura1,2, Yasuhiro Ishidou1, Katsuyuki Imamura1,2,
Masahiro Yokouchi2, Setsuro Komiya1,2 Shingo Maeda1*, Ichiro Kawamura1,2, Yasuhiro Ishidou1, Katsuyuki Imamura1,2,
Masahiro Yokouchi2, Setsuro Komiya1,2 y
1Department of Medical Joint Materials, Kagoshima University, Kagoshima,
890-8544, Japan; 2Department of Orthopaedic Surgery, Kagoshima University,
Kagoshima, 890-8544, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P41 Background: Loss of TGF-b signaling in mice leads to promoted
hypertrophic conversion of articular chondrocytes, which process is
suggested to be linked to progression of osteoarthritis (OA). However, the
molecular mechanisms by which TGF-b signaling inhibits chondrocyte
maturation remain unclear. We screened for mediators downstream of
TGF-b signaling to inhibit chondrocyte hypertrophy. In summary, our findings provide evidences that the activation of Ca2+
permeable channel supports Ca2+ oscillations in progenitor cells and
therefore promotes the potential of osteoclast differentiation. Materials and methods: We induced choncrocyte differentiation of
ATDC5 cells with BMP-2. A TGF-b type I receptor inhibitor compound
SB431542 was applied to inhibit endogenous TGF-b signaling. Expression
of differentiation markers was evaluated by real-time RT-PCR and
immunoblot. The function of SnoN was studied by stable overexpression
and siRNA-knockdown approaches. Organ culture system using mouse
embryo metatarsal bone was employed to study the roles of TGF-b
signaling and SnoN in chondrocyte maturation. P41 p
Intact Trpv4 and Trpv4R616Q/V620I were equally transduced by retroviral
infection into bone marrow derived hematopoietic cells isolated from WT
mice, and mock-transfection was used as control. The resorptive activity
was significantly increased in Trpv4R616Q/V620I-expressing osteoclasts when
treated with RANKL for 7 days, associating increased NFATc1 and
calcitonin receptor mRNA expression. Noteworthy, the expression of these
differentiation markers was already elevated in Trpv4R616Q/V620I cells
before RANKL treatment, suggesting that the activation of Trpv4
advances osteoclast differentiation through Ca2+-NFATc1 pathway. Accordingly, basal [Ca2+]i, analyzed in progenitor cells treated with RANKL
for 24 hr, increased 2 fold in intact Trpv4 (p < 0.05) and 3 fold in
Trpv4R616Q/V620I (p < 0.01) compared to controls. Although spontaneous
Ca2+ oscillations were absent in control progenitor cells, Trpv4R616Q/V620I
progenitor cells already displayed irregular oscillatory pattern. 2+ P41
TGF-b signaling induces SnoN to suppress BMP-induced hypertrophic
maturation of chondrocytes
Shingo Maeda1*, Ichiro Kawamura1,2, Yasuhiro Ishidou1, Katsuyuki Imamura1,2,
Masahiro Yokouchi2, Setsuro Komiya1,2
1Department of Medical Joint Materials, Kagoshima University, Kagoshima,
890-8544, Japan; 2Department of Orthopaedic Surgery, Kagoshima University,
Kagoshima, 890-8544, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P41 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Since
the region of these substitutions at the trans-membrane pore domain is
perfectly conserved between species, we created a mutant of the mouse
Trpv4 (Trpv4R616Q/V620I) and characterized it on Ca2+ signaling especially in
the occurrences of oscillations at the initial step of osteoclast differentiation More patients had indeterminate QFT result after entracept treatment but
negative QFT result after adalimumab therapy (p<0.05). In current study,
none of patients with positive or indeterminate QFT result received
preventive INH treatment and none of them had evidence of non-
tuberculosis mycobacterium infection. y
Conclusion: The overall frequency of LTBI in patients with RA was 11.6%
in this study. Although history of anti-TB treatment and negative BCG
scar were risk factors for LTBI, other factors still need to be considered
due to limited sample size in current study. Further regular follow up
should be done. the occurrences of oscillations at the initial step of osteoclast differentiation. Intact Trpv4 and Trpv4R616Q/V620I were equally transduced by retroviral Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 pathways; Ca2+ oscillations evoked by the release of Ca2+ from the
endoplasmic reticulum, and/or Ca2+ entry from the extracellular fluid. The
latter is carried out by the plasmamembrane localized Ca2+ permeable
channel such as “transient receptor potentials (Trps)”. Trpv4-deficient mice
show an increased bone mass due to impaired osteoclast maturation,
because Trpv4 mediates Ca2+ influx at the late stage of osteoclast
differentiation and hereby regulates Ca2+ signaling [1]. Furthermore,
substitutions of amino acids R616Q/V620I of Trpv4 have been discovered as
gain of function mutations resulting in increased Ca2+ transport [2]. Since
the region of these substitutions at the trans-membrane pore domain is
perfectly conserved between species, we created a mutant of the mouse
Trpv4 (Trpv4R616Q/V620I) and characterized it on Ca2+ signaling especially in
the occurrences of oscillations at the initial step of osteoclast differentiation. Intact Trpv4 and Trpv4R616Q/V620I were equally transduced by retroviral
infection into bone marrow derived hematopoietic cells isolated from WT
mice, and mock-transfection was used as control. The resorptive activity
was significantly increased in Trpv4R616Q/V620I-expressing osteoclasts when
treated with RANKL for 7 days, associating increased NFATc1 and
calcitonin receptor mRNA expression. Noteworthy, the expression of these
differentiation markers was already elevated in Trpv4R616Q/V620I cells
before RANKL treatment, suggesting that the activation of Trpv4
advances osteoclast differentiation through Ca2+-NFATc1 pathway. Accordingly, basal [Ca2+]i, analyzed in progenitor cells treated with RANKL
for 24 hr, increased 2 fold in intact Trpv4 (p < 0.05) and 3 fold in
Trpv4R616Q/V620I (p < 0.01) compared to controls. Although spontaneous
Ca2+ oscillations were absent in control progenitor cells, Trpv4R616Q/V620I
progenitor cells already displayed irregular oscillatory pattern. In summary our findings provide evidences that the activation of Ca2+ pathways; Ca2+ oscillations evoked by the release of Ca2+ from the
endoplasmic reticulum, and/or Ca2+ entry from the extracellular fluid. The
latter is carried out by the plasmamembrane localized Ca2+ permeable
channel such as “transient receptor potentials (Trps)”. Trpv4-deficient mice
show an increased bone mass due to impaired osteoclast maturation,
because Trpv4 mediates Ca2+ influx at the late stage of osteoclast
differentiation and hereby regulates Ca2+ signaling [1]. Furthermore,
substitutions of amino acids R616Q/V620I of Trpv4 have been discovered as
gain of function mutations resulting in increased Ca2+ transport [2]. References 1. Masuyama R, Vriens J, Voets T, Karashima Y, Owsianik G, Vennekens R,
Lieben L, Torrekens S, Moermans K, Vanden Bosch A, et al: TRPV4-mediated
calcium influx regulates terminal differentiation of osteoclasts. Cell Metab
2008, 8:257-265. 2. Rock MJ, Prenen J, Funari VA, Funari TL, Merriman B, Nelson SF,
Lachman RS, Wilcox WR, Reyno S, Quadrelli R, et al: Gain-of-function
mutations in TRPV4 cause autosomal dominant brachyolmia. Nat Genet
2008, 40:999-1003. g
g
y
Results: BMP-induced expression of Col10a1 gene, a specific marker for
hypertrophic chondrocytes, was further up-regulated dramatically, upon
treatment with SB431542. In metatarsal bone organ culture, zone of calcified
matured chondrocytes was expanded upon SB431542 application. Expression of Id1 gene, the direct target of BMP Smads, was enhanced by
SB431542, although the phosphorylation (activated status) of BMP-Smads-1/
5/8 was not influenced by SB431542 application. Therefore, BMP signaling
seemed to be blocked by TGF-b signaling at the level beneath the
phosphorylation process of BMP-Smads. We evaluated expression profile of
BMP signal-inhibitors, and found that SnoN was the only gene which
expression was induced upon TGF-b treatment, while was inhibited by
SB431542 application. Indeed, knockdown of SnoN resulted in enhanced
hypertrophic maturation of ATDC5 cells, and overexpression of SnoN
suppressed it. To evaluate in vivo contribution of SnoN in cartilage cell
hypertrophy, we studied expression of SnoN protein by immunohisto-
chemistry. In mouse growth plate, SnoN was present only in prehy-
pertrophic chondrocytes, but excluded from hypertrophic zone. In human
OA specimens, SnoN was positive around ectopic hypertrophic chond-
rocytes of moderate OA cartilages, whereas SnoN was not detected in
severe-graded OA cartilages. These data support the idea that SnoN inhibits
hypertrophic conversion of chondrocytes in vivo, as well as in vitro. P43 Neutrophils are the source of IL-17 and RANKL in zymosan induced
arthritis
* Results: We found a significant reduction in the clinical signs of arthritis,
indicated by an increase of paw swelling and a decrease in grip strength,
in IL1-/-IL6-/-hTNFtg mice when compared to their hTNFtg littermates. In
line with these findings we observed a significant decrease in synovial
inflammation in IL1-/-IL6-/-hTNFtg mice when compared to hTNFtg
animals. Moreover, the number of synovial TRAP+ osteoclasts was
markedly diminished in IL1-/-IL6-/-hTNFtg mice and reduced osteoclast
formation, was accompanied by significantly less subchondral bone
erosions. Additionally, we found a conserved articular cartilage structure
showing almost no cartilage degradation in IL1-/-IL6-/-hTNFtg mice
compared to their hTNFtg littermates. In IL1-/-IL6-/-hTNFtg mice clinical,
as well as, histological signs of disease, including joint inflammation,
bone destruction and cartilage damage were also significantly diminished
when compared to IL6-/-hTNFtg mice. However, by comparing IL1-/-IL6-/-
hTNFtg mice with IL1-/- hTNFtg mice we found a similar reduction on
synovial inflammation, as well as subchondral bone erosions and articular
cartilage destruction. Viktoriya I Milanova*, Nina D Ivanovska, Petya A Dimitrova Department of Immunology, Institute of Microbiology, Bulgarian Academy of
Sciences, Sofia, 1113, Bulgaria Department of Immunology, Institute of Microbiology, Bulgarian Academy of
Sciences, Sofia, 1113, Bulgaria g
Arthritis Research & Therapy 2012, 14(Suppl 1):P46 Background: Rheumatoid arthritis (RA) is a systemic inflammatory disease
affecting cartilage and bone. Recently, much attention on the role of
neutrophils in the pathology of RA has been paid. However, the capability
of RA neutrophils from periphery and bone marrow (BM) to produce
cytokines like IL-17 and IFN-g has not been well understood. Our aim is to
analyze neutrophil distribution in BM, blood and synovium and to
elucidate IL-17, IL-4 and IFN-g production and surface expression of RANKL
on peripheral and synovial neutrophils during the progression of zymosan-
induced arthritis (ZIA). Materials and methods: In the present study BALB/c and SCID mice were
injected intra-articularly with zymosan. Cells from BM, periphery and
synovium were collected at day 7 and day 30 of ZIA and the frequencies of
Ly6G+CD11b+ neutrophils and surface expression of RANKL and CD69 on
them were evaluated by flow cytometry. In some experiments peripheral
neutrophils were isolated at day 7 of ZIA, re-stimulated in vitro with
zymosan in the presence or the absence of IL-17, then fixed, permeabilized
and used for flow cytometry analyses of IL-17, IL-4 and IFN-g intracellular
levels and of surface RANKL expression. Apoptosis of cultured neutrophils
was detected by annexin/propidium iodide kit. P44 Combined depletion of interleukin-1 and interleukin-6 does not exceed
single depletion of interleukin -1 in TNF-mediated arthritis
Silvia Hayer*, B Niederreiter, J Smolen, K Redlich
Department of Internal Medicine III, Division of Rheumatology, Medical
University of Vienna, Vienna, Austria
Arthritis Research & Therapy 2012, 14(Suppl 1):P44 Combined depletion of interleukin-1 and interleukin-6 does not exceed
single depletion of interleukin -1 in TNF-mediated arthritis
Silvia Hayer*, B Niederreiter, J Smolen, K Redlich References 1. Amano T, Yamasaki S, Yagishita N, Tsuchimochi K, Shin H, Kawahara K,
Aratani S, Fujita H, Zhang L, Ikeda R, Fujii R, Miura N, Komiya S, Nishioka K,
Maruyama I, Fukamizu A, Nakajima T: Synoviolin/Hrd1, an E3 ubiquitin
ligase, as a novel pathogenic factor for arthropathy. Genes Dev 2003,
17(19):2436-2449. y
,
,
,
Department of Internal Medicine III, Division of Rheumatology, Medical
University of Vienna, Vienna, Austria
Arthritis Research & Therapy 2012, 14(Suppl 1):P44 y
,
,
,
Department of Internal Medicine III, Division of Rheumatology, Medical
University of Vienna, Vienna, Austria
Arthritis Research & Therapy 2012, 14(Suppl 1):P44 2. 2. Yamasaki S, Yagishita N, Sasaki T, Nakazawa M, Kato Y, Yamadera T, Bae E,
Toriyama S, Ikeda R, Zhang L, Fujitani K, Yoo E, Tsuchimochi K, Ohta T,
Araya N, Fujita H, Aratani S, Eguchi K, Komiya S, Maruyama I, Higashi N,
Sato M, Senoo H, Ochi T, Yokoyama S, Amano T, Kim J, Gay S, Fukamizu A,
Nishioka K, Tanaka K, Nakajima T: Cytoplasmic destruction of p53 by the
endoplasmic reticulum-resident ubiquitin ligase ‘Synoviolin’. EMBO J
2007, 26(1):113-122. Background: Previous studies demonstrated a regulatory role of interleukin
1 (IL-1) in inflammatory cartilage damage and bone destruction in human
tumor necrosis factor transgenic (hTNFtg) mice, an animal model for
Rheumatoid Arthritis (RA). Moreover, blocking of IL-6 has been shown to
reduce local bone erosions in this model. Therefore we wanted to
investigate the effect of a combined depletion of IL-1 and IL-6 on the
development and severity of inflammatory, erosive arthritis. 3. Yagishita N, Yamasaki S, Nishioka K, Nakajima T: Synoviolin, protein folding
and the maintenance of joint homeostasis. Nat Clin Pract Rheumatol 2008,
4(2):91-97. Methods: We first crossed IL1a and ß deficient (IL1-/-) mice with IL6-/-
mice to generate IL1-/-IL6-/- double knockout mice. We next intercrossed
these animals with arthritogenic hTNFtg mice to receive IL1-/-IL6-/-
hTNFtg mice. We weekly assessed clinical signs of arthritis in hTNFtg,
IL1-/-hTNFtg mice, IL6-/-hTNFtg mice and IL1-/-IL6-/-hTNFtg mice starting
from week 4 after birth until week 16. We stained decalcified paw
sections from all 4 genotypes with hematoxylin&eosin to determine the
amount of inflammatory synovial pannus formation, with tartrate-
resistant acid phosphatase (TRAP) to evaluate the number of synovial
osteoclasts and the occurrence of subchondral bone erosions, with
toluidine-blue to assess articular cartilage damage. Quantitative analysis
of histopathological changes were performed using the Osteomeasure
Software System. 4. P43
STAT3 is critical to promote inflammatory cytokines and RANKL
expression in inflammatory arthritis
* Takeshi Miyamoto1*, Tomoaki Mori1, Akihiko Yoshimura2,
Toshiaki Toyama1 y
1Department of Orthopedic Surgery, Keio University School of Medicine,
Shinjuku, Tokyo, 160-8582, Japan; 2Department of Immunology, Keio
University School of Medicine, Shinjuku, Tokyo, 160-8582, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P43 Rheumatoid arthritis (RA) causes sever joint damage and significant
disability of daily living. The symptoms of RA patients are mainly from
chronic inflammation and continuous joint destruction, however, the
mechanisms underlying how inflammation and joint destruction in RA
develop and are sustained chronically remain largely unclear. In this study,
we show that signal transducer and activator of transcription 3 (STAT3)
plays a critical role in both chronic inflammation and joint destruction in
RA. We found that inflammatory cytokines, such as IL-1b, TNFa and IL-6,
activated STAT3 either directly or indirectly and induced expression of
inflammatory cytokines, further activating STAT3. STAT3 activation also
induced expression of receptor activator of nuclear factor kappa B ligand
(RANKL), an essential cytokine for osteoclast differentiation. STAT3
knockout or pharmacological inhibition resulted in significant reduction of
the expression of both inflammatory cytokines and RANKL in vitro. STAT3
inhibition was also effective in treating an RA model, collagen induced
arthritis (CIA), in vivo through significant reduction in expression of
inflammatory cytokines and RANKL, inhibiting both inflammation and joint
destruction. Thus our data provide new insight into pathogenesis of RA
and provide evidence that inflammatory cytokines induce a cytokine
amplification loop via STAT3 that promotes sustained inflammation and
joint destruction. Conclusions: Our results suggest that SnoN suppresses hypertrophic
transition of chondrocytes, as a mediator of TGF-b signaling, to prevent
the progression of OA. P42
Activation of TRPV4 promotes osteoclasts differentiation
Ritsuko Masuyama
Department of Cell Biology, Nagasaki University Graduate School of
Biomedical Sciences, Nagasaki, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P42 P42
Activation of TRPV4 promotes osteoclasts differentiation
Ritsuko Masuyama
Department of Cell Biology, Nagasaki University Graduate School of
Biomedical Sciences, Nagasaki, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P42 Osteoclast differentiation is critically dependent on cellular calcium (Ca2+)
signaling. Intracellular Ca2+ concentration ([Ca2+]i) is regulated by two flux Page 39 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Neutrophils are the source of IL-17 and RANKL in zymosan induced
arthritis
* The ability of peripheral
neutrophils to affect RANKL or IL-17-induced osteoclast differention of
bone marrow precursors in vitro was evaluated after TRAP staining of cell
co-cultures. Conclusion: The phenotype of IL1-/-IL6-/-hTNFtg mice does not differ
from IL1-/-hTNFtg animals indicating no synergistic effects when IL-1 and
IL-6 is simultaneously blocked in TNF-mediated arthritis. References Suzuki A, Yamada R, Chang X, Tokuhiro S, Sawada T, Suzuki M, Nagasaki M,
Nakayama-Hamada M, Kawaida R, Ono M, Ohtsuki M, Furukawa H,
Yoshino S, Yukioka M, Tohma S, Matsubara T, Wakitani S, Teshima R,
Nishioka Y, Sekine A, Iida A, Takahashi A, Tsunoda T, Nakamura Y,
Yamamoto K: Functional haplotypes of PADI4, encoding citrullinating
enzyme peptidylarginine deiminase 4, are associated with rheumatoid
arthritis. Nat Genet 2003, 34(4):395-402. RA synoviocytes could associate for ER stress and explore the crosstalk of
ubiquitination and citrullination. RA synoviocytes could associate for ER stress and explore the crosstalk of
ubiquitination and citrullination. RA synoviocytes could associate for ER stress and explore the crosstalk of
ubiquitination and citrullination. RA synoviocytes could associate for ER stress and explore the crosstalk of
ubiquitination and citrullination. Maintenance of mitochondrial DNA copy number is essential for
osteoclast survival
* Cw4 (12.8% vs. 36.2% in control, p > 0.05) met reliably more rare in
HLA-A locus. Tsuyoshi Miyazaki1*, Shuuichi Mori1, Kazuhiro Shigemoto1, Nils-Goran Larsson2,
Takeshi Nakamura3, Shigekaki Kato3, Tomoki Nakashima4, Hiroshi Takayanagi4,
Sakae Tanaka5 The highest indicator of risk was established for A25 (RR = 26.6), then for
B22 (RR = 8.7), B16 (RR = 4.0), B27 (RR = 2.8), B18 and A10 (RR = 2.7). Results showed that antigens A25 and A28 (p < 0.001), have major effect,
while the B16, B18, B22, B27 - additive contribution to the predisposition
to the RA among Uzbek women. 1Department of Geriatric Medicine, Tokyo Metropolitan Geriatric Hospital and
Institute of Gerontology, Tokyo 173-0015, Japan; 2Division of Metabolic
Diseases, Karolinska Institute, Stockholm, Sweden; 3Institute of Molecular and
Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan;
4Department of Cell Signaling, Graduate School of Medical and Dental
Sciences, Tokyo Medical and Dental University, Tokyo 113-8549, Japan;
5Department of Orthopaedic Surgery, Faculty of Medicine, The University of
Tokyo, Tokyo 113-0033, Japan g
Analysis of results in different clinical RA forms revealed association of slowly
progressing articular form with antigens: A25 (p < 0.001, RR = 25.2); A28 (p <
0.01, RR = 6.7); whether A10, B16, B27, B22 were not significant (p > 0.05). Fast progressing articular-visceral form development was associated with
HLA-A28, A25, B16, B27, and significance of association was established only
for A28 (p < 0.001, RR = 7.6). The important moment in our investigation
seems to be the association of RA showed unfavorable development in
Uzbek women with antigens HLA-B16 which is a split of antigen B8 and
antigen B27, being marker of rheumatoid diseases, that correlates with
identical research in different populations. y
y
p
Arthritis Research & Therapy 2012, 14(Suppl 1):P49 y
y
p
Arthritis Research & Therapy 2012, 14(Suppl 1):P49 y
y
p
Arthritis Research & Therapy 2012, 14(Suppl 1):P49 Background: There is accumulating evidence that osteoclasts, the primary
cells responsible for bone resorption, are involved in bone and joint
destruction in rheumatoid arthritis. Bone resorption is highly regulated by
mature osteoclast function as well as osteoclastogenesis. The life span of
mature osteoclasts is relatively short both in vitro and in vivo, and once
differentiated, they rapidly die in the absence of supporting cell or growth
factors. P48
SNP algorithms for prediction of efficacy and adverse events of
abatacept (ABT) p
James E Middleton1*, Tsukasa Matsubara1,2, Keiko Funahashi1,2, Satoru Koyano1,
Takafumi Hagiwara2, Takako Miura2, Kosuke Okuda2, Takeshi Nakamura2,
Mitsuyoshi Iwahashi3, Tomomi Tsuru4, Shoichi Uchimura5, Shigeru Honjo6
1Research Institute of Joint Diseases, Kobe, Japan; 2Matsubara Mayflower
Hospital, Kato, Japan; 3Higashi-Hiroshima Memorial Hospital, Higashi-
Hiroshima, Japan; 4PS Clinic, Fukuoka, Japan; 5Kanzaki Municipal General
Hospital, Japan; 6Honjo Rheumatism Clinic, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P48 James E Middleton1*, Tsukasa Matsubara1,2, Keiko Funahashi1,2, Satoru Koyano1,
Takafumi Hagiwara2, Takako Miura2, Kosuke Okuda2, Takeshi Nakamura2,
Mitsuyoshi Iwahashi3, Tomomi Tsuru4, Shoichi Uchimura5, Shigeru Honjo6
1Research Institute of Joint Diseases, Kobe, Japan; 2Matsubara Mayflower
Hospital, Kato, Japan; 3Higashi-Hiroshima Memorial Hospital, Higashi-
Hiroshima, Japan; 4PS Clinic, Fukuoka, Japan; 5Kanzaki Municipal General
Hospital, Japan; 6Honjo Rheumatism Clinic, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P48 Materials and methods: To address this question, we generated
osteoclast-specific Tfam conditional knock-out (cKO) mice by mating Tfamfl/fl
mice with cathepsin K-Cre transgenic mice, in which the Cre recombinase
gene is knocked into the cathepsin K locus and specifically expressed in
mature osteoclasts. The in vivo effects of Tfam deficiency on bone
metabolism were examined by histological and histomorphometric analysis. The survival and bone-resorbing activity of Tfam cKO osteoclasts were
determined by in vitro survival assay and pit formation assay, respectively. Materials and methods: To address this question, we generated
osteoclast-specific Tfam conditional knock-out (cKO) mice by mating Tfamfl/fl
mice with cathepsin K-Cre transgenic mice, in which the Cre recombinase
gene is knocked into the cathepsin K locus and specifically expressed in
mature osteoclasts. The in vivo effects of Tfam deficiency on bone
metabolism were examined by histological and histomorphometric analysis. The survival and bone-resorbing activity of Tfam cKO osteoclasts were
determined by in vitro survival assay and pit formation assay, respectively. Results: The expression level of Tfam, mtDNA copy number, and cellular
ATP level were markedly reduced in osteoclasts derived from Tfam cKO
mice. The body size of Tfam cKO mice was smaller than that of the
control mice, although trabecular bone volume remained unchanged by
Tfam deficiency. However, histological sections of proximal tibia and
lumbar spine of Tfam cKO mice showed significantly decreased osteoclast
number. Interestingly, Tfam cKO osteoclasts exhibited increased bone-
resorbing activity in spite of their pro-apoptotic tendency. The functions of the post-translational modifications in rheumatoid
arthritis Satoko Aratani1*, Naoko Yagishita2, Teruhisa Kanazawa1, Fukami Nakajima1,
Yoshihisa Yamano2, Kusuki Nishioka1, Toshihiro Nakajima1
1Institute of Medical Science, Tokyo Medical University, Shinjuku-ku, Tokyo,
160-8402, Japan; 2Institute of Medical Science, St. Marianna University School
of Medicine, Kawasaki, Kanagawa, 216-8512, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P45 Results: The development of inflammatory process in SCID mice after
zymosan injection was related to increased frequencies of Ly6G+CD11b+
neutrophils in periphery and synovium along with elevated IL-17
production in plasma and serum. We observed that arthritic neutrophils
collected at day 7 of disease have higher IL-17, IL-4 and IFN-g intracellular
levels than healthy cells. Exogenous IL-17 increased the cytokine and
RANKL expression on healthy and arthritic neutrophils in vitro. While
neutrophils were able to inhibit RANKL-induced osteoclast differentiation,
they increased the number of TRAP positive mature osteoclasts in the
presence of IL-17. Rheumatoid Arthritis (RA) is a chronic inflammatory joint disease and
characterized by synovial hyperplasia. We previously cloned an E3
ubiquitin ligase, Synoviolin, as a regulatory factor of cell proliferation [1]. It suggested that endoplasmic reticulum (ER) associated degradation
system (ERAD) via Synoviolin has important roles for overgrowth of
synoviocytes [2,3]. Meanwhile, it is known that autoantibodies to
citrullinated proteins are specific for RA and good markers for RA. Peptidyl-Arginine Deiminases 4 (PADI4) is identified as the RA-susceptible
gene [3]. However functions of citrulinated proteins are unclear. In this
study, we hypothesize that the accumulation of citrullinated proteins in Conclusions: We suggest that Ly6G+CD11b+ peripheral neutrophils that are
positive for IL-17, IL-4, IFN-g and RANKL can migrate to the synovium where
they can affect inflammatory and destructive processes. Our study displays
new aspect of the role of neutrophils in the pathology of RA and provides
diverse ground for the development of novel therapeutic strategies. Page 40 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Quad chip technology. Case-control analyses between 598,821 SNPs and
responsiveness or occurrence of adverse events were examined by Fisher’s
exact test. We selected 10 SNPs associated with ABT-responsiveness,
remission, and adverse events (p < 0.0001). P49 In HLA-A locus (17.4% vs. 4.9% in control); B18 were met in 9.3% vs. 3.7%
in control, (p > 0.05); B22 (10.5% vs. 1.3% in control, p > 0.05); B27
(15.1% vs. 8.9% in control, p > 0.05). P47 P47
Role of HLA-antigens class 1 in the development of rheumatoid
arthritis in Uzbek women
Nazima N Mirakhmedova1*, Mavlyuda I Mirzakhanova2
1Institute of Immunology of the Academy of Sciences of the Republic of
Uzbekistan, Tashkent, Uzbekistan; 2Tashkent Medical Academy, Tashkent,
Uzbekistan
Arthritis Research & Therapy 2012, 14(Suppl 1):P47 P47
Role of HLA-antigens class 1 in the development of rheumatoid
arthritis in Uzbek women
*
2 Role of HLA-antigens class 1 in the development of rheumatoid
arthritis in Uzbek women
1*
2 Nazima N Mirakhmedova1*, Mavlyuda I Mirzakhanova2
1Institute of Immunology of the Academy of Sciences of the Republic of
Uzbekistan, Tashkent, Uzbekistan; 2Tashkent Medical Academy, Tashkent,
Uzbekistan
Arthritis Research & Therapy 2012 14(Suppl 1):P47 Nazima N Mirakhmedova1*, Mavlyuda I Mirzakhanova2
1Institute of Immunology of the Academy of Sciences of the Republic of
Uzbekistan, Tashkent, Uzbekistan; 2Tashkent Medical Academy, Tashkent,
Uzbekistan Nazima N Mirakhmedova1*, Mavlyuda I Mirzakhanova2
1Institute of Immunology of the Academy of Sciences of the Republic of
Uzbekistan, Tashkent, Uzbekistan; 2Tashkent Medical Academy, Tashkent,
Uzbekistan Arthritis Research & Therapy 2012, 14(Suppl 1):P47 According to the multiple studies women suffer from rheumatoid arthritis
(RA) three times more often than men. The women seem to be ill at the age
of more active working activity that results in early disability. The great
attention is paid to the hereditary factors, particularly, to HLA-system, in the
RA development. In this connection the question about early diagnosis and
primary prevention of rheumatoid arthritis remain to be important. Consequently, we studied distribution of HLA I class antigens (A, B, C) in
86 Uzbek women with RA. HLA were identified with 2 step standard
microlymphocytotoxicity test using antileucocyte HLA-antisera (St-Petersburg,
Russia) and rabbit complement. Control group consist of 301 healthy random
Uzbeks. In current study 39 antigens were expressed. Higher frequency was
found for A25 (15.1% vs. 0.7% in control), A28 (22.1% vs. 4.9%, respectively)
with p < 0.001. Antigen A19 (3.5% vs. 11.9% in control, p < 0.01). Results: Accuracy, specificity, and sensitivity of the algorithm for
responsiveness of abatacept ranged from 90-96%. For remission,
accuracy, specificity and sensitivity of the algorithm ranged from 91-97%. For adverse events, accuracy, specificity and sensitivity of the algorithm
ranged from 95-100%. It is therefore suggested that the SNP algorithms
can predict responders and adverse events prior to the initiation of
treatment with abatacept. Conclusions: These highly accurate algorithms using SNP analysis may
be useful in the prediction of responsiveness and adverse events before
treatment with abatacept, and in this way can contribute to future tailor-
made treatment with biologic agents. Maintenance of mitochondrial DNA copy number is essential for
osteoclast survival
* Mitochondria is known as powerhouse of cell because they generate
most of the cell’s supply of adenosine triphosphate (ATP), used as a source
of chemical energy. In addition to supplying cellular energy, mitochondria
are involved in a range of other processes, such as signaling, cellular
differentiation, cell growth, and cell death. Transcription and replication of
mitochondrial DNA (mtDNA) are important steps in mitochondrial
biogenesis and mitochondrial transcription factor A (Tfam) is essential for
mtDNA transcription and replication. However, the functional significance of
mitochondria has not been established in osteoclastic bone resorption. Thus, the results of our investigation show important contribution of HLA
in predisposition to rheumatoid arthritis in Uzbek women. The functions of the post-translational modifications in rheumatoid
arthritis We scored the relationship
between each SNP and responsiveness, the estimated total score of 10
SNPs (estimated scoring in each SNP was as follows: homo allele in the
majority in responders: +1 point, hetero allele: 0 points, and homo allele in
the majority of non-responders: -1 point), and then examined relationships
between responders and non-responders, remission and non-remission,
and occurrence of adverse events, plus or minus, and the total score. Over expression of toll-like receptors in peripheral blood and synovial
fluid monocytes of enthesitis related arthritis category of juvenile
idiopathic arthritis (JIA-ERA) patients contributes to secretion of
inflammatory mediators Fibromyalgia (FM) is a highly populated chronic pain disease, which has
unique characteristics including generalized or widespread allodynia and
female prevalence of gender difference. Many FM patients are common
with Sjögren’s syndrome. Pilocarpine, a non-selective muscarinic
receptor agonist, is used clinically as a drug that promptes the secretion
of salvia for dry eyes and mouth. Otherwise, pilocarpine has been
shown to possess antinociceptive effect, which maybe caused by vagal
afferents activation. The experimental FM mice exposed to intermittent
cold stress (ICS) showed sustained abnormal pain, such as mechanical
allodynia and hyperalgesia to nociceptive thermal stimuli for up to 19
days, but those given constant cold stress (CCS) did not. The abnormal
pain was bilateral (generalized), female-predominant and specific for A-
delta and A-beta, but not C-fiber-stimuli. In ICS mice, intraperitoneal or
oral administration of pilocarpine showed potent anti-hyperalgesic
effects in doses without excess salivation at post-stress day5 (P5). The
anti-hyperagesic effects last for more than 1 h, but disappear at 24 h. Daily administration of pilocarpine showed equivalent anti-hyperalgesic
effects without tolerance. These findings suggest that pilocarpine
possesses a beneficial effect for the pain treatment of FM patients with
dry eyes and mouth symptoms. y
Arpita Myles*, Amita Aggarwal Arpita Myles*, Amita Aggarwal
Department of Clinical Immunology, Sanjay Gandhi Postgraduate Institute of
Medical Sciences Lucknow-226014, India
Arthritis Research & Therapy 2012, 14(Suppl 1):P50 Arpita Myles*, Amita Aggarwal
Department of Clinical Immunology, Sanjay Gandhi Postgraduate Institute of
Medical Sciences Lucknow-226014, India
Arthritis Research & Therapy 2012, 14(Suppl 1):P50 Arpita Myles , Amita Aggarwal
Department of Clinical Immunology, Sanjay Gandhi Postgraduate Institute of
Medical Sciences Lucknow-226014, India Background: TLRs 2, 4 and 9 have been implicated in murine models
and human patients of arthritis, but the other TLRs are not well-
investigated. Thus, we studied TLR expression and signaling and effect of
TLR ligand stimulation in peripheral blood (PB) and synovial fluid (SF)
monocytes (MC) of ERA patients. y
Methods: Levels of TLR2, TLR4 and TLR9 were measured by flow
cytometry in ERA PBMC (n = 26), paired SFMC (n = 13) and healthy PBMC
(n = 19) Real time PCR was done for TLRs 1-9 and their adaptors IRAK1,
IRAK4, TRIF, TRAF3, TRAF6. PBMC and SFMC were stimulated with ligands
for TLR1 (pam3-cys), 2 (peptidoglycan), 3 (polyI:C), 4 (LPS), 5 (flagellin)
and 6 (zymosan). Levels of IL-6, IL-8 and MMP3 (ng/ml) were measured in
the culture supernatants. Over expression of toll-like receptors in peripheral blood and synovial
fluid monocytes of enthesitis related arthritis category of juvenile
idiopathic arthritis (JIA-ERA) patients contributes to secretion of
inflammatory mediators p
Results: ERA PBMC had higher MFI of TLR2 [295.5(48.1-598) vs 179(68.7-
442); p < 0.05] and TLR4 [448(178-2581) vs 402(229-569); p < 0.05]
compared to controls. Intracellular TLR9 expression showed no significant
difference between both groups. In paired samples, SFMC had higher MFI
of both TLR2 [485(141-1683) vs 353(180-598); p < 0.05] and TLR4 [1016
(42.4-3159) vs 513(193-2581); p < 0.05] compared to PBMC. Difference in
TLR9 expression was not significant [1]. Patient PBMC (compared to
healthy control) and SFMC (compared to corresponding PBMC) had
higher RNA expression of TLRs1, 2, 3, 4, 5 and 6 and downstream
adaptors. Patients PBMC produced significantly higher IL-6 (13.51 vs 6.54)
and MMP3 (61 vs 32.9) as compared to controls on stimulation by LPS. With peptidoglycan also IL-6 (30.58 vs 10.84) and MMP-3 (102.54 vs
49.45) was higher than controls. Patient PBMCs produced more IL-6 and
IL-8 compared to healthy PBMCs on stimulation with Pam3-cys, poly I:C,
flagellin and zymosan. In paired samples, SFMCs showed a trend towards
higher IL-6 and IL-8 production compared to PBMCs (Table 1). Acknowledgements: The research described in this article was
supported in part by MEXT KAKENHI (17109015 to Hiroshi Ueda) and
Health Labor Sciences Research Grants from the Ministry of Health, Labor
and Welfare of Japan (to Hiroshi Ueda): “Research on Allergic disease and
Immunology” also supported this work. References 1. Nishiyori M, Ueda H: Prolonged gabapentin analgesia in an experimental
mouse model of fibromyalgia. Mol Pain 2008, 4:52. 1. Nishiyori M, Ueda H: Prolonged gabapentin analgesia in an experimental
mouse model of fibromyalgia. Mol Pain 2008, 4:52. y
2. Nishiyori M, Nagai J, Nakazawa T, Ueda H: Absence of morphine analgesia
and its underlying descending serotonergic activation in an experimental
mouse model of fibromyalgia. Neurosci Lett 2010, 472:184-187. 2. Nishiyori M, Nagai J, Nakazawa T, Ueda H: Absence of morphine analgesia
and its underlying descending serotonergic activation in an experimental
mouse model of fibromyalgia. Neurosci Lett 2010, 472:184-187. P51
Pilocarpine suppresses hyperalgesia induced by intermittent cold
stress (ICS) as an experimental fibromyalgia model in mice
Jun Nagai*, Michiko Nishiyori, Hiroshi Ueda
Division of Molecular Pharmacology and Neurosciences, Nagasaki University
Graduate School of Biomedical Sciences, Nagasaki 852-8521, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P51 P51
Pilocarpine suppresses hyperalgesia induced by intermittent cold
stress (ICS) as an experimental fibromyalgia model in mice
Jun Nagai*, Michiko Nishiyori, Hiroshi Ueda
Division of Molecular Pharmacology and Neurosciences, Nagasaki University
Graduate School of Biomedical Sciences, Nagasaki 852-8521, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P51 P51 P51 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 indicating that there may be an inverse correlation between osteoclast
survival vs bone resorption. Further investigation of mitochondria in
bone-resorbing osteoclasts will give us new insights into the molecular
mechanism regulating bone homeostasis. Application of tetraspanin CD81 RNAi for diagnosis and therapy of
rheumatoid arthritis (RA)
Tohru Nakanishi1*, Yuji Arai2, Hiroki Mori1, Toshihiro Nakajima3,
Toshikazu Kubo2
1Shujitsu University School of Pharmacy, Okayama, 703-8516 Japan;
2Department of Orthopaedics, Kyoto Prefectural University of Medicine,
Kyoto, 602-8566 Japan; 3Institute of Medical Science, Tokyo Medical
University, Shinjuku, 160-8402 Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P52 P48
SNP algorithms for prediction of efficacy and adverse events of
abatacept (ABT) C
l
i
Thi
t d
d
t
t
th t Tf
KO
t
l
t Background: Abatacept (ABT), a CTLA4-Ig fusion protein, which inhibits the
binding of CD28 and CD80 agents targeted to T-cells, is a relatively new
biological agent for RA treatment in Japan. However, there is no method for
prediction of responders, non-responders, or adverse events which can occur
during treatment. We established SNP algorithms for prediction of
responders (R) or non-responders (NR), and adverse events in ABT-treated
patients. determined by in vitro survival assay and pit formation assay, respectively. Results: The expression level of Tfam, mtDNA copy number, and cellular
ATP level were markedly reduced in osteoclasts derived from Tfam cKO
mice. The body size of Tfam cKO mice was smaller than that of the
control mice, although trabecular bone volume remained unchanged by
Tfam deficiency. However, histological sections of proximal tibia and
lumbar spine of Tfam cKO mice showed significantly decreased osteoclast
number. Interestingly, Tfam cKO osteoclasts exhibited increased bone-
resorbing activity in spite of their pro-apoptotic tendency. Materials and methods: Forty-six RA patients treated with ABT were
included in this study. Efficacy was assessed by DAS28 (CRP) at 48 weeks
after the initial treatment. Any adverse events that may have been related
to ABT administration and observed at 48 weeks of this long-term
administration and during phase II were considered to be side effects. Genome-wide SNP genotyping was performed by Illumina Human610- Conclusions: This study demonstrates that Tfam cKO osteoclasts
exhibited increased bone resorption with accelerated apoptosis, Page 41 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 P52 P52
Application of tetraspanin CD81 RNAi for diagnosis and therapy of
rheumatoid arthritis (RA)
Tohru Nakanishi1*, Yuji Arai2, Hiroki Mori1, Toshihiro Nakajima3,
Toshikazu Kubo2
1Shujitsu University School of Pharmacy, Okayama, 703-8516 Japan;
2Department of Orthopaedics, Kyoto Prefectural University of Medicine,
Kyoto, 602-8566 Japan; 3Institute of Medical Science, Tokyo Medical
University, Shinjuku, 160-8402 Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P52 Application of tetraspanin CD81 RNAi for diagnosis and therapy of
rheumatoid arthritis (RA) Conclusion: Increased TLR expression and signaling on PBMC and SFMC
from JIA-ERA patients may exacerbate disease by upregulating IL-6, IL-8
and MMP-3 in response to microbial/ endogenous ligands. TLR pathway
is a potential therapeutic target in these patients. Reference 1. Myles A, Aggarwal A: Expression of Toll-like receptors 2 and 4 is
increased in peripheral blood and synovial fluid monocytes of patients
with enthesitis-related arthritis subtype of juvenile idiopathic arthritis. Rheumatology 2011, 50:481-488. Table 1(abstract P50) Production of IL-6 and IL-8 [median (range) ng/ml] by PBMCs and SFMCs upon TLR ligand
stimulation
Cultured with IL-6
IL-8
Normal PBMC (n = 5) ERA PBMC (n = 7) ERA SFMC (n = 3) Normal PBMC (n = 5) ERA PBMC (n = 7) ERA SFMC (n = 3)
Medium
4.4 (1.5-5.4)
7.6 (3-16.6) *
18 (9.3-24.2)
10 (4.3-12.6)
12.6 (8.1-35.7)
10 (4.3-12.6)
TNF
13.6 (9.6-14.8)
16 (12-35)
21(18-30)
30.8 (15.8-36.3)
34.4 (30.8-46.1)
31 (15.8-36.3)
Pam3cys
15.1 (13.3-19.6)
44 (26-62) **
53 (28-71)
37.1 (11.4-41.8)
106 (42.6-147.6) **
37.1 (11.4-42)
PolyI:C
13.1 (1.3-25.8)
28 (24-4) *
46 (45-65)
33.6 (31.1-56.3)
126(78-167) *
34(31.1-56.3)
Flagellin
14.1 (6.7-23.5)
34.9 (15-39.2) *
52.6 (40-53.8)#
35.2 (16.1-84.2)
115 (73-162) **
35 (16.1-84.2)
Zymosan
14.7 (8.8-36.3)
34 (28.4-39) *
48 (39.8-56.1)#
56 (12.6-89.6)
106 (103-163) **
56 (12.6-90)
p < 0.05** 0.01 ERA PBMC versus control PBMC p <#0.05 ERA SFMC versus ERA PBMC. Production of IL-6 and IL-8 [median (range) ng/ml] by PBMCs and SFMCs upon TLR ligand Table 1(abstract P50) Production of IL-6 and IL-8 [median (range) ng/ml] by PBMCs and SFMCs u
stimulation Page 42 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Figure 1(abstract P52) . Receptor activator of NF-B ligand (RANKL; also known as TNFSF11), a TNF
family molecule, and its receptor RANK (TNFRSF11A) are key regulators of
osteoclast differentiation and function. Aberrant expression of RANKL
explains why autoimmune diseases, cancers, leukemia and periodontal
disease result in systemic and local bone loss. In particular, RANKL is the
pathogenic factor that cause bone and cartilage destruction in arthritis. Inhibition of RANKL function by the natural decoy receptor osteoprotegerin
(OPG; also known as TNFRSF11B) or anti-RANKL antibody prevents bone loss
in postmenopausal osteoporosis, cancer metastases and arthritis. RANKL also
regulates T cell/dendritic cell communications, dendritic cell survival and
lymph node organogenesis. Intriguingly, RANKL and RANK play an essential
role in the maturation of mammary glands in pregnancy and lactation. Bone
homeostasis depends on the coordination of osteoclastic bone resorption
and osteoblastic bone formation. We reported that RANKL induces
osteoclast differentiation through activating a transcriptional programme
mediated by the master transcription factor nuclear factor of activated
T cells (NFAT) c1. Reference Although it is well accepted that the RANKL-NFATc1
pathway is crucially important for osteoclast differentiation, little is known
about the major cellular source of RANKL in the skeletal tissue. RANKL has
been postulated to be mainly expressed by osteoblasts and bone marrow
stromal cells. However, here we show that osteocytes embedded within the
bone matrix are the critical source of RANKL in bone remodeling. Osteocytes, the most abundant cell type in bone, are thought to orchestrate
bone homeostasis by regulating both osteoclastic bone resorption and
osteoblastic bone formation, but in vivo evidence and the molecular basis
for the regulation has not been sufficiently demonstrated. Using a newly
established method for the isolation of high-purity dentin matrix protein
1-positive osteocytes from bone, we have found that osteocytes express a
much higher amount of RANKL and have a much greater capacity to
support osteoclast formation than osteoblasts and bone marrow stromal
cells. The crucial role of RANKL expressed by osteocytes was validated by
the severe osteopetrotic phenotype observed in mice lacking RANKL
specifically in osteocytes. Thus, we provide in vivo evidence for the key role
of osteocyte-derived RANKL in bone homeostasis, establishing a molecular
basis for osteocyte regulation of bone resorption. CD81 belomgs to a family of cell-surface protein (tetraspanin) which has four
transmembrane domains and two outer-membrane loops.Under the DNA
chip analysis, we found several genes highly expressed in rheumatoid
arthritis (RA) synoviocytes comparing with the expression in OA or normal
synoviocytes. Among these genes, tetraspanin CD81 was shown to be
involved in the progression of RA through the promotion of Synoviolin
expression.Synoviolin is already known as one of the important progressive
elements of RA in synoviocytes. We also showed Synoviolin and CD81 highly
distributed in RA tissues. The therapeutic effect of small interfering RNA targeting CD81 (siCD81)
was examined by in vivo electroporation method. Treatment with siCD81
significantly ameliorated paw swelling of collagen-induced arthritic (CIA)
rats. In histological examination, hypertrophy of synovium, bone erosion,
and degeneration of articular cartilage were minder in rats treated with
siCD81 than in the control group and the non-specific siRNA group. Expression of synoviolin, a rheumatoid regulator, was also suppressed by
siCD81 [1]. These results showed that siCD81 would become effective
tools for treatment of RA. In addition, siCD81 reduced the amount of
CD81 in synovial fluid indicating that quantitative analysis of CD81 opens
up the novel and highly sensitive diagnosis for RA. P53
The crucial role of osteocyte-derived RANKL in bone homeostasis
Tomoki Nakashima1,2*, Mikihito Hayashi1,2, Hiroshi Takayanagi1,2
1Department of Cell Signaling, Graduate School of Medical and Dental
Sciences, Tokyo Medical and Dental University, Yushima 1-5-45, Tokyo, Japan;
2Japan Science and Technology Agency (JST), ERATO, Takayanagi
Osteonetwork Project, Yushima 1-5-45, Tokyo, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P53 An essential role of IBζ in the transcriptional program in Th17
development
* An essential role of IBζ in the transcriptional program in Th17
development
* Surgeons, New York, NY 10032, USA Kazuo Okamoto*, Masatsugu Oh-hora, Hiroshi Takayanagi
Department of Cell Signaling, Tokyo Medical and Dental University, Tokyo,
Japan. 2GCOE Program, Tokyo, Japan. 3JST, ERATO, Takayanagi Osteonetwork
Project, Tokyo, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P56 Arthritis Research & Therapy 2012, 14(Suppl 1):P54 Regulation of irreversible cell lineage commitment depends on a delicate
balance between positive and negative regulators, which comprise a
sophisticated network of transcription factors. Receptor activator of nuclear
factor-B ligand (RANKL) stimulates the differentiation of bone-resorbing
osteoclasts through the induction of nuclear factor of activated T-cells c1
(NFATc1), the essential transcription factor for osteoclastogenesis. Osteoclast-specific robust induction of NFATc1 is achieved through an
autoamplification mechanism, in which NFATc1 is constantly activated by
calcium signaling while the negative regulators of NFATc1 are being
suppressed. However, it has been unclear how such negative regulators are
repressed during osteoclastogenesis. Here we show that B lymphocyte-
induced maturation protein-1 (Blimp1; encoded by Prdm1), which is induced
by RANKL through NFATc1 during osteoclastogenesis, functions as a
transcriptional repressor of anti-osteoclastogenic genes such as Irf8 and
Mafb. Overexpression of Blimp1 leads to an increase in osteoclast formation
and Prdm1-deficient osteoclast precursor cells do not undergo osteoclast
differentiation efficiently. The importance of Blimp1 in bone homeostasis is
underscored by the observation that mice with an osteoclast-specific
deficiency in the Prdm1 gene exhibit a high bone mass phenotype owing to
a decreased number of osteoclasts. Thus, NFATc1 choreographs the cell fate
determination of the osteoclast lineage by inducing the repression of
negative regulators as well as its effect on positive regulators. IL-17-producing helper T (Th17) cells are a distinct T cell subset
characterized by its pathological role in autoimmune diseases. Our group
previously showed that Th17 cells function as osteoclastogenic helper
T cells in bone destruction associated with inflammation, and that
inhibition of Th17 development has the potential of a beneficial impact
on bone diseases including rheumatoid arthritis (RA) [1]. It is therefore
important to comprehend the molecular mechanism underlying Th17 T cells in bone destruction associated with inflammation, and that
inhibition of Th17 development has the potential of a beneficial impact
on bone diseases including rheumatoid arthritis (RA) [1]. It is therefore
important to comprehend the molecular mechanism underlying Th17
development in order to develop ideal therapeutic strategies against RA. P54
Active repression by Blimp1 play an important role in osteoclast
differentiation
* Keizo Nishikawa1*, Tomoki Nakashima2,3,4, Mikihito Hayashi2,3,4,
Takanobu Fukunaga2,3,4, Shigeaki Kato5,6, Tatsuhiko Kodama7,
Satoru Takahashi8, Kathryn Calame9, Hiroshi Takayanagi2,3,4
1Laboratory of Cellular Dynamics Immunology Frontier Research Center,
Osaka University, Yamada-oka 3-1, Suita, Osaka 565-0871, Japan; 2Department
of Cell Signaling, Graduate School, Tokyo Medical and Dental University,
Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8549, Japan; 3Global Center of
Excellence Program, International Research Center for Molecular Science in
Tooth and Bone Diseases, Japan; 4Japan Science and Technology Agency,
ERATO, TakayanagiOsteonetwork Project, Yushima 1-5-45, Bunkyo-ku, Tokyo
113-8549, Japan; 5Institute of Molecular and Cellular Biosciences, Graduate
School of Medicine, University of Tokyo, Tokyo 113-0032, Japan; 6Japan
Science and Technology Agency, ERATO, Kato Nuclear Complex, Saitama
332-0012, Japan; 7Department of Molecular Biology and Medicine,
Research Center for Advanced Science and Technology, University of Tokyo,
Komaba 4-6-1, Meguro-ku, Tokyo 153-8904, Japan; 8Institute of Basic Medical
Sciences and Laboratory Animal Resource Center, University of Tsukuba,
Tennodai 1-1-1, Tsukuba 305-8575, Japan; 9Department of Biochemistry and
Molecular Biophysics, Columbia University College of Physicians and
Surgeons, New York, NY 10032, USA
Arthritis Research & Therapy 2012, 14(Suppl 1):P54 P56 P56
An essential role of IBζ in the transcriptional program in Th17
development
Kazuo Okamoto*, Masatsugu Oh-hora, Hiroshi Takayanagi
Department of Cell Signaling, Tokyo Medical and Dental University, Tokyo,
Japan. 2GCOE Program, Tokyo, Japan. 3JST, ERATO, Takayanagi Osteonetwork
Project, Tokyo, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P56 1.
Sato K, Suematsu A, Okamoto K, Yamaguchi A, Morishita Y, Kadono Y,
Tanaka S, Kodama T, Akira S, Iwakura Y, Cua DJ, Takayanagi H: Th17
functions as an osteoclastogenic helper T cell subset that links T cell
activation and bone destruction. J Exp Med 2006, 203:2673-2682.
2.
Okamoto K, Iwai Y, Oh-Hora M, Yamamoto M, Morio T, Aoki K, Ohya K,
Jetten AM, Akira S, Muta T, Takayanagi H: IBζ regulates TH17
development by cooperating with ROR nuclear receptors. Nature 2010,
464:1381-1385. Reference 1. Nakagawa Shuji, Arai Yuji, Mori Hiroki, Matsushita Yumi, Kubo Toshikazu,
Nakanishi Tohru: Small interfering RNA targeting CD81 ameliorated
arthritis in rats. Biochem Biophys Res Commun 2009, 388:467-472. P53
The crucial role of osteocyte-derived RANKL in bone homeostasis
Tomoki Nakashima1,2*, Mikihito Hayashi1,2, Hiroshi Takayanagi1,2
1Department of Cell Signaling, Graduate School of Medical and Dental
Sciences, Tokyo Medical and Dental University, Yushima 1-5-45, Tokyo, Japan;
2Japan Science and Technology Agency (JST), ERATO, Takayanagi
Osteonetwork Project, Yushima 1-5-45, Tokyo, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P53 Page 43 of 54 Page 43 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 whose expression was screened, the expression of Tks5, an adaptor protein
with the phox homology (PX) domain with multiple Src homology 3
domains, was induced during osteoclastogenesis. Tks5 was localized in the
podosomes and fusing membranes of osteoclasts, and reducing its
expression impaired both formation of circumferential podosomes and
osteoclast fusion without altering osteoclast differentiation. In addition, the
expression of a deletion mutant of the PX domain abrogated circumferential
podosome formation as well as osteoclast fusion, suggesting that Tks5-
dependent circumferential podosomes function as fusion machinery during
osteoclastogenesis. As Tks5 is known to promote the formation of
podosomes/invadopodia in transformed/cancer cells, we tested if these cells
also have the potential to fuse with osteoclasts. Among the cells tested,
B16F0 melanoma cells formed circumferential podosomes with Tks5
accumulation in the presence of RANKL, TGFb and TNFa. Co-culture of
B16F0 melanoma cells with osteoclasts in an inflammatory milieu promoted
increased formation of melanoma-osteoclast hybrid cells. Our results
revealed a previously unknown mechanism of regulation of both
circumferential podosome formation and cell-cell fusion by Tks5. An essential role of IBζ in the transcriptional program in Th17
development
* IL-6 and TGF-b induce Th17 development, in which the orphan nuclear
receptors RORgt and RORa play an indispensable role. We found that
the expression of a nuclear IB family member, IBζ (encoded by the
Nfkbiz gene), was upregulated by the combination of IL-6 and TGF-b,
but independently of RORgt [2]. Not only Nfkbiz-/- mice but also Rag2-/-
mice transferred with Nfkbiz-/- CD4+ T cells were highly resistant to
experimental autoimmune encephalomyelitis, which is a mouse model
of multiple sclerosis. Nfkbiz-/- mice were also protected from the
activation of osteoclastogenesis and bone destruction in a LPS-induced
model of inflammatory bone destruction. When activated in vitro under
Th17-polarizing conditions, IL-17 production in Nfkbiz-/- T cells was
markedly reduced compared to WT cells. Notably, the expression of
RORgt and RORa was comparable between WT and Nfkbiz-/- T cells. Thus, it is unlikely that ROR nuclear receptors function downstream of
IBζ or vice versa. P57 Polyarthritis with ARF was observed in 40.7% of
patients, 25 (14.4%) of patients with recurrent ARF articular syndrome
manifested primarily arthralgia. In addition, 6.5% in patients with RF were
observed asymptomatic sacroiliitis stage I-II (Dale), 7 of patients are men
and 5 of them are women. P59
Association of microRNA-221/222 and -323-3p with rheumatoid arthritis
via predictions using the human TNF transgenic mouse model
Ioannis Pandis1*, Caroline Ospelt2, Niki Karagianni1,3, Maria Denis3,
Martin Reczko4, Carme Camps5, Artemis Hatzigeorgiou4, Jiannis Ragoussis5,6,
Steffen Gay2, George Kollias1
1Institute of Immunology, Biomedical Sciences Research Center “Alexander
Fleming”, Vari, Greece; 2Center of Experimental Rheumatology, University
Hospital Zurich and Zurich Center of Integrative Human Physiology, Zurich,
Switzerland; 3Biomedcode Hellas SA, Vari, Greece; 4Institute of Molecular
Oncology, Biomedical Sciences Research Center “Alexander Fleming”, Vari,
Greece; 5The Wellcome Trust Centre for Human Genetics, University of P59
Association of microRNA-221/222 and -323-3p with rheumatoid arthritis
via predictions using the human TNF transgenic mouse model
Ioannis Pandis1*, Caroline Ospelt2, Niki Karagianni1,3, Maria Denis3,
Martin Reczko4, Carme Camps5, Artemis Hatzigeorgiou4, Jiannis Ragoussis5,6,
Steffen Gay2, George Kollias1
1I
i
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l
Bi
di
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“Al
d Association of microRNA-221/222 and -323-3p with rheumatoid arthritis
via predictions using the human TNF transgenic mouse model Ioannis Pandis1*, Caroline Ospelt2, Niki Karagianni1,3, Maria Denis3,
Martin Reczko4, Carme Camps5, Artemis Hatzigeorgiou4, Jiannis Ragoussis5,6,
Steffen Gay2, George Kollias1 1Institute of Immunology, Biomedical Sciences Research Center “Alexander
Fleming”, Vari, Greece; 2Center of Experimental Rheumatology, University
Hospital Zurich and Zurich Center of Integrative Human Physiology, Zurich,
Switzerland; 3Biomedcode Hellas SA, Vari, Greece; 4Institute of Molecular
Oncology, Biomedical Sciences Research Center “Alexander Fleming”, Vari,
Greece; 5The Wellcome Trust Centre for Human Genetics, University of
Oxford, Oxford, UK; 6Institute of Molecular Biology & Genetics, Biomedical
Sciences Research Center “Alexander Fleming”, Vari, Greece
Arthritis Research & Therapy 2012, 14(Suppl 1):P59 Background: MicroRNAs (miRs), a class of small non-coding RNA
molecules, act as posttranscriptional regulators and are involved in a
plethora of cellular functions. miRs have attracted a great deal of attention
as potential therapeutic targets, as the sequence-specific mode in which
they act, allows the simultaneous targeting of multiple target genes, often
members of the same biological pathway(s) [1]. Previous studies have
demonstrated that miRs are dysregulated and functionally involved in
rheumatoid arthritis (RA) [2-9]. P57 P57
Features of rheumatic fever in adult patients in modern Kyrgyzstan
Nazgul A Omurzakova1*, Aynagul S Djumagulova1, Raisa I Rudenko1,
Kusuki Nishioka2, Toshihiro Nakajima2
1National Center of Cardiology and Internal Medicine, Bishkek, Kyrgyz
Republic; 2Institute of Medical Science, Tokyo Medical University, Tokyo,
Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P57 Arthritis Research & Therapy 2012, 14(Suppl 1):P57 Arthritis Research & Therapy 2012, 14(Suppl 1):P57 Objective: Study of peculiarities of rheumatic fever (RF) in adult patients. Materials and methods: We have studied prospectively for 5 years 200
patients (56 men and 144 women) with acute rheumatic fever (ARF in 27)
and recurrent ARF (in 173) at the age of 15-40 years (average age ± 24,5
7 years). Clinical and laboratory (ESR, antistreptolysin-O (ASL-O) and CRP)
and instrumental (ECG, ECG monitoring daily, 2-D echocardiography
color) studies conducted. The diagnosis of ARF was verified according to
the WHO diagnostic criteria in the modification of Jones’ criteria, AHA
(1999) and WHF (2008). Conclusion: We found that smoking induces the expression of ligands of
the activating immune receptor NKG2D in murine as well as in human
joints. Since dysregulated expression of NKG2D ligands has been
previously implicated in induction of autoimmune responses, continuous
excess of NKG2D ligands in joints of smokers might be a trigger for the
development of RA in susceptible individuals. P59 Results: We found that predisposing factors for the development of ARF
was the presence of tonzillopharingitis (47.5%), while carriers of group A
streptococcus (GAS) was 38.0% among patients examined. Clinical
symptoms of carditis with echocardiographic signs of valvulitis occurred in
196 (98.0%) patients. In 54 (27.5%) of them installed valvulitis mitral valve. Valvulitis aortic valve was detected in 24 (12.2%) patients. In 118 (60.2%)
patients observed at the same time valvulitis mitral and aortic valves, while
in 22 (39.2%) patients are men and 92 (63.8%) patients are women. In 18
(66.6%) patients with ARF was observed mitral valve prolapse (MVP), in 6
(22.2%) were in men, 12 (44.4%) in women. In 9 (4.5%) patients with ARF
proceeded pancarditis (endocarditis, myocarditis and pericarditis). Signs of
coronaritis with typical anginal pain with ECG signs of ischemia,
arrhythmias, heart block were observed in 12 (6.0%) patients with RF. Verification of diagnosis was carried out using the angiography of coronary
arteries. The symptoms of coronaritis in this patients disappeared after
anti-inflammatory therapy. P58 Materials and methods: miR expression in SFs from TghuTNF and WT
control mice were determined by deep sequencing and the arthritic profile
was established by pairwise comparisons. qRT-PCR analysis was utilised for
profile validation, miR and gene quantitation in patient SFs. Dysregulated
miR target genes and pathways were predicted via bioinformatic algorithms. Results: Deep sequencing demonstrated that TghuTNF-SFs exhibit a distinct
pathogenic profile with 22 significantly upregulated and 30 significantly
downregulated miRs (fold change>1.5, p-value<0.05). qRT-PCR validation
assays confirmed the dysregulation of miR-223, miR-146a and miR-155
previously associated with human RA pathology, as well as that of miR-221/
222 and miR-323-3p. Notably, the latter were also found significantly
upregulated in patient RASFs, suggesting their association with human RA
pathology. Bioinformatic analysis suggested Wnt/Cadherin signaling as the
most significant pathway targets of miR-221/222 and miR-323-3p and
CSNK1A1 and BTRC, the negative regulators of b-catenin, amongst predicted
gene targets. qRT-PCR assays confirmed the downregulation of these genes
in RASFs, validating our hypothesis that the newly identified miRs may
function to modulate Wnt/Cadherin signaling. Arthritis Research & Therapy 2012, 14(Suppl 1):P58 Background: While different studies confirmed an increased risk for
smokers to develop rheumatoid arthritis (RA), the mechanisms behind this
phenomenon are not known up to now. In all probability, smoking induces
expression or post-translational modification of immune activating proteins
which then initiate an autoimmune reaction in individuals with a susceptible
genetic background. To identify these triggering molecules we screened
joints of mice that were exposed to cigarette smoke for differences of gene
expression and verified our results in synovial tissues of human smokers. y
Methods: C57BL/6 mice were exposed to cigarette smoke (n = 6) or room
air (n = 8) in a whole body exposure chamber for 3 weeks. Protein and
mRNA was isolated from murine ankle joints and from synovial tissues
obtained from smoking (n = 4) and non smoking (n = 5) RA patients
undergoing joint replacement surgery. Tissues were further analysed by
Affymetrix microarrays, Real-time PCR or immunoblotting. Conclusions: In this study, by performing comparative analyses between an
established mouse model of arthritis and RA patient biopsies, we identified
novel dysregulated miRs in RASFs potentially involved in pathways
important for the pathogenic phenotype of these cells and highlighting the
value of such cross-species comparative approaches [13]. Tks5-dependent formation of circumferential podosomes mediates
cell-cell fusion
* Page 44 of 54 Page 44 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
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http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 were 3.2 fold higher in joints of smoke-exposed mice compared to control
mice (dCT 12.5 +/-0.3 versus 14.2 +/-0.4, p = 0.03). Upregulation of H60
protein after smoke exposure was also seen in immunoblotting experiments. Since H60 is not expressed in humans, we analysed expression of the 7
human NKG2D ligands RAET1E, RAET1G, MICA, MICB, and ULBP1-3 in
synovial tissues of RA patients. Transcripts of ULBP1-3 were not detectable in
synovial tissues and there was no difference in the expression levels of
RAET1G and RAET1E in synovial tissues of smokers compared to non
smokers. However, expression levels of MICA and MICB were 2.3 and 2.8 fold
higher in synovial tissues of smokers than in non-smokers (dCT 11.3 +/-0.2
versus 10.1 +/-0.3, p = 0.03 and dCT 10.8 +/-0.3 versus 9.3 +/-0.5, p = 0.03). Conclusion: We found that smoking induces the expression of ligands of
the activating immune receptor NKG2D in murine as well as in human
joints. Since dysregulated expression of NKG2D ligands has been
previously implicated in induction of autoimmune responses, continuous
excess of NKG2D ligands in joints of smokers might be a trigger for the
development of RA in susceptible individuals. were 3.2 fold higher in joints of smoke-exposed mice compared to control
mice (dCT 12.5 +/-0.3 versus 14.2 +/-0.4, p = 0.03). Upregulation of H60
protein after smoke exposure was also seen in immunoblotting experiments. Since H60 is not expressed in humans, we analysed expression of the 7
human NKG2D ligands RAET1E, RAET1G, MICA, MICB, and ULBP1-3 in
synovial tissues of RA patients. Transcripts of ULBP1-3 were not detectable in
synovial tissues and there was no difference in the expression levels of
RAET1G and RAET1E in synovial tissues of smokers compared to non
smokers. However, expression levels of MICA and MICB were 2.3 and 2.8 fold
higher in synovial tissues of smokers than in non-smokers (dCT 11.3 +/-0.2
versus 10.1 +/-0.3, p = 0.03 and dCT 10.8 +/-0.3 versus 9.3 +/-0.5, p = 0.03). Conclusion: We found that smoking induces the expression of ligands of
the activating immune receptor NKG2D in murine as well as in human
joints. Tks5-dependent formation of circumferential podosomes mediates
cell-cell fusion
* In the absence of IL-6 and TGF-b, neither the ROR nuclear receptors nor
IBζ induced Th17 development efficiently. However, when IBζ was
overexpressed, either RORgt or RORa strongly induced IL-17 production,
even in the absence of exogenous polarizing cytokines. In cooperation
with RORgt and RORa, IBζ enhanced Il17a expression by directly binding
to the regulatory region of the Il17a gene. In addition, the expression of
Il17f, Il21 and Il23r mRNA was decreased in Nfkbiz-/- T cells. IBζ also
bound to the promoter or the enhancer region of these genes in Th17
cells. Our study demonstrates the essential role of IBζ in Th17
development, and points to a molecular basis for a novel therapeutic
strategy against autoimmune disease. Tsukasa Oikawa1*, Masaaki Oyama2, Hiroko Kozuka-Hata2, Shunsuke Uehara3,
Nobuyuki Udagawa3, Hideyuki Saya4,5, Koichi Matsuo1
1Laboratory of Cell and Tissue Biology, Institute for Integral Medical Research,
School of Medicine, Keio University, Shinanomachi 35, Shinjuku-ku, Tokyo
160-8582, Japan; 2Medical Proteomics Laboratory, Institute of Medical
Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639,
Japan; 3Department of Biochemistry, Matsumoto Dental University, 1780
Gobara, Hiro-oka, Shiojiri, Nagano 399-0781, Japan; 4Division of Gene
Regulation, Institute for Advanced Medical Research, School of Medicine,
Keio University, Shinanomachi 35, Shinjuku-ku, Tokyo 160-8582, Japan;
5CREST, Japan Science and Technology Agency, Tokyo, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P55 1. Sato K, Suematsu A, Okamoto K, Yamaguchi A, Morishita Y, Kadono Y,
Tanaka S, Kodama T, Akira S, Iwakura Y, Cua DJ, Takayanagi H: Th17
functions as an osteoclastogenic helper T cell subset that links T cell
activation and bone destruction. J Exp Med 2006, 203:2673-2682. 1. Sato K, Suematsu A, Okamoto K, Yamaguchi A, Morishita Y, Kadono Y,
Tanaka S, Kodama T, Akira S, Iwakura Y, Cua DJ, Takayanagi H: Th17
functions as an osteoclastogenic helper T cell subset that links T cell
activation and bone destruction. J Exp Med 2006, 203:2673-2682. Multinucleation of osteoclasts during osteoclastogenesis requires dynamic
rearrangement of the plasma membrane and cytoskeleton, and this process
involves numerous previously characterized factors. However, the
mechanism underlying osteoclast fusion remains obscure. Live-imaging
analysis of osteoclastogenesis revealed that the products of PI3-kinase are
enriched at the sites of osteoclast fusion. Among the downstream molecules 2. Okamoto K, Iwai Y, Oh-Hora M, Yamamoto M, Morio T, Aoki K, Ohya K,
Jetten AM, Akira S, Muta T, Takayanagi H: IBζ regulates TH17
development by cooperating with ROR nuclear receptors. Nature 2010,
464:1381-1385. Tks5-dependent formation of circumferential podosomes mediates
cell-cell fusion
* Since dysregulated expression of NKG2D ligands has been
previously implicated in induction of autoimmune responses, continuous
excess of NKG2D ligands in joints of smokers might be a trigger for the
development of RA in susceptible individuals. were 3.2 fold higher in joints of smoke-exposed mice compared to control
mice (dCT 12.5 +/-0.3 versus 14.2 +/-0.4, p = 0.03). Upregulation of H60
protein after smoke exposure was also seen in immunoblotting experiments. Since H60 is not expressed in humans, we analysed expression of the 7
human NKG2D ligands RAET1E, RAET1G, MICA, MICB, and ULBP1-3 in
synovial tissues of RA patients. Transcripts of ULBP1-3 were not detectable in
synovial tissues and there was no difference in the expression levels of
RAET1G and RAET1E in synovial tissues of smokers compared to non
smokers. However, expression levels of MICA and MICB were 2.3 and 2.8 fold
higher in synovial tissues of smokers than in non-smokers (dCT 11.3 +/-0.2
versus 10.1 +/-0.3, p = 0.03 and dCT 10.8 +/-0.3 versus 9.3 +/-0.5, p = 0.03). P57 In this study we sought to identify novel
miR associations in synovial fibroblasts (SFs), a key pathogenic cell type in
RA [10,11], by performing miR expression profiling on cells isolated from
the human TNF transgenic mouse model (TghuTNF, Tg197) [12] and
patients biopsies. Conclusion: The reducing of clinical manifestations of ARF in adult led to
gypo-diagnostics of disease, a consequence of which was the formation
of rheumatic heart disease. P58
Smoking induces expression of ligands of the immune receptor NKG2D
Caroline Ospelt1*, Giovanni G Camici2, Fabia Brentano1, Peter Künzler1,
Christoph Kolling3, Renate E Gay1, Beat A Michel1, Steffen Gay1
1Center of Experimental Rheumatology and Center of Integrative Human
Physiology (ZIHP), University Hospital, Zurich, Switzerland; 2Institute of
Physiology, University of Zurich, Switzerland; 3Schulthess Clinic, Zurich,
Switzerland
Arthritis Research & Therapy 2012, 14(Suppl 1):P58 Methotrexate alone and methotrexate combined with etanercept in
treatment of rheumatoid arthritis
* Tomoo Sato1, Ryoji Fujii1*, Koji Konomi2, Naoko Yagishita1, Satoko Aratani1,3,
Natsumi Araya1, Hiroyuki Aono2, Kazuo Yudoh1, Noboru Suzuki1,
Moroe Beppu1, Yoshihisa Yamano1, Kusuki Nishioka3, Toshihiro Nakajima1,3,4
1St. Marianna University School of Medicine, Kawasaki, Japan; 2Santen
Pharmaceutical Co., Ltd., Osaka, Japan; 3Institute of Medical Science, Tokyo
Medical University, Tokyo, Japan; 4Misato Marine Hospital, Kochi, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P61 Tomoo Sato1, Ryoji Fujii1*, Koji Konomi2, Naoko Yagishita1, Satoko Aratani1,3,
Natsumi Araya1, Hiroyuki Aono2, Kazuo Yudoh1, Noboru Suzuki1,
Moroe Beppu1, Yoshihisa Yamano1, Kusuki Nishioka3, Toshihiro Nakajima1,3,4
1St. Marianna University School of Medicine, Kawasaki, Japan; 2Santen
Pharmaceutical Co., Ltd., Osaka, Japan; 3Institute of Medical Science, Tokyo
Medical University, Tokyo, Japan; 4Misato Marine Hospital, Kochi, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P61 Tomoo Sato1, Ryoji Fujii1 , Koji Konomi2, Naoko Yagishita1, Satoko Aratani1,3,
Natsumi Araya1, Hiroyuki Aono2, Kazuo Yudoh1, Noboru Suzuki1,
Moroe Beppu1, Yoshihisa Yamano1, Kusuki Nishioka3, Toshihiro Nakajima1,3,4
1St. Marianna University School of Medicine, Kawasaki, Japan; 2Santen
Pharmaceutical Co., Ltd., Osaka, Japan; 3Institute of Medical Science, Tokyo
Medical University, Tokyo, Japan; 4Misato Marine Hospital, Kochi, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P61 Sylejman Rexhepi1*, Mjellma Rexhepi1, Blerta Rexhepi2, Vjollca Sahatçiu-Meka3,
Vigan Mahmutaj2 Sylejman Rexhepi1*, Mjellma Rexhepi1, Blerta Rexhepi2, Vjollca Sahatçiu-Meka3,
Vigan Mahmutaj2 1Rheumatology Department, University of Prishtina, Prishtina, Kosova, 10000;
2Private Clinic “Rheuma”, Prishtina, Kosova, 10000; 3Physical Medicine
Department, University of Prishtina, Kosova, 10000
Arthritis Research & Therapy 2012, 14(Suppl 1):P60 Objectives: The aim of this study is to evaluate the efficacy and safety of
methotrexate (MTX) alone and combined therapy of Etanercept (ETN) and
methotrexate (MTX), in patients with rheumatoid arthritis (RA). Background: The bone and cartilage destruction seen inrheumatoid
arthritis (RA) is caused by synovial pannus formation, which is characterized
by aberrant proliferation of synovial fibroblasts. Inhibition of synovial
proliferation has recently been reported to be a promising therapeutic
strategy for RA.However, the specific mechanism underlyingdysregulated
proliferation of synovial fibroblasts remains unclear. Methods: Patients with RA were treated in combination with ETN (with
doses of 25 mg subcutaneously twice weekly), with oral MTX (doses up to
20 mg weekly), and alone MTX (doses up to 20 mg weekly) in period of two
years, in Rheumatology Department of Internal Clinic in Prishtina. Clinical
response was assessed using American College of Rheumatology (ACR)
criteria and the Disease Activity Score (DAS28) in 60 patients with RA. References 1. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS: The
American Rheumatism Association 1987 revised criteria for the
classification of rheumatoid arthritis. Arthritis Rheum 1988, 31:315-324. 2. Matsumoto AK, Bathon J, Bingham III CO: Rheumatoid arthritis treatment. Available from Johns Hopkins Arthritis Center.[http://www.hopkins-
arthritis.org/arthritis-info/rheumatoid-arthritis/rheum_treat.html]. 3. Felson DT, Anderson JJ, Boers M, Bombardier C, Furst D, Goldsmith C, et al:
American College of Rheumatology preliminary definition of
improvement in rheumatoid arthritis. Arthritis Rheum 1995, 38:727-35. 4. Prevoo ML, van ‘t Hof MA, Kuper HH, van Leeuwen MA, van de Putte LB,
van Riel PL: Modified disease activity scores that include twenty-eight-
joint counts: development and validation in a prospective longitudinal
study of patients with rheumatoid arthritis. Arthritis Rheum 1995, 38:44-8. 5. Pincus T, Sokka T, Wolfe F: Premature mortality in patients with
rheumatoid arthritis: evolving concepts. Arthritis Rheum 2001, 44:1234-6. 6. Bathon JM, Martin RW, et al: A comparison treatment of etanercept and
methotrexate in patients with early rheumatoid arthritis. N Engl J Med
2000, 343:1586-93. 1. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS: The
American Rheumatism Association 1987 revised criteria for the
classification of rheumatoid arthritis. Arthritis Rheum 1988, 31:315-324. 7. Bluml S, Bonelli M, Niederreiter B, Puchner A, Mayr G, Hayer S, Koenders MI,
van den Berg WB, Smolen J, Redlich K: Essential role for micro-RNA 155 in
the pathogenesis of autoimmune arthritis. Arthritis Rheum 2011, 63(5):1281-8. 2. Matsumoto AK, Bathon J, Bingham III CO: Rheumatoid arthritis treatment. Available from Johns Hopkins Arthritis Center.[http://www.hopkins-
arthritis.org/arthritis-info/rheumatoid-arthritis/rheum_treat.html]. 8. Kurowska-Stolarska M, Alivernini S, Ballantine LE, Asquith DL, Millar NL,
Gilchrist DS, Reilly J, Ierna M, Fraser AR, Stolarski B, et al: MicroRNA-155 as a
proinflammatory regulator in clinical and experimental arthritis. Proc Natl
Acad Sci USA 2011, 108(27):11193-8. 3. Felson DT, Anderson JJ, Boers M, Bombardier C, Furst D, Goldsmith C, et al:
American College of Rheumatology preliminary definition of
improvement in rheumatoid arthritis. Arthritis Rheum 1995, 38:727-35. 9. Nakasa T, Shibuya H, Nagata Y, Niimoto T, Ochi M: The inhibitory effect of
microRNA-146 expression on bone destruction in arthritis. Arthritis Rheum
2011, 63(6):1582-90. 4. Prevoo ML, van ‘t Hof MA, Kuper HH, van Leeuwen MA, van de Putte LB,
van Riel PL: Modified disease activity scores that include twenty-eight-
joint counts: development and validation in a prospective longitudinal
study of patients with rheumatoid arthritis. Arthritis Rheum 1995, 38:44-8. 10. P61 P61
SPACIA1/SAAL1: a newly identified gene associated with aberrant
proliferation of synovial fibroblasts
Tomoo Sato1, Ryoji Fujii1*, Koji Konomi2, Naoko Yagishita1, Satoko Aratani1,3,
Natsumi Araya1, Hiroyuki Aono2, Kazuo Yudoh1, Noboru Suzuki1,
Moroe Beppu1, Yoshihisa Yamano1, Kusuki Nishioka3, Toshihiro Nakajima1,3,4
1St. Marianna University School of Medicine, Kawasaki, Japan; 2Santen
Pharmaceutical Co., Ltd., Osaka, Japan; 3Institute of Medical Science, Tokyo
Medical University, Tokyo, Japan; 4Misato Marine Hospital, Kochi, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P61 References 1. Montgomery RL, van Rooij E: microRNA Regulation as a Therapeutic
Strategy for Cardiovascular Disease. Curr Drug Targets 2010, 11(8):936-942. 2. Murata K, Yoshitomi H, Tanida S, Ishikawa M, Nishitani K, Ito H, Nakamura T:
Plasma and synovial fluid microRNAs as potential biomarkers of
rheumatoid arthritis and osteoarthritis. Arthritis Res Ther 2010, 12(3):R86. 3. Pauley KM, Satoh M, Chan AL, Bubb MR, Reeves WH, Chan EK: Upregulated
miR-146a expression in peripheral blood mononuclear cells from
rheumatoid arthritis patients. Arthritis Res Ther 2008, 10(4):R101. 4. Kawano S, Nakamachi Y: miR-124a as a key regulator of proliferation and
MCP-1 secretion in synoviocytes from patients with rheumatoid arthritis. Ann Rheum Dis 2011, 70:I88-I91. Stanczyk J, Ospelt C, Karouzakis E, Filer A, Raza K, Kolling C, Gay R, 5. Stanczyk J, Ospelt C, Karouzakis E, Filer A, Raza K, Kolling C, Gay R,
Buckley CD, Tak PP, Gay S, et al: Altered expression of MicroRNA-203 in
rheumatoid arthritis synovial fibroblasts and its role in fibroblast
activation. Arthritis Rheum 2011, 63(2):373-381. Conclusions: According to our results we can conclude that ETN in
combination with MTX reduced disease activity, slowed radiographic
progression and improved clinical manifestations more effectively than
MTX alone within period of 2 years. During the treatment, no serious
adverse events were noticed with combination treatment of ETN and
MTX. 6. Stanczyk J, Pedrioli DM, Brentano F, Sanchez-Pernaute O, Kolling C, Gay RE,
Detmar M, Gay S, Kyburz D: Altered expression of MicroRNA in synovial
fibroblasts and synovial tissue in rheumatoid arthritis. Arthritis Rheum
2008, 58(4):1001-1009. 6. Stanczyk J, Pedrioli DM, Brentano F, Sanchez-Pernaute O, Kolling C, Gay RE,
Detmar M, Gay S, Kyburz D: Altered expression of MicroRNA in synovial
fibroblasts and synovial tissue in rheumatoid arthritis. Arthritis Rheum
2008, 58(4):1001-1009. P58 Results: Since data from microarray experiments had shown increased levels
of the immune receptor NKG2D ligand histocompatibility 60 (H60) after
cigarette smoke exposure, we measured H60 expression levels by Real-time
PCR in ankle joints of smoke exposed and control mice. H60 transcript levels Acknowledgements: This project was funded by the Masterswitch Project
(HEALTH-F2-2008-223404), EURO-RA RTN (HPRN-CT-2002-00255) and IMI Page 45 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 BtCure (grant agreement No 115142) grants to GK and SG. JR was supported
by the Wellcome Trust grant 075491/Z/04. In SG also received funding from
IAR-EPALINGES. with combined therapy (ETN plus MTX) and 50 or 83.3% of patients with
monotherapy (MTX). The group of combined therapy (ETN+MTX) after the
treatment resulted with improvement of acute phase reactants as
erythrocyte sedimentation rate (ESR) for the first hour (41.1 vs. 10.3 mm/
hour) and C-reactive protein (CRP) (40.8 vs. 6 mg/liter) comparing to the
group treated with MTX alone there were no significant changes (ESR: 45.7
vs. 34.3 mm/hour; CRP: 48 vs. 24 mg/liter). Before treatment the severity of
the disease was high, where in group with combined therapy (ETN plus
MTX) DAS28 was 5.32, and in the group with monotherapy of MTX DAS28
was 5.90. After 2 years of treatment we had significant changes in the
results of DAS28, where in group treated with ETN plus MTX DAS28 was
2.12 ± 0.15, while in the group of patients treated with MTX DAS28 were
3.75 ± 0.39 (t = 13.03; df = 58; p < 0.0001). The group with combined
therapy showed less radiographic progression comparing to the group of
monotherapy (p < 0.05). 1.
Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS: The
American Rheumatism Association 1987 revised criteria for the
classification of rheumatoid arthritis. Arthritis Rheum 1988, 31:315-324.
2.
Matsumoto AK, Bathon J, Bingham III CO: Rheumatoid arthritis treatment.
Available from Johns Hopkins Arthritis Center.[http://www.hopkins-
arthritis.org/arthritis-info/rheumatoid-arthritis/rheum_treat.html].
3.
Felson DT, Anderson JJ, Boers M, Bombardier C, Furst D, Goldsmith C, et al:
American College of Rheumatology preliminary definition of
improvement in rheumatoid arthritis. Arthritis Rheum 1995, 38:727-35.
4.
Prevoo ML, van ‘t Hof MA, Kuper HH, van Leeuwen MA, van de Putte LB,
van Riel PL: Modified disease activity scores that include twenty-eight-
joint counts: development and validation in a prospective longitudinal
study of patients with rheumatoid arthritis. Arthritis Rheum 1995, 38:44-8.
5.
Pincus T, Sokka T, Wolfe F: Premature mortality in patients with
rheumatoid arthritis: evolving concepts. Arthritis Rheum 2001, 44:1234-6.
6.
Bathon JM, Martin RW, et al: A comparison treatment of etanercept and
methotrexate in patients with early rheumatoid arthritis. N Engl J Med
2000, 343:1586-93. References Armaka M, Apostolaki M, Jacques P, Kontoyiannis DL, Elewaut D, Kollias G:
Mesenchymal cell targeting by TNF as a common pathogenic principle
in chronic inflammatory joint and intestinal diseases. J Exp Med 2008,
205(2):331-337. y
p
,
5. Pincus T, Sokka T, Wolfe F: Premature mortality in patients with
rheumatoid arthritis: evolving concepts. Arthritis Rheum 2001, 44:1234-6. 6. Bathon JM, Martin RW, et al: A comparison treatment of etanercept and
methotrexate in patients with early rheumatoid arthritis. N Engl J Med
2000, 343:1586-93. 11. Lefevre S, Knedla A, Tennie C, Kampmann A, Wunrau C, Dinser R, Korb A,
Schnaker E-M, Tarner IH, Robbins PD, et al: Synovial fibroblasts spread
rheumatoid arthritis to unaffected joints. Nat Med 2009, 15(12):1414-1420. ff
b
l
l
l 7. Weinblatt ME, Kreer JM, Bankhurst AS: A trial of etanercept, a recombinant
tumor necrosis factor receptor: Fc fusion protein, in patients with
rheumatoid arthritis receiving methotrexate. N Engl J Med 1999,
340(4):253-9, The first DBPCS (89 patients) with etanercept which showed
the efficacy of etanercept when used as combination treatment with
methtrexate over 6 months of treatment. 7. Weinblatt ME, Kreer JM, Bankhurst AS: A trial of etanercept, a recombinant
tumor necrosis factor receptor: Fc fusion protein, in patients with
rheumatoid arthritis receiving methotrexate. N Engl J Med 1999,
340(4):253-9, The first DBPCS (89 patients) with etanercept which showed
the efficacy of etanercept when used as combination treatment with
methtrexate over 6 months of treatment. 12. Keffer J, Probert L, Cazlaris H, Georgopoulos S, Kaslaris E, Kioussis D,
Kollias G: Transgenic mice expressing human tumour necrosis factor: a
predictive genetic model of arthritis. EMBO J 1991, 10(13):4025-4031. 13
K lli
G P
d ki P A
ill F V
d ld
k MJ H l d hl R 13. Kollias G, Papadaki P, Apparailly F, Vervoordeldonk MJ, Holmdahl R,
Baumans V, Desaintes C, Di Santo J, Distler J, Garside P, et al: Animal
models for arthritis: innovative tools for prevention and treatment. Ann
Rheum Dis 2011, 70(8):1357-1362. P61 Two cases of multiple-drug-resistant adult-onset Still’s disease
treated successfully with tocilizumab - the relationship between
interleukin 6 and 18 Background: The GI Randomized Event and Safety Open-Label NSAID
Study (GI-REASONS) was a novel prospective, randomized, open-label,
blinded end point (PROBE) study that measured adjudicated clinical
outcomes throughout the GI tract. It was designed to assess if celecoxib
use in patients with osteoarthritis (OA) at moderate GI risk (≥55 y) is
associated with a lower incidence of clinically significant upper and lower
GI events compared to nsNSAIDs, with/without proton-pump inhibitors
(PPIs), in standard US clinical practice. Kojiro Sato*, Akinori Yamamoto, Yoshihiro Yoshida, Toshihide Mimura
Department of Rheumatology and Applied Immunology, Saitama Medical
University, Saitama 350-0495, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P62 Kojiro Sato*, Akinori Yamamoto, Yoshihiro Yoshida, Toshihide Mimura
Department of Rheumatology and Applied Immunology, Saitama Medical
University, Saitama 350-0495, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P62 Background: Adult-onset Still’s disease (AOSD) is an inflammatory disease of
unknown cause characterized by a high spiking fever, arthritis and
evanescent rash. The mainstay of treatment is glucocorticoids with or
without immunosuppressants. Recently, biologics such as anti-tumor necrosis
factor (TNF) antibodies have also been tried in certain refractory cases. Materials and methods: 8067 OA patients were randomized 1:1 for 6-mos
with celecoxib or a nonselective (ns)NSAID, stratified by H pylori status. The
primary end point was a composite of adjudicated clinically significant
upper and lower GI events. Aspirin use was not permitted. Treatment doses
could be adjusted per US prescribing information. Patients randomized to
the nsNSAID arm could switch between nsNSAIDs; however, crossover
between treatment arms was not allowed. PPIs and histamine-2 receptor
antagonists (H2RAs) were prescribed at the providers’ discretion. y
Results: We have had two cases of AOSD which were treated successfully
with anti-interleukin (IL-) 6-receptor antibody, tocilizumab (TOC). (Case 1)
A 36-year-old woman who was diagnosed 8 years previously, and had
been treated with various DMARDs plus etanercept (ETA) or adalimumab,
presented with a high spiky fever and elevated liver enzymes. After
excluding infection, she was treated with TOC. (Case 2) A 26-year-old
man with new-onset AOSD, which was shown to be resistant to multiple
immunosuppressants including infliximab and ETA, was treated with TOC
starting 7 months after the diagnosis. In both cases, serum IL-18 was
extremely high, and TOC promptly improved clinical symptoms and liver
function. The high level of serum ferritin also became normalized. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Results: We identified a novel gene named SPACIA1/SAAL1 (synoviocyte
proliferation-associated in CIA 1/serum amyloid A-like 1)that was associated
with aberrant proliferation of synovial fibroblasts. Immunohistochemical-
analysis indicated that SPACIA1/SAAL1 was strongly expressed in the foot
joints of mice with CIA and in the thickened synovial lining of the human RA
synovium. Transfection of siRNA targeting SPACIA1/SAAL1into RA synovial
fibroblastscould inhibit tumor necrosis factor (TNF)a-induced proliferation
more effectively thanit could inhibit serum-induced proliferation.In addition,
the antiproliferative effect of SPACIA1/SAAL1 siRNA was caused byinhibition of
cell cycle progression and not by induction of apoptosis.We established
transgenic (Tg) mice that overexpressed SPACIA1/SAAL1. These Tg mice did
not spontaneously develop arthritis or cancer. However,inducing CIA
causedgreatersynovial proliferation and worse diseasein Tg mice thanin wild-
type mice. Next, we cultured human monocytes derived from healthy controls with
or without the presence of IL-6 and/or IL-18 in vitro. The level of ferritin
in the supernatant was significantly increased only when both IL-6 and
IL-18 were added, indicating that IL-6 and IL-18 have a synergistic effect
on the production of ferritin (Figure 1). Next, we cultured human monocytes derived from healthy controls with
or without the presence of IL-6 and/or IL-18 in vitro. The level of ferritin
in the supernatant was significantly increased only when both IL-6 and
IL-18 were added, indicating that IL-6 and IL-18 have a synergistic effect
on the production of ferritin (Figure 1). Conclusion: TOC can be a first-line biologic applicable against multiple-
drug-resistant AOSD. If an IL-18 blocker is developed, however, it may be
even more beneficial in that it may block the cascade of inflammation at
a point further upstream. P63
GI-REASONS: a novel 6-month, prospective, randomized, open-label,
blinded end point (PROBE) trial
Byron Cryer1*, Chunming Li2, Lee S Simon3, Gurkirpal Singh4,
Martin J Stillman5, Manuela Berger2
1University of Texas Southwestern Medical Center, Dallas, TX, USA; 2Pfizer Inc. New York, NY, USA; 3SDG, LLC, Cambridge, MA, USA; 4Stanford University,
Palo Alto, CA, USA; 5Hennepin County Medical Center, Minneapolis, MN, USA
Arthritis Research & Therapy 2012, 14(Suppl 1):P63 1Institute of Experimental Musculoskeletal Medicine, University Muenster, 48149,
Muenster, Germany; 2Department of Anesthesiology and Intensive Care
Medicine, Medical University Hannover, 30625, Hannover, Germany; 3Institute of
Immunology, Biomedical Sciences Research Center, Vari, 16672, Greece
Arthritis Research & Therapy 2012, 14(Suppl 1):P64 P62 Two cases of multiple-drug-resistant adult-onset Still’s disease
treated successfully with tocilizumab - the relationship between
interleukin 6 and 18
* P63 Conclusion: SPACIA1/SAAL1 plays an important role in the aberrant
proliferation of synovial fibroblasts under inflammatory conditions. Methotrexate alone and methotrexate combined with etanercept in
treatment of rheumatoid arthritis
* Radiographic changes were measured in the beginning and at the end of
the study with Sharp Score. Objective: We aimed toidentify and characterize genesthat are involved in
the aberrant proliferation of synovial fibroblasts. Methods: Microarray analysiswas performed to identifythe genes that had
upregulated expression inmice with collagen-induced arthritis (CIA). The
effect of candidate genes on the proliferation of synovial fibroblasts was
screened using antisense oligodeoxynucleotides and small interfering RNAs
(siRNAs). Results: Of total number of 60 patients (10 of them were males and 50
were females) with mean age of 57.63, 10 or 16.6% of patients were treated Page 46 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Two cases of multiple-drug-resistant adult-onset Still’s disease
treated successfully with tocilizumab - the relationship between
interleukin 6 and 18 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Page 47 of 54 Table 1(abstract P63) Clinically significant upper and lower GI events: primary analysis ract P63) Clinically significant upper and lower GI events: primary analysis Table 1(abstract P63) Clinically significant upper and lower GI events: primary analysis
Celecoxib
nsNSAID
N
Patients With Event n (%)
N
Patients With Event n (%)
All patients
4035
54 (1.3)
4032
98 (2.4)
H pylori status
Positive
1401
25 (1.8)
1386
34 (2.5)
Negative
2634
29 (1.1)
2646
64 (2.4)
OR (95% CI); P value
1.82 (1.31-2.55); p = 0.0003 Table 1(abstract P63) Clinically significant upper and lower GI events: primary analysis
Celecoxib
nsNSAID The results achieved and their novelty: On the systemic and local levels
an approach was applied allowing consideration of nitrogen oxide
metabolism disorders as an important part of the pathogenesis of
rheumatoid arthritis. A number of new data were obtained concerning the
relationship of nitrogen oxide metabolism and C-reactive protein
formation, clinical course of rheumatoid arthritis. For the first time a
complex approach was suggested for the pathogenic justification of
simvastatin use in the scheme of conventional treatment to increase the
therapy efficiency, to achieve stable early remission in patients with
rheumatoid arthritis. It was proved that an important mechanism of
increasing the therapeutic efficiency of simvastatin was its action on the
system of endothelial function in blood and joint fluid. It was suggested
that one should include assessment of blood and joint fluid for nitrogen
oxide, nitrate diaphorase and nitrate reductase in the algorithm of
investigation and dynamic observation, choice of tactics and therapy
efficiency assessment. Background: Syndecan-4, a member of a syndecan family of transme-
mbrane heparansulfate proteoglycans has been recently associated with
cell-matrix-adhesion, cell-migration, differentiation and proliferation, but its
specific function in inflammatory pathologies remains unclear. We used the
human TNFalpha transgenic mouse (hTNFtg) to analyse the expression and
function of syndecan-4 in chronic-destructive-arthritis and answer the
question whether inhibition of syndecan-4 by specific antibodies may
prevent cartilagedestruction and/or improve the phenotype after onset of
the disease in this animal model of human RA. Methods: Expression of syndecan-4 was investigated by immunohisto-
chemistry in the hind-paws of 8-weeks/12-weeks old hTNFtg mice and wild
type controls. In addition, synovial fibroblasts were isolated and analysed for
syndecan-4-expression by RT-PCR. For functional analyses, we generated
blocking-antibodies against syndecan-4. Two cases of multiple-drug-resistant adult-onset Still’s disease
treated successfully with tocilizumab - the relationship between
interleukin 6 and 18 Interestingly, especially in case 2, the level of IL-18 remained high after
the administration of TOC, suggesting that IL-18 is located either
upstream of, or at the same level as, IL-6 in the pathogenesis of AOSD. g
(
2
)
p
p
Results: 4035 celecoxib and 4032 nsNSAID patients were randomized and
included in the ITT analyses. Baseline demographics were similar. Overall,
significantly more nsNSAID users met the primary end point at 6 mos (OR,
1.82; 95% CI 1.31-2.55; p = 0.0003; Table 1). The most commonly used
nsNSAIDs were meloxicam (42%), naproxen (21%), diclofenac (20%) and
nabumetone (14%). 2596 celecoxib (64.3%) and 2611 (64.8%) nsNSAID users
completed the study. 189 patients were lost to follow-up (LTFU; 2.1%
celecoxib and 2.6% nsNSAID). Attributing the primary end point to all LTFU
patients (worst-case sensitivity analysis), celecoxib remained superior (OR
1.46; 95% CI 1.18-1.82; p = 0.0006). AEs, SAEs and discontinuations were
similar in both treatment groups. 23% of celecoxib and 24% of nsNSAID
patients used a PPI (p = NS). Moderate to severe abdominal symptoms were
experienced by 94 (2.3%) celecoxib and 138 (3.4%) nsNSAID patients (P<.01). Conclusion: Celecoxib use had a lower risk of clinically significant upper
and lower GI events than nsNSAIDs. A major strength of this study is its
PROBE design. Simple inclusion and exclusion criteria allowed for a broad
patient population of moderate GI risk. Switching among nsNSAIDs and
allowing for dose adjustments, along with use of PPIs and H2RAs as
needed, more closely reflects daily clinical practice. GI-REASONS
demonstrates the improved GI safety profile of celecoxib throughout the
GI tract in patients treated in a “real-world” setting. Figure 1(abstract P62) The level of ferritin in the supernatant of
monocytes cultured with or without the presence of IL-6 and/or
IL-18 (10 ng/mL each). Athanasios Stratis1*, Katja Neugebauer1, Mareike Frohling1, Peter Paruzel1,
Berno Dankbar1, Corinna Wemeyer1, Christoph Cromme1, Lars Godmann1,
Jessica Bertrand1, Adelheide Korb1, Frank Echtermeyer2, George Kollias3,
Thomas Pap1 Figure 1(abstract P62) The level of ferritin in the supernatant of
monocytes cultured with or without the presence of IL-6 and/or
IL-18 (10 ng/mL each). Two cases of multiple-drug-resistant adult-onset Still’s disease
treated successfully with tocilizumab - the relationship between
interleukin 6 and 18 To investigate their effect on
TNFalpha mediated-destructive-arthritis, hTNFtg mice were injected with the
antibodies or with IgG-control twice weekly for 4-weeks in a preventive
manner (age-4-to-8-weeks) and for disease treatment of joint destruction
(age-8-to-12-weeks) into their hind paws. Evaluation of disease severity
included clinical parameters (weight, arthritis-score, grip-strength) as well as
histomorphometric analysis of toluidin-blue-stained paraffin sections. Practical value: Obtained new data are necessary for increasing the
pharmacotherapy efficacy in patients with rheumatoid arthritis taking into
account the metabolic activity of NO-synthetase mechanism in blood and
synovial fluid. An algorithm was suggested for screening observation and
differentiated management of patients with rheumatoid arthritis taking
account of severity of nitrogen oxide metabolism disorders. A differentiated
approach was worked out and justified of simvastatin prescription both to
increase the efficacy of treatment taking into account the clinical activity of
the disease and to correct metabolic disorders in patients with rheumatoid
arthritis. Results: As seen in immunohistochemistry, there was a strong expression of
syndecan-4 in the synovial membranes of hTNFtg mice, whereas only
negligible staining for syndecan-4 was found in synovial tissues of wild type
animals. In vitro, synovial fibroblasts isolated from hTNFtg mice showed
more than 30-fold higher expression of syndecan-4 than wild type controls. Administration of the anti-syndecan-4 antibodies but not of IgG-control in
preventive treated 4-week-old hTNFtg mice clearly ameliorated the clinical
signs of arthritis and protected the treated joints from cartilage damage. At
histomorphometric analysis, this was evident for all analysed parameters but
seen most prominently for area of distained cartilage. Metabolic syndrome in Indian patients with rheumatoid arthritis and its
correlation with disease activity
1*
1
2
3 Significantly reduced cartilage damage in the anti-syndecan-4 treated
hTNFtg mice was accompanied by a striking reduction in the expression
of MMP-3. The treatment with antisyndecan-4 in 8-week-old hTNFtg mice
after onset of arthritis clearly ameliorated the jointdestruction, and
improved cartilage-damage. The treatment also showed a clear reduction
of inflammation in the paws compared to the untreated animals. Aman Sharma1*, Nilesh Bhilave1, Kusum Sharma2, Indu Varma3
1Internal Medicine, PGIMER, Chandigarh, India,160012; 2Medical Microbiology,
PGIMER, Chandigarh, India,160012; 3Biochemistry, PGIMER, Chandigarh, India,
160012 Aman Sharma1*, Nilesh Bhilave1, Kusum Sharma2, Indu Varma3
1Internal Medicine, PGIMER, Chandigarh, India,160012; 2Medical Microbiology,
PGIMER, Chandigarh, India,160012; 3Biochemistry, PGIMER, Chandigarh, India,
160012 Conclusions: Our findings indicate that syndecan-4 is involved
prominently in fibroblast-mediated cartilagedamage in hTNFtg mice by
regulating the exression of disease-relevant MMPs. More importantly, the
data suggest that inhibition of syndecan-4 not only prevens cartilage
damage, but also reduces the severity after onset of the disease. Background: Increased prevalence of metabolic syndromein rheumatoid
arthritis (RA) has been reported from American and European populations
but it has not been studied in Indian patients with RA. Objectives: The main objective of our study was to assess the prevalence
of the metabolic syndrome in Asian-Indian patients with rheumatoid
arthritis and also to studyits correlation with disease activity. P65
Clinical-experimental assessment of simvastatin efficiency in the
treatment of rheumatoid arthritis
Rikhikhon N Tadjikhodjaeva*, Nargiza G Khabibullaeva
Tashkent Medical Academy, Tashkent, Uzbekistan
Arthritis Research & Therapy 2012, 14(Suppl 1):P65 P65 Methods: This was a prospective case control study in which 114 patients
diagnosed to have rheumatoid arthritis of more than 1 year duration and
114 healthy age (± 5 years) and sex matched controls were included. Height,
weight, body mass index, blood pressure and waist circumference of the
patients were measured at the enrolment visit. Venous samples were taken
after eight hours of overnight fasting for the estimation of serum
cholesterol, triglycerides and plasma glucose levels. Metabolic syndrome
was diagnosed according to Adult Treatment Panel III criteria [1] and the
consensus definition of the metabolic syndrome for adult Asian patients [2]. The disease activity was assessed by DAS 28. Subject of the inquiry: 35 patients with rheumatoid arthritis, 50 mature
male rats of mixed population. Subject of the inquiry: 35 patients with rheumatoid arthritis, 50 mature
male rats of mixed population. Aim of the inquiry: Clinical-experimental assessment of simvastatin
efficiency and pathogenic justification of its inclusion into the complex
treatment for therapy optimization in patients with rheumatoid arthritis. Methods of investigation: clinical-laboratory, biochemical - determination
of total cholesterol, low and high density lipoproteins, triglycerides,
calculation of atherogenic coefficient in blood serum of patients with
rheumatoid arthritis and in experimental animals. Results: The mean age of patients with RA and control group was 44.8 and
43.2 years (p <0.36) respectively. The mean duration of RA was 6.5 years. Though the mean BMI was similar in both the groups(25.5 and 24.2), there
was a statistically highly significant difference in mean waist circumference
(92.1 cm and 81.2 cm, p < 0.001) and diastolic blood pressure(80.5 and 75.3 Page 48 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 y
References 1. Expert Panel on Detection, Evaluation, and Treatment of High Blood
CholesterolIn Adults (Adult Treatment Panel III). Executive summary of
the third report of The National Cholesterol Education Program (NCEP). JAMA 2001, 285:2486-97. 2. Mishra A, Chowbey P, Makkar BM, Vikram NK, Wasir JS, Joshi SR, et al:
Consensus statement for diagnosis of obesity, abdominal obesity and
the metabolic syndrome for Asian Indians and recommendations for
physical activity, medical and surgical management. J Assoc Physician
India 2009, 57:163-7. 2. Mishra A, Chowbey P, Makkar BM, Vikram NK, Wasir JS, Joshi SR, et al:
Consensus statement for diagnosis of obesity, abdominal obesity and
the metabolic syndrome for Asian Indians and recommendations for
physical activity, medical and surgical management. J Assoc Physician
India 2009, 57:163-7. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 known to induce osteoclast formation when injected onto calvaria in mice. Unexpectedly, we observed that mice injected with LPS up-regulate OPG
and down-regulate RANKLlevels in peripheral blood. known to induce osteoclast formation when injected onto calvaria in mice. Unexpectedly, we observed that mice injected with LPS up-regulate OPG
and down-regulate RANKLlevels in peripheral blood. mm Hg, p < 0.001) in patients with RA as compared to controls.Metabolic
syndrome was present in 36 patients and 17 controls (p < 0.05) according to
the Adult Treatment Panel III criteria and in 40 patients and 18 controls (p <
0.01) according to the consensus definition of the metabolic syndrome for
adult Asian patients. There was no significant correlation between the
metabolic syndrome and disease activity as measured by DAS-28 using both
the criteria. In the present study, we examined whether OPG is induced by microbial
infection of various kinds, and the sites and significance of OPG production
in infected mice. Wild-type mice infected withSalmonella, Staphylococcus,
Mycobacteriaor influenza virus showed increase in OPG levels in peripheral
blood. We also found that the levels of OPG in serum of human patients
infected with M. tuberculosis and M. avium were significantly increased. Moreover, injection of mice with LPS induced OPG production specifically in
lymph nodes, especially in high endothelial venule(HEV)cells, but not in
other organs. OPG production was suppressed in c-Fos-deficient mice and
enhanced in Fra-1 transgenic mice, indicating that OPG production is
regulated by AP-1 transcription factors.Loss of OPG in mice did not affect
either their survival or Salmonella proliferation in spleen and liver after
infection with virulent strains of Salmonella. Interestingly, however, when
wild-type mice were infected with an avirulentSalmonella strain, which can
induce OPG, osteoclast development was suppressed and bone mineral
density was increased. These data reveal for the first time that lymph nodes
protect bones from infection-induced bone loss through OPG production. Conclusions: Indian patients with RA have increased prevalence of
metabolic syndrome as compared to their age and sex matched healthy
controls, but there is no significant correlation between metabolic
syndrome and disease activity. P68 P68
Expression patterns and function of chromatin protein HMGB2 during
mesenchymal stem cell differentiation
Noboru Taniguchi1,2*, Beatriz Caramés2, Yasuhiko Kawakami3, Martin Lotz2
1Hakuaikai Kaisei Hospital, Obihiro, Hokkaido, Japan; 2The Scripps Research
Institute, La Jolla, CA, USA; 3University of Minnesota, Minneapolis, MN, USA
Arthritis Research & Therapy 2012, 14(Suppl 1):P68 P69
Age features of metabolic syndrome and cardiovascular disorders in
gout
h k
*
h
b
d
lf Background: To maintain the bone strength and functions, the balance
between bone resorption and bone formation has to be tightly regulated. However, under certain pathological conditions, including osteoporosis and
rheumatoid arthritis, the equilibrium gets disrupted, resulting in a severe
bone loss. Recent studies have shown that signaling molecules involved in
the unfolded protein response (UPR) are potentially involved in the coupling
of bone resorption and bone formation [1-3]. In the present study, we
investigated the roles of UPR mediator, the IRE1a-XBP1 pathway in osteoblast
differentiation. j
y,
j
Arthritis Research & Therapy 2012, 14(Suppl 1):P69 Materials and methods: Are surveyed 76 gout patients, middle age
equaled 56.6 ± 7.5 year. Have been distributed on 3 groups: more Materials and methods: Are surveyed 76 gout patients, middle age
equaled 56.6 ± 7.5 year. Have been distributed on 3 groups: more Table 1(abstract P69) Frequency of revealing of signs of
metabolic syndrome at gout patients (n = 76)
Sign
Frequency
CW > 102 cm
48 (63.2%)
SBP > 140 mm Hg and/or DBP > 90 mm Hg
50 (65.8%)
TG ≥120 mg/dl
22 (29%)
Glucose ≥110 mg/dl
32 (42.1%)
HDL-cholesterol < 50 mg/dl
58 (76.3%)
CW - circle waist; TG - triglycerides; SBP - systolic blood pressure; DBP -
diastolic blood pressure; HDL - high density lipoproteides. Materials and methods: To induce osteoblast differentiation in vitro, we
used recombinant human BMP-2 and mouse embryonic fibroblasts (MEFs)
obtained from wild-type and Ire1-/- embryos. Small interfering RNA-
mediated gene silencing was used to suppress the expression of the target
molecules of IRE1 (XBP1 and TRAF2) in wild-type MEFs. Osteoblast
differentiation was evaluated by analyzing the expression levels of the
transcripts for osteoblast differentiation markers (Runx2, Osterix, Osteoclacin
and type I collagen) and alkaline-phosphatase activity. yp
g
p
p
y
Results: We found that UPR is induced during osteoblast differentiation in
in vitro and ex vivo experiments. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 younger 50, from 50 to 60 and more senior 60 years. Metabolic syndrome
was diagnosed by criteria Adult Treatment Panel III (National Institute of
Health, USA) [1]. Serum level of Uric Acid defined by colorimetric enzyme
method, glucose - by glucose oxidize method, cholesterol, triglycerides
and high density lipoproteides-cholesterol - by colorimetric method [2]. Low and very low density lipoproteides-cholesterol defined by “WT
Friedewald Equation” (1972) [3]. younger 50, from 50 to 60 and more senior 60 years. Metabolic syndrome
was diagnosed by criteria Adult Treatment Panel III (National Institute of
Health, USA) [1]. Serum level of Uric Acid defined by colorimetric enzyme
method, glucose - by glucose oxidize method, cholesterol, triglycerides
and high density lipoproteides-cholesterol - by colorimetric method [2]. Low and very low density lipoproteides-cholesterol defined by “WT
Friedewald Equation” (1972) [3]. changes in osteoarthritis (OA). The expression of chromatin protein
HMGB2 is restricted to the SZ, which contains cells expressing
mesenchymal stem cell (MSC) markers [1]. Aging-related loss of HMGB2
and gene deletion are associated with reduced SZ cellularity and early
onset OA [2]. This study addressed HMGB2 expression patterns in MSC
and its role during differentiation. HMGB2 was detected at higher levels in human MSC as compared to human
articular chondrocytes and its expression declined during chondrogenic
differentiation of MSC (Figure 1). Lentiviral HMGB2 transduction of MSC
suppressed chondrogenesis as reflected by an inhibition of Col2a1 and
Col10a1 expression. Conversely, in bone marrow MSC from Hmgb2-/- mice,
Col10a1 was more strongly expressed than in wildtype MSC. This is
consistent with in vivo results from mouse growth plates showing that
Hmgb2 is expressed in proliferating and prehypertrophic zones but not in
hypertrophic cartilage where Col10a1 is strongly expressed. Osteogenesis
was also accelerated in Hmgb2-/- MSC. The expression of Runx2, which
plays a major role in late stage chondrocyte differentiation, was enhanced in
Hmgb2-/- MSC and HMGB2 negatively regulated the stimulatory effect of
Wnt/b-catenin signaling on the Runx2 proximal promoter. Results: Metabolic syndrome has been diagnosed at 46 (60.5%) patients. Middle age patients with presence of metabolic syndrome has made 55.7
± 4.7, without - 57.9 ± 8.3 year. Conclusions: At the same time we have not revealed age distinctions in
occurrence of metabolic syndrome at patients with primary gout,
however frequency of IHD of gout patients naturally increased with the
years - from 38% to 68%. Osteoprotegerin induction in response to microbial infection
Yasunari Takada*, Koichi Matsuo Noboru Taniguchi1,2*, Beatriz Caramés2, Yasuhiko Kawakami3, Martin Lotz2
1Hakuaikai Kaisei Hospital, Obihiro, Hokkaido, Japan; 2The Scripps Research
Institute, La Jolla, CA, USA; 3University of Minnesota, Minneapolis, MN, USA
Arthritis Research & Therapy 2012, 14(Suppl 1):P68 Noboru Taniguchi1,2*, Beatriz Caramés2, Yasuhiko Kawakami3, Martin Lotz2
1Hakuaikai Kaisei Hospital, Obihiro, Hokkaido, Japan; 2The Scripps Research
Institute, La Jolla, CA, USA; 3University of Minnesota, Minneapolis, MN, USA
Arthritis Research & Therapy 2012, 14(Suppl 1):P68 Laboratory of Cell and Tissue Biology, Keio Univ. School of Medicine, Tokyo
160-8582 Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P67 Laboratory of Cell and Tissue Biology, Keio Univ. School of Medicine, Tokyo
160-8582 Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P67 p
thritis Research & Therapy 2012, 14(Suppl 1):P67 The superficial zone (SZ) of articular cartilage is critical in maintaining
tissue function and homeostasis and represents the site of the earliest Osteoprotegeirn (OPG) is an endogenous decoy receptor for RANKL, which is
a cytokine essential for osteoclast differentiation. Lipopolysaccharide (LPS) is Figure 1(abstract P68) HMGB2 expression during chondrogenesis of human MSC. Immunohistochemistry shows that HMGB2 is expressed at days 1
and 3, but that expression is reduced at days 7, 14 upon induction of chondrogenesis. SO: safranin O staining. Figure 1(abstract P68) HMGB2 expression during chondrogenesis of human MSC. Immunohistochemistry shows that HMGB2 is expressed at days 1
and 3, but that expression is reduced at days 7, 14 upon induction of chondrogenesis. SO: safranin O staining. Figure 1(abstract P68) HMGB2 expression during chondrogenesis of human MSC. Immunohistochemistr
and 3, but that expression is reduced at days 7, 14 upon induction of chondrogenesis. SO: safranin O staining Figure 1(abstract P68) HMGB2 expression during chondrogenesis of human MSC. Immunohistochemistry shows that HMGB2 is expressed at days 1
and 3, but that expression is reduced at days 7, 14 upon induction of chondrogenesis. SO: safranin O staining. Page 49 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 P70 Unfolded protein response mediator, the IRE1a-XBP1 pathway is
involved in osteoblast differentiation Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Patients of the senior age groups the increase
in frequency of hypertension and IHD while patients of younger age have
obesity, hypertriglyceridemia and hyperglycemia is more often noted. Acknowledgements: Research grants were received from APLAR. References 1. Executive summary of the Third Report of the National Cholesterol
Education Program (NCEP) Expert Panel on Detection, Evaluation, and
Treatment of High Blood Cholesterol in Adults (Adult Treatment
Panel III). JAMA 2001, 285(19):2486-2497. These results demonstrate that HMGB2 expression is inversely correlated
with the differentiation status of MSC and that HMGB2 suppresses
chondrogenic differentiation. The aging-related loss of HMGB2 in articular
cartilage may represent a mechanism responsible for the decline in adult
cartilage stem cell populations. 2. Rifai N, Bachorik PS, Albers JJ: Lipids, lipoproteins and apolipoproteins. Tietz Textbook of Clinical Chemistry Philadelphia: W.B. Saunders Company:
Burtis CA, Ashwood ER , 3 1999, 809-861. 2. Rifai N, Bachorik PS, Albers JJ: Lipids, lipoproteins and apolipoproteins. Tietz Textbook of Clinical Chemistry Philadelphia: W.B. Saunders Company:
Burtis CA, Ashwood ER , 3 1999, 809-861. Unfolded protein response mediator, the IRE1a-XBP1 pathway is
involved in osteoblast differentiation Takahide Tohmonda1,2*, Kazuhiro Chiba2, Yoshiaki Toyama2,
Keisuke Horiuchi1,2 1Department of Anti-aging Orthopedic Research, School of Medicine, Keio
University, Tokyo, Japan; 2Department of Orthopedic Surgery, School of
Medicine, Keio University, Tokyo, Japan
h
h
h
l 1Department of Anti-aging Orthopedic Research, School of Medicine, Keio
University, Tokyo, Japan; 2Department of Orthopedic Surgery, School of
Medicine, Keio University, Tokyo, Japan P69
Age features of metabolic syndrome and cardiovascular disorders in
gout
Surayo Shukurova*, Khisrav Toirov, Nabijon Hamidov, Dilfuza Jonnazarova
AvicennaTajik State Medical University, Tajikistan
Arthritis Research & Therapy 2012, 14(Suppl 1):P69
l
d
h d
d
ddl References 3. Friedewald WT, Levy RS, Fredrickson DS: Estimation of the concentration
of low-density lipoprotein cholesterol in plasma, without use of the
preparative ultracentrifuge. Clin Chem 1972, 18:499-502. 3. Friedewald WT, Levy RS, Fredrickson DS: Estimation of the concentration
of low-density lipoprotein cholesterol in plasma, without use of the
preparative ultracentrifuge. Clin Chem 1972, 18:499-502. 1. Dowthwaite GP, Bishop JC, Redman SN, Khan IM, Rooney P, Evans DJ,
Haughton L, Bayram Z, Boyer S, Thomson B, et al: The surface of articular
cartilage contains a progenitor cell population. J Cell Sci 2004, 117(Pt
6):889-897. 2. Taniguchi N, Carames B, Ronfani L, Ulmer U, Komiya S, Bianchi ME, Lotz M:
Aging-related loss of the chromatin protein HMGB2 in articular cartilage
is linked to reduced cellularity and osteoarthritis. Proc Natl Acad Sci USA
2009, 106(4):1181-1186. Resistance to morphine analgesia and its underlying mechanisms in an
experimental mouse model of fibromyalgia
* Purpose: The aim of the present study was to determine the brain areas
associated with fibromyalgia, and whether pretreatment regional cerebral
blood flow (rCBF) can predict response to gabapentin treatment. Hitoshi Uchida , Michiko Nishiyori, Hiroshi Ueda
Division of Molecular Pharmacology and Neuroscience, Nagasaki University
Graduate School of Biomedical Sciences, Nagasaki 852-8521, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P71 Methods: A total of 29 women with fibromyalgia and 10 healthy women
without pain matched for age were finally enrolled in the study. Technetium-99 m ethyl cysteinate dimer single photon emission
computed tomography (99 mTc-ECD SPECT) was performed in the
fibromyalgia patients and controls. A voxel-by-voxel group analysis was
performed using SPM2. After treatment with gabapentin, 16 patients
were considered “responders”, with decrease in pain of greater than 50%
as evaluated by visual analogue scale (VAS). The remaining 13 patients
were considered “poor responders”. Fibromyalgia (FM) is a common condition with generalized or widespread
allodynia that affects at least 2% of the US, European and Japanese
populations. Although the etiology of this disease remains poorly
understood, physical and psychological stressors have been assumed to play
a role in the development of FM. Previously, we have established an
experimental mouse model of FM pain, using intermittent cold stress (ICS)
exposure. This model was found to produce mechanical allodynia and
thermal hyperalgesia in a female-predominant manner, as often observed in
FM patients. In contrast, exposure to constant cold stress produced a
transient allodynia. Importantly, we found that anticonvulsant agent
gabapentin, especially when injected intracerebroventricularly, exerts
powerful anti-allodynic and anti-hyperalgesic effects in the ICS-exposed
mice. In this study, we found that ICS model mice show morphine
resistance, as often observed in FM patients. To be concrete, systemic or
intracerebroventricular, but not intrathecal or intraplantar, injection of Results: Compared to control subjects, we observed rCBF abnormalities in
fibromyalgia
including
hypoperfusion
in
the
left
culmen
and
hyperperfusion in the right precentral gyrus, right posterior cingulate, right
superior occipital gyrus, right cuneus, left inferior parietal lobule, right
middle temporal gyrus, left postcentral gyrus, and left superior parietal
lobule (Table 1, Figure 1). References 1. Nishiyori M, Ueda H: Prolonged gabapentin analgesia in an experimental
mouse model of fibromyalgia. Mol Pain 2008, 4:52. 1. Nishiyori M, Ueda H: Prolonged gabapentin analgesia in an experimental
mouse model of fibromyalgia. Mol Pain 2008, 4:52. 1. Nishiyori M, Ueda H: Prolonged gabapentin analgesia in an experimental
mouse model of fibromyalgia. Mol Pain 2008, 4:52. 2. Nishiyori M, Nagai J, Nakazawa T, Ueda H: Absence of morphine analgesia
and its underlying descending serotonergic activation in an experimental
mouse model of fibromyalgia. Neurosci Lett 2010, 472:184-187. 2. Nishiyori M, Nagai J, Nakazawa T, Ueda H: Absence of morphine analgesia
and its underlying descending serotonergic activation in an experimental
mouse model of fibromyalgia. Neurosci Lett 2010, 472:184-187. 1. Takayanagi H, Kim S, Matsuo K, Suzuki H, Suzuki T, Sato K, Yokochi T, Oda H,
Nakamura K, Ida N, Wagner EF, Taniguchi T: RANKL maintains bone
homeostasis through c-Fos-dependent induction of interferon-beta. Nature 2002, 416:744-9. 1. Takayanagi H, Kim S, Matsuo K, Suzuki H, Suzuki T, Sato K, Yokochi T, Oda H,
Nakamura K, Ida N, Wagner EF, Taniguchi T: RANKL maintains bone
homeostasis through c-Fos-dependent induction of interferon-beta. Nature 2002, 416:744-9. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 morphine caused no significant analgesia in the ICS-exposed mice. In
addition, we found that intracerebroventricularly administrated morphine
increases the 5-hydroxytryptamine turnover ratio in the dorsal half of the
spinal cord of control mice, but not in the ICS-exposed mice. These findings
indicate that ICS model well reflects pathological and pharmacotherapeutic
features of FM pain, and the loss of descending serotonergic activation
seems to be a crucial mechanism underlying the absence of morphine-
induced analgesia in the ICS model. silenced MEFs were defective in BMP2-induced osteoblast differentiation,
indicating that the IRE1a-XBP1 pathway is essential for the maturation of
osteoblasts. Furthermore, we found that UPR induces transcription of Osterix
(a transcription factor indispensable for bone formation) via the IRE1a-XBP1
pathway, and that XBP1 directly binds to the promoter region of the Osterix
gene and functions as a transcription factor. Taken together, the present
study indicates that the UPR induced during osteoblast differentiation
stimulates Osterix transcription through the IRE1a-XBP1 pathway. References Conclusions: The present study shows that the IRE1a-XBP1 pathway is a
critical component of osteoblast differentiation. Since the IRE1a-XBP1 is
also involved in the production of a potent regulator for osteoclast
differentiation, interferon beta [1,2], the IRE1a-XBP1 pathway may be an
attractive molecular target in modulating the equilibrium between bone
formation and bone resorption under pathological conditions. References sults are listed by clusters.value, Z score, Talairach coordinates of peak voxel, and anatomic localization are provided for each cluster P72 2. Smith JA, Turner MJ, DeLay ML, Klenk EI, Sowders DP, Colbert RA:
Endoplasmic reticulum stress and the unfolded protein response are
linked to synergistic IFN-beta induction via X-box binding protein 1. Eur
J Immunol 2008, 38:1194-203. 2. Smith JA, Turner MJ, DeLay ML, Klenk EI, Sowders DP, Colbert RA:
Endoplasmic reticulum stress and the unfolded protein response are
linked to synergistic IFN-beta induction via X-box binding protein 1. Eur
J Immunol 2008, 38:1194-203. Chie Usui1*, Kotaro Hatta1, Nagafumi Doi2, Atsushi Nakanishi3,
Hiroyuki Nakamura4, Kusuki Nishioka5 Chie Usui1*, Kotaro Hatta1, Nagafumi Doi2, Atsushi Nakanishi3,
Hiroyuki Nakamura4, Kusuki Nishioka5 1Department of Psychiatry, Juntendo University School of Medicine, 2-1-1
Hongo, Bunkyo-Ku, Tokyo 113-8421, Japan; 2Ibaraki Prefectural Tomobe
Hospital, 654 asahi-cho, kasama-city, Ibaraki 309-1717, Japan; 3Department of
Radiology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-Ku,
Tokyo 113-8421, Japan; 4Department of Environmental and Preventive
Medicine, Graduate School of Medical Science, Kanazawa University, kakuma-
cho, Kanazawa-city, Kanazawa 920-1192, Japan; 5Institute of Innovative
Medical Science and Education, Tokyo Medical University, 6-1-1 Shinjyuku,
Shinjyuku-ku, Tokyo 160-8402, Japan 3. Murakami T, Saito A, Hino S, Kondo S, Kanemoto S, Chihara K, Sekiya H,
Tsumagari K, Ochiai K, Yoshinaga K, Saitoh M, Nishimura R, Yoneda T,
Kou I, Furuichi T, Ikegawa S, Ikawa M, Okabe M, Wanaka A, Imaizumi K:
Signalling mediated by the endoplasmic reticulum stress transducer
OASIS is involved in bone formation. Nat Cell Biol 2009, 11:1205-11. 3. Murakami T, Saito A, Hino S, Kondo S, Kanemoto S, Chihara K, Sekiya H,
Tsumagari K, Ochiai K, Yoshinaga K, Saitoh M, Nishimura R, Yoneda T,
Kou I, Furuichi T, Ikegawa S, Ikawa M, Okabe M, Wanaka A, Imaizumi K:
Signalling mediated by the endoplasmic reticulum stress transducer
OASIS is involved in bone formation. Nat Cell Biol 2009, 11:1205-11. P69
Age features of metabolic syndrome and cardiovascular disorders in
gout
h k
*
h
b
d
lf Most importantly, Ire-/- MEFs and Xbp1- Frequency of revealing of signs metabolic syndrome at gout patients depending on age, n (%) Table 2(abstract P69) Frequency of revealing of signs metabolic syndrome at gout patients depending on age, n (%)
Sign
Age groups
<50 y (n = 26)
50-60 y (n = 26)
>60 y (n = 24)
CW > 102 cm
22 (84.6%)
20 (76.9%)
6 (25%)
SBP > 140 mm Hg and/or DBP > 90 mm Hg
20 (76.9%)
14 (53.8%)
20 (83.3%)
TG ≥120 mg/dl
8 (30.8%)
10 (38.45%)
4 (16.7%)
Glucose ≥110 mg/dl
14 (53.85%)
14 (53.85%)
4 (16.7%)
HDL-cholesterol < 50 mg/dl
14 (53.85%)
24 (92.3%)
20 (83.3%) Table 2(abstract P69) Frequency of revealing of signs metabolic syndrome at gout patients depe Page 50 of 54 Page 50 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Results are listed by clusters.value, Z score, Talairach coordinates of peak voxel, and anatomic localization are provided for each cluster. P71 Arthritis Research & Therapy 2012, 14(Suppl 1):P72 Arthritis Research & Therapy 2012, 14(Suppl 1):P72 P71
Resistance to morphine analgesia and its underlying mechanisms in an
experimental mouse model of fibromyalgia
Hitoshi Uchida*, Michiko Nishiyori, Hiroshi Ueda
Division of Molecular Pharmacology and Neuroscience, Nagasaki University
Graduate School of Biomedical Sciences, Nagasaki 852-8521, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P71 Resistance to morphine analgesia and its underlying mechanisms in an
experimental mouse model of fibromyalgia
* Compared to responders, poor responders
exhibited hyperperfusion in the right middle temporal gyrus, left middle
frontal gyrus, left superior frontal gyrus, right postcentral gyrus, right
precuneus, right cingulate, left middle occipital gyrus, and left declive Table 1(abstract P72) Regions of significant hyperperfusion and hypoperfusion in the FM group
Z score
x(mm)
y(mm)
z(mm)
Localisation
Hyperperfusion
134
4.55
66
-10
30
R Precentral Gyrus
262
4.16
2
-62
14
R Posterior Cingulate
824
3.98
36
-82
32
R Superior Occipital Gyrus
429
3.95
18
-96
-6
R Cuneus
220
3.57
50
-38
52
L Inferior Parietal Lobule
55
3.54
52
-46
6
R Middle Temporal Gyrus
113
3.52
-30
-42
68
L Postcentral Gyrus
3.74
-14
-74
56
L Superior Parietal Lobule
709
4.66
-2
56
-22
L Superior Frontal Gyrus
Hypoperfusion
1111
4.38
-12
-32
-18
L Culmen
Results are listed by clusters.value, Z score, Talairach coordinates of peak voxel, and anatomic localization are provided for each cluster. Table 1(abstract P72) Regions of significant hyperperfusion and hypoperfusion in the FM group Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Page 51 of 54 Figure 1(abstract P72) Comparison of rCBF between patients with FM and age-matched healthy controls. Maximum intensity projections of SPM2
results from comparison of rCBF between patients with FM and age-matched healthy controls. a, b The FM patient group exhibited significant
hypoperfusion in the left culmen. c, d The FM patient group exhibited significant hyperperfusion in the right precentral gyrus, right posterior cingulate,
right superior occipital gyrus, right cuneus, left inferior parietal lobule, right middle temporal gyrus, left postcentral gyrus, and left superior parietal lobule. Height threshold is < 0.001, corrected for multiple comparison. Figure 1(abstract P72) Comparison of rCBF between patients with FM and age-matched healthy controls. Maximum intensity projections of SPM2
results from comparison of rCBF between patients with FM and age-matched healthy controls. a, b The FM patient group exhibited significant
hypoperfusion in the left culmen. c, d The FM patient group exhibited significant hyperperfusion in the right precentral gyrus, right posterior cingulate,
right superior occipital gyrus, right cuneus, left inferior parietal lobule, right middle temporal gyrus, left postcentral gyrus, and left superior parietal lobule. Height threshold is < 0.001, corrected for multiple comparison. P75 Results: Many genes were regulated within 6 hours of OA surgery
(compared to sham surgery) including Adamts5, Mmp3, IL1b, Ccl2, activin
and TNF-stimulated gene 6 (Tsg6). Mmp13 was not regulated at this early
time point. Of the 47 genes studied, all gene responses were strongly
suppressed if the joint was immobilised (by prolonged anaesthesia). Joint
immobilisation by sciatic neurectomy also suppressed a number of genes
(approx. 50%) including Adamts5, and protected the joints from cartilage
degradation at 12 weeks. The symptoms of rheumatoid arthritis (RA) are based on the many
processes; chronic inflammation, overgrowth of synovial cells, bone and
joint destruction and fibrosis. To clarify the mechanism of outgrowth of
synovial cells, we carried out immunoscreening using anti-rheumatoid
synovial cell antibody, and cloned ‘Synoviolin’. Synoviolin, a mammalian
homolog of Hrd1p/Der3p, is endoplasmic reticulum (ER)-resident E3
ubiquitin ligases with a RING motif, and is involved in ER-associated
degradation (ERAD). Synoviolin is highly expressed in synoviocytes of
patients with RA. Overexpression of synoviolin in transgenic mice leads to
advanced arthropathy caused by reduced apoptosis of synoviocytes. We postulate that the hyperactivation of the ERAD pathway by
overexpression of synoviolin results in prevention of ER-stress-induced
apoptosis leading to synovial hyperplasia. Indeed, synoviolin+/- knockout
mice showed resistance to the development of collagen-induced arthritis
owing to enhanced apoptosis of synovial cells. In addition, Synoviolin
ubiquitinates and sequesters the tumor suppressor p53 in the cytoplasm,
thereby negatively regulating its biological functions in transcription, cell-
cycle regulation and apoptosis by targeting it for proteasomal degradation. Therefore Synoviolin regulates, not only apoptosis in response to ER stress,
but also a p53-dependent apoptotic pathway. These studies indicate that
Synoviolin is one of the causative factors of arthropathy. Further analysis
using gene targeting approaches showed that in addition to its role in RA,
Synoviolin is essential for embryogenesis. Synoviolin deficient (syno-/-) mice
exhibited severe anemia caused by enhancement of apoptosis in fetal liver,
and the results suggested that the liver is sensitive organ for Synoviolin. Conclusion: Pathogenic protease expression occurs rapidly upon induction
of OA in mice (within 6 h) and is highly mechanosensitive. Suppression of
Adamts5 also occurs following sciatic neurectomy in which the joint is
immobilised but the mice are able to bear weight (they walk with a
‘splinted’ knee). Reference 1. Yamanishi S, Iizumi T, Watanabe E, et al: Implications for induction of
autoimmunity via activation of B-1 cells by Helicobacter pylori urease. Infect Immun 2006, 74(1):248-56. 1. Yamanishi S, Iizumi T, Watanabe E, et al: Implications for induction of
autoimmunity via activation of B-1 cells by Helicobacter pylori urease. Infect Immun 2006, 74(1):248-56. Materials and methods: Destabilisation of the medial meniscus or sham
surgery was performed in 10 week old male mice. Joints were immobilised
either by prolonged anaesthesia (for a max of 4 h to examine gene
expression changes) or by sciatic neurectomy (for 4-6 h or for 12 weeks). mRNA was extracted from whole joints at 4-6 h following induction of OA. A microarray was performed and 47 genes validated by RT-PCR. Joints were
examined histologically after 12 weeks forcartilage damage. P75
Importance of E3 ubiquitin ligase Synoviolin in fibrogenesis
Naoko Yagishita1, Daisuke Hasegawa1, Satoko Aratani1,2, Yoshihisa Yamano1,
Toshihiro Nakajima1,2,3*
1Institute of Medical Science, St.Marianna University School of Medicine,
Kawasaki, Kanagawa 216-8512 Japan; 2Institute of Medical Science, Tokyo
Medical University, Shinjuku-ku, Tokyo 160-8402 Japan; 3Bayside Misato
Medical Center, Kochi 781-0112 Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P75 Resistance to morphine analgesia and its underlying mechanisms in an
experimental mouse model of fibromyalgia
* Table 2(abstract P72) Regions of significant hyperperfusion in the poor responder group compared to the responder
group
Z score
x(mm)
y(mm)
z(mm)
Localisation
Hyperperfusion
1260
4.08
42
-62
16
R Middle Temporal Gyrus
95
3.88
-46
6
50
L Middle Frontal Gyrus
95
3.88
-20
38
52
L Superior Frontal Gyrus
69
3.67
56
-12
56
R Postcentral Gyrus
578
3.67
14
-76
28
R Preuneus
59
3.58
4
20
36
R Cingulate
70
3.54
-20
-80
4
L Middle Occipital Lobule
77
3.51
-20
-80
-26
L Declive
Results are listed by clusters. value, Z score, Talairach coordinates of peak voxel, and anatomic localization are provided for each cluster. attentional areas in fibromyalgia patients. Furthermore, hyperperfusion in
these areas was strongly predictive of poor response to gabapentin. Reference 1. Usui C, Hatta K, Doi N, Nakanishi A, Nakamura H, Nishioka K, Arai H: Brain
perfusion in fibromyalgia patients and its differences between responders
and poor responders to gabapentin. Arthritis Res Ther 2010, 12:R64. Conclusion: The present study revealed brain regions with significant
hyperperfusion associated with the default-mode network, in addition to
abnormalities in the sensory dimension of pain processing and affective- Page 52 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 Methods: 292 patients with rheumatic diseases were recruited through
outpatient rheumatology clinics between 2005-2008. The study was
approved by the Second Hospital of Shanxi Medical University Ethics
Committees, and all participating patients signed an informed consent form. The description of this study is 3-fold: to evaluate the relationship between
Hp and rheumatic diseases, to assess the relationship between Hp and
rheumatoid arthritis (RA), to explore the relationship between Hp and
ankylosing spondylitis (AS). Pathogenic protease expression in murine OA is critically dependent
upon mechanical joint loading
* Committees, and all participating patients signed an informed consent form. The description of this study is 3-fold: to evaluate the relationship between
Hp and rheumatic diseases, to assess the relationship between Hp and
rheumatoid arthritis (RA), to explore the relationship between Hp and
ankylosing spondylitis (AS). Annika Burleigh*, Anastasios Chanalaris, Jeremy Saklatvala, Tonia Vincent
Department of cell signalling, Kennedy Institute of Rheumatology, Oxford
University, London W6 8LH, UK
Arthritis Research & Therapy 2012, 14(Suppl 1):P73 Results: Patients of rheumatic diseases were significantly more likely to be
Hp infection than health control (89 vs 42%, P < 0.01). The study revealed
that 88% of RA patients and 90% AS patients suffer from Hp infection. RA
patients carried a diagnosis of Hp, a higher prevalence of the value of CRP
was associated with the DAS28(Disease Activity Scor-28) (r = 0.287,P =
0.034). AS patients carried a diagnosis of Hp, a higher prevalence of the
value of MMP-3(matrix metalloproteinase-3, MMP-3) was associated with the
BASDI(Bath AS Disease Activity Index) (r = 0.435,P = 0.009). Background: Once considered a passive disease of ‘wear and tear’ of the
joint, osteoarthritis (OA) is now known to be driven by the expression and
activation of specific proteases that degrade the extracellular matrix of
articular cartilage. Such proteases include aggrecanases, principally
adistintegrin and metalloproteinase (Adamts) 4 and 5, and collagenases
which are members of the matrix metalloproteinase (Mmp) family. In mice,
Adamts5 and Mmp13 are considered to be the critical proteases in disease,
as mice in which these proteases have been knocked out are protected
from developing OA [1,2]. What drives these proteases in vivo is unknown,
but one possibility is that mechanical factors alone are sufficient to lead to
their expression and activation. Conclusions: Patients of RA and AS are associated with a high
prevalence of Hp infection rate. Hp infection may be play an important
role in RA and AS. Next steps: Further investigation with other rheumatic diseases are
planned. p
To test this hypothesis we investigated the effects of joint immobilisation
on protease expression and the course of disease in mice with surgically
induced OA. P73 Pathogenic protease expression in murine OA is critically dependent
upon mechanical joint loading
Annika Burleigh*, Anastasios Chanalaris, Jeremy Saklatvala, Tonia Vincent
Department of cell signalling, Kennedy Institute of Rheumatology, Oxford
University, London W6 8LH, UK
Arthritis Research & Therapy 2012, 14(Suppl 1):P73 P75 This suggests that dynamic flexion of the destabilised knee
joint is important for induction of proteases and subsequent disease.The
pathway by which joint cells sense and respond to these mechanical signals
could represent a novel target for disease intervention. References 1. Glasson SS, et al: Deletion of active ADAMTS5 prevents cartilage
degradation in a murine model of osteoarthritis. Nature 2005,
434(7033):644-8. Glasson SS, et al: Deletion of active ADAMTS5 prevents cartilage 2. Little CB, et al: Matrix metalloproteinase 13-deficient mice are resistant to
osteoarthritic cartilage erosion but not chondrocyte hypertrophy or
osteophyte development. Arthritis Rheum 2009, 60(12):3723-33. P77 AJAK inhibitor, tofacitinibreduces IL-6 and matrix metalloproteinase-3
productionin rheumatoid arthritis with suppressed cartilage destruction
Kunihiro Yamaoka1*, Satoshi Kubo1, Koshiro Sonomoto1, Keisuke Maeshima2,
Yoshiya Tanaka1 P76
Human retrovirus promotes the plasticity of regulatory T cells into
T helper type 1-like cells through the T-bet transcriptional activation
in neuroinflammatory disease
Yoshihisa Yamano
Department of Rare Diseases Research, Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P76 y
1The First Department of Internal Medicine, University of Occupational and
Environmental Health, Japan, Kitakyushu, Japan, 807-8555; 2Department of
Internal Medicine I, Faculty of Medicine, Oita University, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P77 y
1The First Department of Internal Medicine, University of Occupational and
Environmental Health, Japan, Kitakyushu, Japan, 807-8555; 2Department of
Internal Medicine I, Faculty of Medicine, Oita University, Japan
Arthritis Research & Therapy 2012, 14(Suppl 1):P77 Background: Tofacitinib, targeting Janus kiase (JAK) has gained attention
as anorally available new disease modifying anti-rheumatic drug with
high clinical efficacy against rheumatoid arthritis (RA). While the clinical
trial has progressed and the wide usage of tofacitinib is conceivable in
the near future, the precise mechanism of action in RA patients remains
to be solved. Recently, it has become increasingly clear that some committed effecter and
regulatory T (Treg) cells are not stable, and the plasticity of these T-cells may
be related to the pathogenesis of autoimmunity and inflammatory diseases
[1]. However, the precise mechanisms that allow for T cell plasticity have not
yet been clearly understood. Human T-lymphotropic virus type 1 (HTLV-1) is
a retrovirus that is associated with multiorgan inflammatorydisorders such
as HTLV-1- associated myelopathy (HAM/TSP), HTLV-1- associated
arthropathy (HAAP), uveitis, Sjögren syndrome, and polymyositis [2-5]. HTLV-
1-infected T cells may contribute to development of these disorders, since
the number of HTLV-1-infected T cells circulating in the peripheral blood is
higher in patients [6]. HTLV-1 mainly infects CD4+ T helper (Th) cells that
play central roles in adaptive immune responses. Based on their functions,
patterns of cytokine secretion, and expression of specific transcription
factors and chemokine receptors, Th cells differentiated from naïve CD4+
T-cells are classified into 4 major lineages: Th1, Th2, Th17, and T regulatory
(Treg) cells. P77 We recently demonstrated that CD4+CD25+CCR4+ T cells, which
mainly include suppressive T-cell subsets such as Treg and Th2 under
healthy conditions, are the predominant viral reservoir of HTLV-1 in both
adult T-cell leukemia/lymphoma (ATL) and HAM/TSP [7]. Interestingly, T-cells
of this subset become Th1-like cells with overproduction of IFN-g in HAM/
TSP, suggesting that HTLV-1 may intracellularly induce Tcell plasticity from
Treg to IFN-g+ T cells [7].In this study, using human T-cell line and HTLV-1
infected CD4+CD25+CCR4+ T-cells of HAM/TSP patients, the virus-encoded
transactivating HTLV-1 Tax protein was demonstrated to induce the IFN-g
production through the expression of T-box 21(Tbx21)/T-bet, a transcription
factor that is known to direct the differentiation of naive CD4+ cells into IFN-
g-expressing Th1 cell. HTLV-1 Tax was also demonstrated to enhance
promoter activity of Tbx21/T-bet cooperatively with transcription factor
Specificity Protein 1 (Sp1). Furthermore, transfer of HTLV-1 tax gene in
CD4+CD25+CCR4+ T-cells using a lentiviral vector resulted in the loss of
regulatory function of these T cells. This is the first report to our knowledge
demonstrating the role of a specific viral product (HTLV-1 Tax) on the
expression of genes associated with T-cell differentiation resulting in
plasticity of Treg cells into Th1-like cells. These results suggest that HTLV-1
infection-induced immune dysregulation may play an important role in the
development and pathogenesis of HTLV-associated immunological
diseasesthrough its interference in the equilibrium maintained among host
immune responses. Materials and methods: Fifteen RA patients enrolled in tofacitinib clinical
trial were randomized to 1, 3, 5 or 10 mg BID for 12 weeks. Serumwas
collected at 0 and 12 weeks for further cytokine measurement by ELISA.To
analyze the effect at the local inflammatory site, synovium and cartilage
from a RA patient undergoing joint replacement was implanted to severe
combined
immunodeficiency
(SCID)
mice
(SCID-huRAg
mouse)
andtofacitinib was administered via osmotic mini-pump and serological and
histological investigation was performed. Results: Background of patients in clinical trial: mean age; 56.4 years, mean
disease duration; 95.1 months, methotrexate (MTX) and tofacitinib were
administered in all patients, median doses were 9.4 mg/week and 4.1 mg
BID, glucocorticoids were administered in 6 patients, median dose was 5.4
mg/day. Baseline characteristics of the disease activity; SDAI 30.0, DAS28
(ESR) 6.3, HAQ 1.1, CRP 21.0 mg/l, ESR 57.1 mm/h, MMP-3 259.3 ng/ml, RF
216.2 U/ml. P77 After 12 weeks treatment, disease activity decreased with
statistical difference (p < 0.05) as follows; SDAI13.8, DAS28(ESR) 4.0, HAQ 0.8,
CRP 8.1 mg/l, ESR 30.9 mm/h, MMP-3 149.9 ng/ml, RF 150.8 U/ml. Among
the multiple cytokines measured, IL-6 and IL-8 tended to decrease, from
52.2 pg/ml to 28.2 pg/ml (p < 0.05) and from 41.7 pg/ml to 29.5 pg/ml (not
significant), respectively. There was a statistically significant correlation
between reduction of IL-6 and reduction of MMP-3. In SCID-huRAg mouse, apparent invasion of RA-derived synoviuminto
cartilage was observed, whileadministration of tofacitinibmarkedly
suppressed invasion. In order to investigate the relevance with our
findings from the patients in the clinical trial, cytokines in SCID-huRAg
mouse serum was measured after administration of tofacitinib for 7 days. Interestingly, tofacitinib significantly decreased production of human IL-6
and IL-8 as well as human MMP-3 from 29.79 pg/ml to 2.89 pg/ml, 17.89
pg/ml to 4.22 pg/ml and 65.96 pg/ml to 33.13 pg/ml respectively. Conclusions: Tofacitinib improved disease activity and suppressed
cartilage destruction with decreased serum IL-6 and IL-8 in both, RA
patients and SCID-huRAg mouse in connection with reduced MMP-3. These results indicate that tofacitinib reduces inflammation by
suppressing IL-6 production and consequently inhibiting cartilage
destruction in the initial several months of administration. Helicobacter pylori infection in rheumatic diseases
Hongyan Wen*, Jing Luo, Junxia Li, Xiaofeng Li Department of Rheumatology, Shanxi Medical University, The Second Department of Rheumatology, Shanxi Medical University, The Second Hospital of Shanxi Medical University, 56 South Xinjian Road, Taiyuan, Shanxi
Province, China Arthritis Research & Therapy 2012, 14(Suppl 1):P74 Arthritis Research & Therapy 2012, 14(Suppl 1):P74 Arthritis Research & Therapy 2012, 14(Suppl 1):P74 y
Thus, this study aimed to explore the involvement of the Synoviolin in
fibrosis process of RA using mice model of liver fibrosis. In CCl4-induced
hepatic injury model, syno+/- mice are resistant to onset of liver fibrosis. The
number of activated HSCs was decreased in syno+/- mice, and some of these
cells showed apoptosis. Furthermore, collagen expression in HSCs was
upregulated by synoviolin overexpression, while synoviolin knockdown led
to reduced collagen expression. Moreover, in syno-/- MEFs, the amounts of
intracellular and secreted mature collagen were significantly decreased, and
procollagen was abnormally accumulated in the endoplasmic reticulum. In Background: Due to a number of factors, Helicobacter pylori (Hp)
infection is increasingly recognized as highly prevalent in many
populations and of increasing health concern. Hp infection has been
associated with digestive diseases and rheumatic diseases[1]. It remains
unclear whether all or part patients of rheumatic diseases should be
routinely screened for Hp infection. We have examined predictors of Hp
infection in rheumatic diseases so as to define who might benefit most
from screening. Page 53 of 54 Page 53 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 conclusion, Synoviolin is involved in not only overgrowth process of synovial
cells but also fibrosis process. References 1. Zhou X, et al: Instability of the transcription factor Foxp3 leads to the
generation of pathogenic memory T cells in vivo. Nat Immunol 2009,
10:1000-1007. 1. Zhou X, et al: Instability of the transcription factor Foxp3 leads to the
generation of pathogenic memory T cells in vivo. Nat Immunol 2009,
10:1000-1007. P78 P78
Regulation of macrophage-mediated chronic inflammation by JAK
inhibitors
Anna Yarilina1*, Kai Xu1, Chunhin Chan1, Lionel B Ivashkiv1,2
1Arthritis and Tissue Degeneration Program, Hospital for Special Surgery,
USA; 2Weill Medical College of Cornell University, New York, NY, 10021 USA
Arthritis Research & Therapy 2012, 14(Suppl 1):P78 Th17 is involved in the pathogenesis of Bechet’s disease via CCL20-
CCR6 axis Hidekata Yasuoka1*, Zhu Chen1,2, Tsutomu Takeuchi1, Masataka Kuwana1
1Department of Internal Medicine, Keio University School of Medicine, Tokyo,
160-8582, Japan; 2Department of Rheumatology and Immunology, Anhui
Provincial Hospital, Hefei, 230001, China
Arthritis Research & Therapy 2012, 14(Suppl 1):P79 Background: Behcet’s disease (BD) is an autoinflammatory disease with a
unique distribution characterized by uveitis, and mucosal and skin lesions,
which are characterized by the prominent infiltration of immune cells such
as lymphocytes and neutrophils. A novel helper T-cell subset Th17, IL-17-
producing helper T cells, has been appreciated [1]. IL-17 is involved in the
induction of a series of chemokines, growth factors, proteases, and
cytokines, and production of IL-17 results in induction of neutrophil
migration and chronic inflammation [2]. Based on these findings, we
hypothesized that Th17 is involved in the pathogenesis of BD. Results: Our study showed that the frequency of anti Ro, and anti nRNP
antibodies in patients with LN WHO class III, IV and V LN weresignificantly
higher compared with patients with class I and II LN. There is no
autoantibodies expression differences between class III, IV and clas V LN. The IFNg/IL4 ratio in patients with classIII and IV LN was significantly
higher than patients with class I,II and class V LN (2.30 ± 0.89 vs 0.94 ±
0.24 and 0.69 ± 0.30, p = 0.000), but the serum level of IL4 in patient
with WHO class III and IV was significantly lower than class V (77.72 ±
40.28 pg/ml vs 145.68 ± 71.21 pg/ml. p = 0.014). The result showed that
the activity of Th1 immune response tent to be higher in patient with
WHO class III and IV LN. The frequency of IFNG 112 allele were higher in
patients with SLE compared with healthy controls (55% vs 25%, p =
0.0471) and the risk to have LN class V in patients with IFNG 112 was 6
times higher compared with patients without these allele (OR 6.1, CI 95%
3.1- 12.4, p = 0.0427). y
Materials and methods: To examine a role of Th17 response in the
pathogenic process of BD, peripheral blood samples from 20 patients with
BD and 14 controls were used to evaluate phenotypic and functional
properties relevant to the Th17 response. Plasma IL-17 and CCL20 levels
were examined using ELISA. The association of autoantibodies expression, Th1/Th2 cytokines
balance and IFNG polymorphism with histological phenotype of
lupusnephritis Kusworini Handono1*, Atma Gunawan2, Singgih Wahono2, Rulli Rosandi2,
Wivina1, Natalina Mallo1, Handono Kalim2 g
g
As expected, both inhibitors abrogated TNF-induced STAT1 activation and
expression of genes encoding inflammatory chemokines (CXCL9, 10, 11 and
CCL5) and ISGs (IFIT1 and 2, IRF7). Interestingly, both compounds attenuated
a late wave of IL-1 induction and nuclear expression of NF-B subunits. Furthermore, ex vivo treatment with inhibitors decreased IL-1 and IL-6
expression in synovial MFs isolated from the patients with arthritis. Next, we
analyzed the effects of JAK inhibitors on TNF-induced osteoclastogenesis
and discovered that both compounds augmented nuclear levels of NFATc1
and cJun, followed by increased formation of TRAP positive multinuclear
cells. Lastly, we examined an in vivo effect of CP on innate immune
response in arthritis using K/BxN serum transfer arthritis model and found
that CP treatment significantly inhibited inflammation and joint swelling. Taken together, our data suggest that JAK inhibitors can affect inflammatory
responses in hMFs and thus, can target both acquired and innate immunity
in RA and other chronic inflammatory diseases. 1Clinical Pathology Department, Medical Faculty Brawijaya University/Dr
Saiful Anwar Hospital Malang, Indonesia; 2Internal Medicine Department,
Medical Faculty Brawijaya University/Dr Saiful Anwar Hospital Malang,
Indonesia Arthritis Research & Therapy 2012, 14(Suppl 1):P80 Background: Racial differences were observed in clinical, serologic and
histologic presentation of lupus nephritis (LN)[1]. It has been suggested
that Th1/Th2 cytokines balance and IFNG polymorphism play important
role in the development of different pathologic pattern of lupus nephritis
(LN )[2-4]. The objective of our study is to determine the association
between autoantibodies expression, Th1/Th2 cytokines balance and IFNG
polymorphisms with pathologic class of LN in Javanese patients. p y
p
p
g
p
Patients and methods: We studied 60 female patients with LN (ARA
criteria,1992), and 20 healthy individual as control. Histopathologic
classification was based on WHO criteria (1995). Anti ds-DNA, anti RO,
anti nRNP and anti Sm autoantibodies were assayed by ELISA.IFNg-IL-4
balance were used to assess Th1/Th2 cytokines balance, IFNg and IL4
serum levels assayed by ELISA. Microsatelitepolymorphisms within the
first intron of the IFNG gene on chromosome 12q24.1 was performed by
DNA sequencing. The association of histopathologic phenotype of LN
with Th1/Th2 balance (IFNg/IL4),and autoantibodies expression were
analysed by Chi-square and Student T test with p < 0.05 is significant. The IFNG allele difference between LN classes were analysed by Chi-
square. Th17 is involved in the pathogenesis of Bechet’s disease via CCL20-
CCR6 axis Conclusions: These results together suggest that Th17 are involved in
the pathogenesis of BD by migrating into the lesions of BD through the
CCL20-CCR6 axis. Cite abstracts in this supplement using the relevant abstract number,
e.g.: Handono et al.: The association of autoantibodies expression, Th1/
Th2 cytokines balance and IFNG polymorphism with histological
phenotype of lupusnephritis. Arthritis Research & Therapy 2012, 14(Suppl
1):P80 Cite abstracts in this supplement using the relevant abstract number,
e.g.: Handono et al.: The association of autoantibodies expression, Th1/
Th2 cytokines balance and IFNG polymorphism with histological
phenotype of lupusnephritis. Arthritis Research & Therapy 2012, 14(Suppl
1):P80 Th17 is involved in the pathogenesis of Bechet’s disease via CCL20-
CCR6 axis Expression levels of RORC mRNA in CD4+
T cells were examined by RT-PCR and CD4+ cells expressing IL-17, CCR6
was examined by flow cytometry. Evaluation of chemotaxis of CD4+ T cells
toward CCL20 was examined by migration assay using TransWell® double
chamber system. Conclusion: The results showed different underlying mechanism of
inflammation in different pathologic class of LN. y
Results: Plasma IL-17 was higher in active BD compared with healthy
controls (P < 0.05). Expression levels of RORC mRNA in peripheral blood
mononuclear cells by RT-PCR and proportion of CD4+ cells expressing
intracellular IL-17 were increased in patients with BD than in controls (P <
0.05 in both comparisons). Expression of chemokine receptor CCR6 was
detected in nearly all IL-17-expressing cells. The proportion of CD4+CCR6+
was higher in BD patients in remission compared those with active
disease (P < 0.05), suggesting that these cells are migrated to the lesions
at active disease phase. In addition, CD4+ T cells from BD patients had
enhanced migration capacity induced by CCL20, than did those from
controls. Finally, CCL20 level was higher in BD patients than in controls
(P < 0.05). 1. Korbet SM, Schwartz MM, Evans J, et al: Severe lupus nephritis: racial
differences in presentation and outcome. J Am Soc Nephrol 2007,
18:244-254. 2. Chan RW-Y, Lai FM-M, Li EK-M, et al: Imbalance of Th1/Th2 transcription
factors in patients with lupus nephritis. Rheumatol 2006, 45:951-957. 2. Chan RW-Y, Lai FM-M, Li EK-M, et al: Imbalance of Th1/Th2 transcription
factors in patients with lupus nephritis. Rheumatol 2006, 45:951-957. 3. Miyake K, Nakashima H, Inoue Y, et al: Genetically determined interferon-g
production influences the histological phenotype of lupus nephritis. Rheumatol 2002, 41:518-24. 3. Miyake K, Nakashima H, Inoue Y, et al: Genetically determined interferon-g
production influences the histological phenotype of lupus nephritis. Rheumatol 2002, 41:518-24. 4. Uhm WS, Na K, Song G-W, Jung SS, Lee T, Park MH, Yoo DH: Cytokine
balance in kidney tissue from lupus nephiritis patients. Rheumatol 2003,
42:935-938. 4. Uhm WS, Na K, Song G-W, Jung SS, Lee T, Park MH, Yoo DH: Cytokine
balance in kidney tissue from lupus nephiritis patients. Rheumatol 2003,
42:935-938. Conclusions: These results together suggest that Th17 are involved in
the pathogenesis of BD by migrating into the lesions of BD through the
CCL20-CCR6 axis. P78
Regulation of macrophage-mediated chronic inflammation by JAK
inhibitors 2. Osame M, et al: HTLV-I associated myelopathy, a new clinical entity. Lancet 1986, 1:1031-1032. Anna Yarilina1*, Kai Xu1, Chunhin Chan1, Lionel B Ivashkiv1,2
1Arthritis and Tissue Degeneration Program, Hospital for Special Surgery,
USA; 2Weill Medical College of Cornell University, New York, NY, 10021 USA
Arthritis Research & Therapy 2012, 14(Suppl 1):P78 3. Nishioka K, et al: Chronic inflammatory arthropathy associated with
HTLV-I. Lancet 1989, 1:441. 4. Mochizuki M, et al: Uveitis associated with human T-cell lymphotropic
virus type I. Am J Ophthalmol 1992, 114:123-129. 5. Nakagawa M, et al: HTLV-I-associated myelopathy: analysis of 213
patients based on clinical featuresand laboratory findings. J Neurovirol
1995, 1:50-61. Small molecule inhibitors of the Janus kinases (JAK) have been developed
as anti-inflammatory and immunosuppressive agents and are currently
subjects of clinical trials. Tofacitinib/CP-690,550 (more potent in inhibiting
JAK3 and JAK1) and Ruxolitinib/INCB-018424 (selective inhibitor of JAK1/2)
have demonstrated clinical efficacy in rheumatoid arthritis (RA), however,
the exact mechanisms that mediate the inhibitory effects of these
compounds are not known. 6. Nagai M, et al: Analysis of HTLV-I proviral load in 202 HAM/TSP patients
and 243 asymptomatic HTLV-I carriers: high proviral load strongly
predisposes to HAM/TSP. J Neurovirol 1998, 4:586-593. 7. Yamano Y, et al: Abnormally high levels of virus-infected IFN-gamma+
CCR4+ CD4+ CD25+ T cells in a retrovirus-associated neuroinflammatory
disorder. PLoS One 2009, 4:e6517. In this study, we examined the effects of CP-690,550 (CP) and INCB-018424
(INCB) on inflammatory responses in human macrophages (hMFs). In our Page 54 of 54 Page 54 of 54 Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 2.
Romagnani S: Human Th18 cells. Arthritis Res Ther 2008, 10:206. Arthritis Research & Therapy 2012, Volume 14 Suppl 1
http://arthritis-research.com/supplements/14/S1 study, we used long term exposure to TNF as a model of chronic
inflammation to investigate mechanisms regulating hMF activation and
functions, and have shown that TNF can activate an IFN-JAK-STAT-
dependent autocrine loop that regulates expression of pro-inflammatory
chemokines and interferon stimulated genes (ISGs), followed by an
increase of NFATc1, that regulates osteoclastogenesis. study, we used long term exposure to TNF as a model of chronic
inflammation to investigate mechanisms regulating hMF activation and
functions, and have shown that TNF can activate an IFN-JAK-STAT-
dependent autocrine loop that regulates expression of pro-inflammatory
chemokines and interferon stimulated genes (ISGs), followed by an
increase of NFATc1, that regulates osteoclastogenesis. 1.
Park H, Li Z, Yang XO, et al: A distinct lineage of CD4 T cells regulates
tissue inflammation by producing interleukin 17. Nat Immunol 2005,
6:1133-1141. The association of autoantibodies expression, Th1/Th2 cytokines
balance and IFNG polymorphism with histological phenotype of
lupusnephritis The risk of LN in patients with certain IFNG allele was calculated
using Odds Ratio. References 1. Park H, Li Z, Yang XO, et al: A distinct lineage of CD4 T cells regulates
tissue inflammation by producing interleukin 17. Nat Immunol 2005,
6:1133-1141. 1. Park H, Li Z, Yang XO, et al: A distinct lineage of CD4 T cells regulates
tissue inflammation by producing interleukin 17. Nat Immunol 2005,
6:1133-1141. 2. Romagnani S: Human Th18 cells. Arthritis Res Ther 2008, 10:206.
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La “subida de clíticos”: modalidad, prominencia y evidencialidad
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Cerrón-Palomino • Cuzco: la piedra donde se posó la lechuza
Lexis XXX.1 (2006): 83-115
La “subida de clíticos”: modalidad, prominencia
y evidencialidad
Fernando Bermúdez
Universidad de Estocolmo
1. Introducción
Una noción central (si no definitoria) de la lingüística cognitiva es que
la forma de una expresión lingüística refleja una organización cognitiva
particular, y que, por lo tanto, las diferencias formales reflejan diferencias semánticas. Cada elección, tanto léxica como gramatical, tiene
valor semántico y el valor de los elementos gramaticales reside, en gran
parte, en la estructura particular que ellos imponen en el contenido
conceptual. La investigación sobre el significado de una construcción
consiste por lo tanto en elucidar cómo la construcción en cuestión construye la escena conceptual que describe.
Esta articulación entre forma gramatical y significado se ha investigado en relación con un número de construcciones (Langacker 1995,
entre muchos otros); sin embargo, poco se ha dicho sobre el alcance semántico de la construcción conocida como subida de clíticos (clitic climbing). Con pocas excepciones, los estudios que tratan el tema —predominantemente dentro de la gramática generativa— investigan las condiciones que permiten (o impiden) la subida de los pronombres átonos,
pero no refieren diferencia semántica alguna entre las construcciones
con y sin subida de clíticos. El objetivo de este trabajo es esclarecer las
diferencias de significado que entraña la así llamada subida de clíticos
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Lexis XXX.1
en castellano, relacionándolas con la estructura que la diferente posición de los pronombres átonos impone sobre la escena conceptual.
El artículo se organiza de la siguiente manera: en la sección 2 se
presenta el fenómeno de la subida de clíticos y se traza un panorama
de los estudios que han tratado el tema. En la sección 3 se presentan
datos que apoyan la hipótesis de que las diferencias en la posición de
los clíticos en las perífrasis verbales entrañan diferencias en la configuración conceptual y por lo tanto significados diferentes. En la sección 4
se resumen los resultados y se presentan las conclusiones.
2. La posición de los pronombres clíticos en castellano
Como es sabido, en las perífrasis verbales, el castellano permite que los
pronombres átonos, semánticamente relacionados con el infinitivo o
gerundio, se unan al verbo auxiliar:
(1)
(2)
a. Pedro debe leer el libro.
b. Pedro debe leerlo.
c. Pedro lo debe leer.
a. Pedro sigue leyendo el libro.
b. Pedro sigue leyéndolo.
c. Pedro lo sigue leyendo.
El fenómeno ha sido estudiado extensamente dentro de la gramática
generativa, donde es conocido como subida de clíticos (clitic climbing, en
adelante SC), ya que el clítico parece haber “subido” desde su posición
“lógica” de complemento del infinitivo o gerundio hasta la posición del
auxiliar, que se encuentra “más arriba” en la estructura de la oración.
En un primer acercamiento podemos decir que los verbos que entran
en las configuraciones de SC pueden en principio clasificarse como sigue:
Verbos modales: {deber, poder, querer, saber, tener que, tratar de, intentar,
atreverse a, pensar, ir a} + INFINITIVO
Esta es la forma de futuro más común del castellano. Para una interpretación modal
del futuro, véase Bermúdez (2005) entre otros. Existen otros verbos lexicales (es decir, en
principio no “funcionales”) que permiten subida de clíticos, que sin embargo son vagamente modales/aspectuales, como “aprender a”.
(3)
(4)
Bermúdez • La “subida de clíticos”
85
Quiere hacerlo / Lo quiere hacer
Va a hacerlo / Lo va a hacer
Verbos aspectuales: {soler, empezar a, terminar de, acabar de, dejar de,
estar por, volver a, llegar a} + INFINITIVO, { estar, seguir, ir, venir, llevar}
+ GERUNDIO.
(5)
(6)
Terminó de hacerlo / Lo terminó de hacer
Está haciéndolo / lo está haciendo
Esta subida de clíticos puede en principio ser ilimitada, dependiendo de la complejidad de la perífrasis verbal, como muestran los ejemplos de (7):
(7)
a. Pedro querría poder volver a empezar a leerla
b. Pedro querría poder volver a empezarla a leer
c. Pedro querría poder volverla a empezar a leer
d. Pedro querría poderla volver a empezar a leer
e. Pedro la querría poder volver a empezar a leer
Varios intentos se han hecho para determinar qué tipo de contexto
permite o impide la SC (Bok-Bennema y Kampers-Manne 1984; Cinque 1999, 2000; Kayne 1989; Luján 1993; Rizzi 1982; Roberts 1987,
Terzi 1996). Dado que estos estudios se inscriben dentro de la gramática generativa, el énfasis está puesto en la estructura sintáctica de los
enunciados y en las restricciones al “movimiento” de los clíticos, ya que,
según esta teoría, los clíticos no deberían poder salir de la cláusula en
la que se generan o, dicho de otra manera, no podrían cruzar ciertas
“barreras” relacionadas con los límites oracionales. Dos tipos de estructura se plantean en los trabajos mencionados: una propuesta es que
las perífrasis verbales que permiten la SC son estructuras biclausales
que, por alguna razón, tienen una estructura “reducida” que permite
el movimiento de los clíticos hacia afuera de la cláusula subordinada
En Bermúdez (2005) hemos argumentado a favor de una interpretación de los
tiempos verbales como marcadores modales y aspectuales/evidenciales. De esta manera,
los verbos que permiten la subida de clíticos podrían verse como una extensión perifrástica de los tiempos verbales, con un tipo análogo de función modal/aspectual/evidencial
(en muchos casos más sutil o elaborada).
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Lexis XXX.1
(Bok-Bennema y Kampers-Manne 1984; Kayne 1989; Luján 1993, Rizzi 1982; Roberts 1987). De hecho, estas propuestas pueden dividirse en dos tipos: unas afirman que la estructura biclausal se mantiene,
pero que la cláusula subordinada es, de alguna manera, “defectiva” (no
posee estructura temporal) y por lo tanto “deja pasar” los clíticos hacia afuera de la cláusula (Lujan 1993). Otras afirman que la estructura
originalmente biclausal se reestructura y se convierte en una estructura
monoclausal, por lo que la subida de clíticos se convierte en un movimiento “permitido” dentro de esta teoría (Rizzi 1982). De esta manera
se explicaría que la cláusula subordinada deba ir en infinitivo o gerundio (formas verbales sin especificación para la categoría tiempo) y que
los clíticos puedan moverse “tan lejos”.
El segundo tipo de propuesta implica un análisis monoclausal de los
verbos modales y aspectuales (Cinque 1999, 2000). La idea es que los
verbos que permiten la SC son elementos funcionales que se insertan
en alguna de las proyecciones funcionales de la oración, explicando
entonces el movimiento de los clíticos ya que éstos no estarían cruzando
ninguna “barrera oracional”. Según esta posición, los verbos que permiten SC son vistos como verdaderos “verbos auxiliares”, que forman
una estructura oracional como la de la Fig. 1.
M(ood)P(hrase)
M
Asp(ect)P
Asp
V(erb)P
Quiero
terminar de
Quiero
terminarte de decir esto
Te quiero terminar de
decirte esto
decir esto
Fig. 1 Proyecciones funcionales y subida de clíticos según Cinque (1999, 2000)
Bermúdez • La “subida de clíticos”
87
Cinque (1999) afirma, además, que existe un ordenamiento universal de proyecciones funcionales y que los verbos de las perífrasis verbales se insertan en el núcleo de estas proyecciones. El orden propuesto
por Cinque es el siguiente:
MoodPspeech act >MoodPevaluative >MoodPevidential >ModPepistemic >TP(Past)
> TP(Future) >MoodPirrealis >ModPalethic >AspPhabitual >AspPrepetitive(I)
>AspPfrequentative(I) >ModPvolitional >AspPcelerative(I) >TP(Anterior) >
AspPterminative >AspPcontinuative >AspPretrospective >AspPproximative >AspPdurative
>AspPgeneric/ progressive >AspPprospective >ModPobligation >ModPpermission/ability
>AspPCompletive >VoiceP >AspPcelerative(II) >AspPrepetitive(II) >AspPfrequentative(II)
De esta manera explica no sólo la SC sino también el hecho de
que (8) sea gramatical mientras (9) no lo es, dado que la modalidad
epistémica está “más arriba” que la volitiva en la jerarquía de proyecciones funcionales.
(8)
(9)
María lo debe querer hacer
* María lo quiere deber hacer
También se explica que la interpretación de poder en (10) sea de
habilidad/permiso, mientras que en (11) es epistémico:
(10)
(11)
María querría poder ir
María podría querer ir
Dejando por un momento esta discusión de lado (volveremos a ella
desde una posición teórica diferente), podemos decir que la mayoría
de los investigadores describe las condiciones en las que la subida es
(im)posible, pero no asocia explícitamente la diferencia estructural causada por la SC con alguna diferencia semántica. Cinque (2000) es claro
en este sentido, y habla de “la total sinonimia de los dos usos”. Otros
señalan a lo sumo una diferencia estilística entre las construcciones con
y sin SC. Explícitamente afirma Berta (2000):
Las dos soluciones —la SC y la no SC— son gramaticalmente correctas
y sintácticamente sinónimas, es decir, son elegibles libremente. […]
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Lexis XXX.1
entre las variantes libres normalmente hay diferencias estilísticas, y parece que en este caso es así también. […] cuando las condiciones de la
SC se dan, es la preferencia del hablante lo que determina la elección
entre ellas (Berta 2000: 86-87, el destacado es nuestro).
Por su parte, los estudios que tratan el tema desde el análisis estadístico de corpus (Davies 1995, Gábor 2002) suelen partir de la misma
hipótesis; esto es, que la diferencia entre las construcciones con y sin SC
es meramente estilística, y, por lo tanto, coinciden en subrayar la mayor
frecuencia de aparición de las construcciones con SC en los corpus orales. De esta manera, sugieren que la SC se relaciona más con un estilo informal, coloquial, distendido, no planeado, de habla, mientras que los
casos sin subida se avienen más a un estilo formal, solemne, planeado.
Una excepción es Bolinger (1949), quien al discutir las construcciones con gerundio, afirma que
Lo fue llamando sugiere una acción repetida, mientras que Fue llamándolo sugiere un solo acto; [...] Lo pasó mirando sugiere un esfuerzo concentrado, mientras que pasó mirándolo refiere un acto casual momentáneo.
Lo estoy haciendo sugiere algo que puede ser interrumpido y luego retomado, mientras que Estoy haciéndolo sugiere algo hecho de una sola vez.
(Bolinger 1949: 259, traducción nuestra)
En este trabajo no nos centraremos en determinar los contextos en
los que la SC puede, no puede, o debe ocurrir, sino que trataremos de
detectar qué diferencias semánticas existen entre las construcciones con
y sin SC y de relacionarlas con la diferente estructura que cada construcción impone a la base conceptual.
3. Subida de clíticos como intensificador de la modalidad
En las secciones siguientes, analizaremos diferentes perífrasis verbales
con y sin SC, y trataremos de detectar las diferencias semánticas que
“Lo fue llamando suggests repeated action, while Fue llamándolo suggests a single act;
[…] Lo pasó mirando suggests a concentrated effort, while Pasó mirándolo refers to a casual
momentary act. Lo estoy haciendo suggests something that might be interrupted and then
resumed, while Estoy haciéndolo suggests something done in a single session.”
Bermúdez • La “subida de clíticos”
89
cada construcción expresa para, luego, tratar de relacionar tal diferencia con la configuración particular que la SC impone a la base conceptual. Por razones de espacio nos restringiremos a analizar tres perífrasis
verbales. Hemos elegido una modal (deber + infinitivo), otra aspectual
(estar + gerundio) y otra que suele presentarse como temporal (el futuro perifrástico ir a + infinitivo). Hemos elegido las dos primeras por su
representatividad dentro de su categoría (modal y aspectual, respectivamente) y por su alta frecuencia de aparición. La elección del futuro perifrástico, por su parte, se apoya, además de en su altísima frecuencia de
aparición, en la intención de dar un apoyo a la hipótesis de que los así
llamados tiempos verbales son mejor descritos como marcadores modales/evidenciales que como deícticos temporales (Bermúdez 2005).
3.1. Deber + Infinitivo
El verbo modal deber posee un significado deóntico y otro epistémico,
tal como puede verse en (12)-(13):
(12)
(13)
Para conseguir un préstamo, debes tener una garantía.
[Claudio pasa conduciendo un coche lujoso] Claudio debe tener
mucho dinero.
(12) significa que se requiere una garantía para poder recibir un
préstamo (necesidad deóntica/obligación). La interpretación de (13) es
‘sobre la base de que se necesita mucho dinero para adquirir coches
lujosos, el hablante infiere que Claudio tiene mucho dinero’ (necesidad
epistémica).
Esta alternancia ha sido abordada de diferentes maneras. Kratzer
(1977) y Perkins (1983) proponen la idea de que existe un significado
básico que está en la base de todos los usos de los verbos modales, ya
El verbo posee además un uso mitigador, íntimamente ligado al significado deóntico, que típicamente aparece con los verbos de lengua:
(i) en México se limita la libertad de expresión, y debo decirle que esto no es nuevo.
Yo desde que era pequeño...
(ii) Para concluir, pues, debo decirles que debemos buscar soluciones de carácter...
El hablante mitiga el hecho de decir algo que puede dañar la imagen del oyente
afirmando que es su deber decirlo, y por lo tanto elude la responsabilidad directa del
acto de enunciación.
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Lexis XXX.1
sean deónticos o epistémicos. Coates (1983), basándose en la teoría de
conjuntos borrosos (Zadeh 1965), propone que existen casos centrales
y periféricos, lo cual explicaría la dificultad de adscribir algunos casos
a uno u otro significado. Sweetser (1982), por su parte, postula que los
casos epistémicos son extensiones metafóricas del significado deóntico.
Según Sweetser, las mismas modalidades a las que están sujetas los actos
mundanos (necesidad, permiso, etc.) pueden aplicarse al mundo de los
razonamientos, dando así origen a la modalidad epistémica. El caso
concreto de deber se explica entonces directamente: el mundo epistémico del razonamiento se conceptualiza en términos del mundo sociofísico, por lo tanto, la obligación en el mundo se reinterpreta como la
conclusión obligada del hablante dado un contexto.
Silva Corvalán (1995), por su parte, postula que existe un significado invariable de los verbos modales (que en el caso de deber sería “X
es requerido”) que es luego interpretado en un contexto determinado,
respecto de un sistema de leyes determinado, lo que daría origen a las
diferentes lecturas (deóntica, epistémica, etc.) Determinados elementos
del contexto situacional o lingüístico, como la presencia/ausencia de
una fuente deóntica o el carácter (in)animado del sujeto, etc., favorecerían en cada caso una lectura deóntica o epistémica del verbo deber.
Ahora bien, al analizar en un corpus concreto los usos modales de
deber con y sin SC obtenemos resultados que señalan que hay una correlación clara entre los casos con y sin SC y el tipo de modalidad invocada
por el verbo deber:
Tabla 1. Deber + INFINITIVO con y sin SC
Significado
deóntico
Significado
epistémico
Total
Clítico deber infinitivo (SC)
ej. Lo debe tener
36% (29)
64% (51)
100%
(80)
deber infinitivo Clítico (No SC)
ej. Debe tenerlo
95% (666)
5% (34)
100%
(100)
El corpus utilizado para este estudio consta de 6,8 millones de palabras. Es un corpus electrónico de lengua oral y se compone de transcripciones de 2041 grabaciones de
conversaciones y entrevistas. El mismo es accesible en http://www.corpusdelespanol.org
Bermúdez • La “subida de clíticos”
91
Los resultados muestran una clara oposición entre las construcciones con y sin SC. En la construcción con SC el verbo deber tiende a
interpretarse como epistémico (64% de los casos), mientras que en la
construcción sin SC el verbo deber tiende a interpretarse como deóntico
(95% de los casos).
Transcribimos algunos ejemplos tomados del corpus para presentar el contraste. Los ejemplos (a) muestran una interpretación deóntica
mientras que los ejemplos (b) exhiben un significado epistémico:
(14)
a) —...y la obligación que tiene un hombre, debe tenerlo también al mismo tiempo la mujer.
b) —Eh... no sé qué otras, tú las debes tener, ¿o no?
—Papá tiene algunas, pero...
(15)
a) —...siempre he pensado que el hombre debe conocerse primero a sí mismo y a su ambiente, y después ...
b) —Usted lo debe conocer mucho.
—Sí, todos los días...
—Ah.
(16)
a) insiste en que el Gobierno Federal debe darle más participaciones a este estado...
b) No sé, es distinto, somos mujeres, también al ladrón le debe
dar un no sé qué. ¿Te acordás de aquel ladrón que...
(17)
a) —Pero no debe pasarme esto... Yo debo odiarlo... Yo...
b) —Y pienso que el bicho ése la debió pasar bien mal para
recibirme así, con tanta cola movida.
Ahora bien, existen otros factores del contexto lingüístico que contribuyen a una interpretación deóntica o epistémica. Un verbo de acción favorece la interpretación deóntica (Silva Corvalán 1995), mientras que la aparición del perfecto favorece la interpretación epistémica.
Esto se refleja en el corpus analizado: 25 de los 29 casos “inesperados”
de SC con interpretación deóntica de la Tabla 1 corresponden a verbos
de acción, como (18):
92
(18)
Lexis XXX.1
Eso no hay derecho; eso no lo debe hacer una mujer. Una mujer
así puede trabajar en su casa.
Y 17 de los 34 casos de interpretación epistémica de estructuras sin
SC son casos en los que el infinitivo está en perfecto:
(19)
—Con razón. A... yo creo que este aparatito debe haberse gastado
ya toda la... ¿eh?
En Bermúdez (2005) proponemos una interpretación evidencial del
pretérito perfecto compuesto en castellano, a saber: “a partir de la evidencia disponible, concluyo/afirmo X”. Este significado evidencial puede explicar el hecho de que muchas de las interpretaciones epistémicas
de construcciones sin SC tengan justamente el tiempo perfecto. Dado
que el perfecto invoca la inferencia como la fuente de la información
expresada por el verbo, el verbo modal deber se interpreta en relación
con el significado del perfecto y por lo tanto tiende a interpretarse epistémicamente, aun en casos sin SC.
La conclusión a la que llegamos es, pues, que la SC es un factor importante que favorece la lectura epistémica de deber. Ahora bien, más
que señalar este hecho estadístico queremos tratar de hallar una explicación al fenómeno: ¿qué es lo que hace que la configuración con subida de clíticos favorezca la lectura epistémica del verbo modal deber?
Antes de responder esta pregunta, debemos caracterizar la estructura de la construcción con el verbo modal deber, con y sin SC. Partimos
de la descripción de Talmy (1988) de los verbos modales en términos
de dinámica de fuerzas (force dynamics). Sweetser (1990) desarrolla ulteriormente las ideas originales de Talmy y afirma que la modalidad
debe entenderse como “la forma lingüística de representar la fuerza y
barreras en general”. El significado de deber podría entonces describirse como una fuerza aplicada sobre un evento, que favorece, o requiere,
la realización de tal evento.
Adoptamos aquí el análisis de Langacker (1999) de la “zona activa”,
y su interpretación de los verbos modales como estructuras de “elevación”. Antes de ver el análisis concreto comenzaremos clarificando
la noción de “zona activa”. Langacker señala que es raro el caso en el
que los participantes de una relación (trayector y marco de referencia, en
Bermúdez • La “subida de clíticos”
93
adelante tr y lm, respectivamente) participen en ella como entidades
totales, como en (20).
(20)
La nave se está acercando a Júpiter.
La Fig. 2 presenta la estructura conceptual de (20):
tr
lm
Fig. 2 Estructura de “La nave se está acercando a Júpiter”
Lo más común es que sea una “zona activa” de los participantes la
que en realidad participa de la relación, como en (21).
(21)
Tu perro mordió a mi gato.
Sólo ciertas partes del perro (dientes, mandíbulas, etc.) están directamente involucradas en la relación de morder al gato, mientras que
otras (la cola o el páncreas) no lo están. Del mismo modo, sólo ciertas
partes del gato participan en la acción. Este procedimiento debe verse
como un tipo de metonimia, dado que una entidad (la zona activa, en
este caso, las partes del perro concretamente involucradas en el proceso
de morder y las partes mordidas del gato) es referida por medio de otra
entidad asociada a ella (en este caso el todo del perro o el gato) cognitivamente más saliente o más accesible. Gráficamente:
za
za
tr
lm
Fig. 3 Zona activa
Las abreviaturas provienen de los términos ingleses trajector y landmark, respectivamente.
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Lexis XXX.1
Esta usual discrepancia entre los participantes elegidos por el hablante y las entidades realmente involucradas en la relación tienen una
explicación cognitiva bastante clara. En palabras de Langacker (1999):
Determinar qué entidades deben explicitarse y adquirir prominencia
como sujeto y objeto de un verbo a menudo involucra una tensión
entre dos objetivos: ser preciso y concreto respecto de qué entidades
realmente participan en la relación perfilada, y por otro lado enfocar
la atención en entidades que son inherentemente salientes o que tengan interés primario para el hablante y el oyente (Langacker 1999: 62,
traducción nuestra).
Dado que por lo general concebimos a perros y gatos como entidades
unitarias y sólo secundariamente nos preocupamos por sus subpartes,
es cognitivamente natural que en (21) seleccionemos la entidad entera
como participante focalizado (trayector y marco, típicamente sujeto y
objeto del verbo), aun cuando sólo son ciertas subpartes de ellos las que
participan activa o pasivamente en el acto de morder.
Ahora bien, la zona activa de la entidad elegida como participante de una relación no necesariamente tiene que ser una parte de ella,
como en el caso de (21); puede muy bien ser una entidad diferente,
de algún modo relacionada con el participante, como en (22). En este
caso, el proceso metonímico queda aún más claro.
(22)
Estoy en la guía telefónica.
Obviamente no soy yo sino ciertos datos relacionados con mi persona (nombre, dirección, número de teléfono) los que están en la guía
telefónica. Esquemáticamente, una situación como esa puede representarse gráficamente así:
tr
za
za
lm
Fig. 4 Zona activa relacionada al participante
Lo que es importante para el análisis de los modales que presenta
remos enseguida es que entre “las cosas relacionadas” con una entidad
Bermúdez • La “subida de clíticos”
95
pueden contarse las actividades o relaciones en las que tal entidad participa. Es decir, es posible que una relación o actividad en la que una
entidad se encuentra involucrada funcione como la zona activa a través de la cual dicha entidad forma parte de otra relación, como en el
diagrama de la Fig. 5.
za
Fig. 5 Relación como zona activa de un participante
Un caso que ejemplifica la estructura de la Fig. 5 es (23).
(23)
Pedro es lento comiendo helado.
En (23), “comiendo helado” especifica la zona activa de Pedro respecto de la relación de “ser lento”. Es sólo al estar involucrado en una
actividad, en este caso comer helado, que una entidad como Pedro puede
ponerse en relación con una escala de rapidez. La estructura conceptual del enunciado (23), representada en la Fig. 6, sería pues una instancia particular de la Fig. 5.
PEDRO
LENTO
COMER HELADO
tr
P
tr
lm
VELOCIDAD
Fig. 6 Estructura de “Pedro es lento comiendo helado”
96
Lexis XXX.1
El hablante establece una relación entre un punto en una escala de
velocidad y una entidad (Pedro), que es el trayector de tal relación y
consecuentemente su sujeto gramatical. Ese trayector, sin embargo, se
relaciona con la escala de velocidad sólo en función de ser el trayector
de una actividad (la de comer helado). Esa actividad, pues, sombreada
en la Fig. 6, es la zona activa de la entidad Pedro respecto de la relación
ser lento.
Para ilustrar el análisis de los modales basado en la noción de “zona
activa”, consideremos ahora el contraste entre los enunciados semánticamente emparentados de (24)-(25):
(24)
(25)
Es necesario que Juan lo haga.
Juan debe hacerlo.
La Fig. 7 representa la estructura conceptual de (24), “Es necesario
que Juan lo haga”.
NECESARIO
JUAN HACERLO
tr
lm
tr
Fig. 7 Diagrama de “Es necesario que Juan lo haga”
La estructura de necesario se articula como una fuerza (necesidad, representada por la doble línea quebrada que penetra en el rectángulo
más pequeño) que se aplica sobre un proceso esquemático, proceso que
es el trayector en la escena. Podemos decir que necesario requiere, obliga
o favorece la realización de ese proceso. Este proceso es elaborado por
la cláusula subjuntiva “que Juan lo haga”, que consecuentemente es el
sujeto de necesario. Nada especial hay en esta estructura. No hay procesos metonímicos como el de (23). La actividad misma, como un todo,
Bermúdez • La “subida de clíticos”
97
entra en relación con la fuerza expresada por necesario. Veamos ahora la
estructura de (25), “Juan debe hacerlo”:
JUAN
J
DEBER1
HACERLO
tr
tr
lm
Fig. 8 Diagrama de “Juan debe hacerlo”
El núcleo general es deber1, que es el que determina el perfil general de la construcción. La doble línea quebrada representa la fuerza
(necesidad) aplicada sobre el proceso, fuerza que es el núcleo de la significación de deber, según apuntábamos anteriormente. Ahora bien, el
trayector de deber ya no es un proceso esquemático (como en el caso
de (24)) sino una entidad concreta (Juan), cuya relación con la fuerza
representada por deber está mediada por el proceso en el que participa
(“hacer algo”). Este proceso esquemático representado por el rectángulo sombreado es la zona activa del trayector con respecto a la fuerza.
Hay además dos relaciones de elaboración, marcadas con flechas: la
frase nominal Juan elabora el trayector de deber y la expresión infinitiva
hacerlo especifica la zona activa. Las líneas punteadas denotan, como es
sabido, correferencia.
La diferencia con el caso de necesario es la elección del trayector.
Esto es, en lugar asignar el status focal de trayector al proceso entero, el
verbo modal deber se lo asigna al participante más saliente del proceso
al que se aplica (típicamente el trayector de ese proceso, en este caso
Juan). Como decíamos anteriormente, éste es un caso de metonimia,
fenómeno común en el lenguaje por el cual dirigimos la atención hacia entidades con alta prominencia y, al mismo tiempo —al invocarlas como punto de referencia de una entidad meta— establecemos un
contacto mental con la entidad meta, en este caso la actividad de Juan:
98
Lexis XXX.1
hacer algo. Resumiendo, analizamos la estructura de los verbos modales como otro caso más de “elevación de sujeto”, otorgándoles una
estructura análoga a otros auxiliares como estar y haber, verbos de elevación como parecer y aspectuales como empezar a, terminar de, etc.
(26)
(27)
(28)
Pedro está trabajando.
Juan parece dormir.
Mi hijo empezó a caminar.
No hay nada extraño en esta posibilidad de “subir el sujeto”. Sabemos que la noción de “subida” es sólo una metáfora que proviene del
paradigma generativista. Desde la perspectiva de la gramática cognitiva,
sujeto y objeto no son otra cosa que la figura primaria y secundaria de
una relación o, dicho de otra manera, trayector y marco de referencia al
nivel de la cláusula. El status de trayector y marco de referencia puede
ser pensado como un reflector que puede ser dirigido hacia diferentes entidades dentro de una escena, otorgándoles así prominencia focal
(Langacker 1995). Nada impide por lo tanto que el reflector se dirija a
los participantes principales (trayector y marco) de un proceso subordinado y que éstos así adquieran prominencia focal. Algunos elementos con prominencia cognitiva intrínseca (como un agente) ejercen una
atracción natural para ser focalizados. En suma, si el status de trayector
no es otra cosa que una cuestión de prominencia, no hay razón que impida que el trayector de deber sea el participante más saliente (el agente)
del proceso subordinado y no el proceso en sí. De hecho, los agentes son
trayectores prototípicos, y no los procesos, por lo cual el agente es un
candidato más natural como trayector que el proceso en su totalidad.
Ahora bien, ¿qué es lo que ocurre en el caso de la subida de clíticos? En
la Fig. 9 vemos el diagrama correspondiente a un enunciado como (29):
(29)
Juan lo debe hacer.
Para un análisis detallado de las estructuras de “elevación de sujeto”, ver Langacker
(1999: 317-360).
Bermúdez • La “subida de clíticos”
JUAN
J
DEBE2
99
HACER
tr
tr
LO
L
lm
lm
Fig. 9 Diagrama de “Juan lo debe hacer”
Lo que muestra el diagrama de la Fig. 9 es que el verbo deber2 no sólo
toma como su trayector al trayector del proceso al que se aplica (Juan)
sino que también toma como marco (landmark, marcado en el diagrama
como lm) al marco del proceso incluido (el pronombre lo). Esto podría
verse como una “subida de objeto a objeto”, posibilidad que Langacker
(1999) no contempla en su exposición de la explicación cognitiva de
las estructuras de elevación. Es decir, en (29) no hay solo una subida de
sujeto a sujeto (Juan, que “sube” de ser sujeto de hacer a ser sujeto de
debe) sino también de objeto a objeto (lo, que “sube” de ser objeto de
hacer a ser objeto de debe).
Tampoco hay nada extraño respecto de esta posibilidad de “subida
de objeto a objeto”. Lo que ocurre en esta subida de objeto a objeto es
que el marco del proceso subordinado adquiere prominencia focal al
nivel de la cláusula principal como marco del verbo modal deber. En
principio, el verbo deber no impone ninguna restricción sobre su marco
(es decir, es en este sentido un predicado transparente (Langacker
1995)); esto permite que en principio cualquier entidad pueda tomar
esta función. Dado que el trayector del proceso subordinado no está
accesible (éste ha sido ya tomado como trayector del modal, según veíamos más arriba), el único candidato posible es el marco del proceso
subordinado.
Llegamos, pues, a la conclusión de que las estructuras con y sin subida de clíticos son diferentes articulaciones de la misma base conceptual.
Como vemos al comparar los diagramas de la Fig. 8 y la Fig. 9, la única
diferencia es la distinta prominencia focal del marco del proceso incluido
100
Lexis XXX.1
(el pronombre lo). En la construcción sin SC, el marco del proceso elaborado por el infinitivo no posee ninguna prominencia al nivel de la
cláusula principal, no es más que uno de los participantes de la estructura subordinada. Por el contrario, en la construcción con SC el marco del
proceso subordinado es un participante (el marco) de la escena principal
regida por el verbo modal deber. De esta diferencia en prominencia debe
deducirse la diferencia semántica entre ambas construcciones.
Si analizamos el diagrama de la Fig. 9, vemos que el verbo modal
deber ha “captado” las entidades relacionadas por el infinitivo. El trayec
tor y el marco de deber son, respectivamente, el trayector y el marco
del infinitivo. Lo que se produce, entonces, a partir de esta diferente
asignación de perfil, es una pérdida de prominencia del evento elaborado por el infinitivo (en el caso de (29), la actividad de hacer), que
queda en el fondo, sin ninguna prominencia: no sólo está en infinitivo,
esto es, referido holísticamente, sino que además ha quedado como un
proceso esquemático, privado de trayector y marco explícitos. O dicho
de otra manera, al tomar tanto el trayector como el marco del proceso
subordinado como propios, el modal deber ha ganado en prominencia a
expensas de la prominencia del infinitivo.
Ahora bien, ¿cuál es la consecuencia semántica de esta ganancia de
prominencia del verbo modal originada por la SC? Simplemente que
el enunciado tiende a interpretarse como más modal. Pero ¿qué significa
“más modal” en nuestro caso concreto del verbo deber?
Como señalábamos anteriormente, la modalidad epistémica es un
tipo “más sofisticado”, más abstracto y más subjetivo de modalidad
(Sweetser 1982). De ahí que las instancias del verbo modal deber con SC
tiendan a interpretarse como epistémicas (“más marcadamente modales”), mientras que aquellas sin SC tiendan a interpretarse como deónticas (“menos marcadamente modales”). La colocación de los clíticos junto
al modal, pues, funciona como un indicador de que el enunciado debe
interpretarse como más subjetiva o abstractamente modal. La diferencia
Un análisis alternativo sería pensar en una operación cognitiva que daría origen
a un verbo compuesto, debe hacer, lo que produciría un reajuste focal de los participantes
(Bermúdez 2004). Dado que el verbo modal no posee trayector ni marco propios, los del
infinitivo pasarían a ser los participantes del predicado complejo. Este análisis alternativo
no alteraría las conclusiones extraídas del análisis que adoptamos en este trabajo, dado
que en ambos casos el infinitivo perdería prominencia inicial.
Bermúdez • La “subida de clíticos”
101
estadística observada en el corpus adquiere así una explicación cognitiva: el diferente perfil impuesto sobre la misma base conceptual provoca
las diferencias de interpretación observadas en el corpus.
En los próximos apartados aplicaremos esta misma idea a otras perífrasis verbales, a saber, el futuro perifrástico “ir a + infinitivo” y el
progresivo “estar + gerundio”.
3.2. Ir a + Infinitivo
En Bermúdez (2005) argumentamos a favor de una interpretación estrictamente modal/evidencial de los tiempos verbales. En ese trabajo, el
significado básico de los tiempos verbales es visto no como la ubicación
temporal de los eventos sino como la especificación combinada de dos
magnitudes: la actitud del hablante frente a lo dicho (modalidad) y la
perspectiva del hablante (evidencialidad/aspecto). La modalidad expresada por los tiempos verbales, por su parte, puede ser deóntica —que
sería el tipo básico—, epistémica, circunstancial, bulética, etc. —que
serían los tipos derivados o subjetivos, más abstractos. En (30) el imperfecto expresa modalidad circunstancial, mientras que en (31) el presente expresa modalidad deóntica.
(30)
(31)
Si sabía esto, te aseguro que no me veías acá.
Te vas de acá inmediatamente.
La perspectiva tiene también un tipo básico o central, la evidencialidad,
y un tipo derivado, o subjetivo, el aspecto. Ambos se relacionan con la
distancia del hablante respecto del evento. La evidencialidad trata de
la distancia del hablante a la fuente de información (evidencia sensorial
directa, endofórica, indirecta, inferida, transmitida, etc.), y el aspecto
trata del punto de vista, de la distancia subjetiva al evento (perspectiva interna, externa, incoativa, terminativa, etc.) En (32) el imperfecto expresa
evidencia indirecta transmitida (alguien me dio la información o la leí en
un horario de llegadas, etc.), en (33) el perfecto compuesto indica inferencia y el pretérito perfecto simple indica perspectiva externa en (34):
(32)
(33)
(34)
El avión llegaba hoy a las 4.32.
Hay muchos policías en la esquina. Algo ha pasado.
No me llames a las 5 porque a esa hora ya me fui.
102
Lexis XXX.1
Dentro de esta interpretación de los tiempos verbales, el futuro es
descrito no como una forma con significado temporal sino como un
modal; su significado es una referencia a la potencialidad —en el mundo actual— del evento al que se asocia.
(35)
Los beneficios que nos va a traer/traerá esta ley son evidentes.
Esto es, en (35), al utilizar el tiempo futuro, el hablante no está hablando de un evento futuro que existe límbicamente en algún reino
metafísico sino de cierta disposición del mundo actual, de cierta potencialidad, de ciertas propiedades del mundo actual, que posee, entre
otras cosas, la ley en cuestión.
De acuerdo con la discusión de los apartados anteriores, esperaríamos encontrar que en las construcciones de ir a + Infinitivo con SC el
componente modal asociado al auxiliar quede en foco y gane en saliencia, adquiriendo ese componente, por lo tanto, un significado más
marcado, más abstracto o más subjetivo.
Este parece ser el caso. Veamos los ejemplos siguientes, extraídos de
nuestro corpus, que ejemplifican la oposición lo vamos a hacer/vamos a
hacerlo:
(36)
(37)
(38)
(39)
Lo estás haciendo muy bien. Vamos a hacerlo ahora con la pierna izquierda: tensiónala. Relájala.
Y ahora, expúlsalo. Muy bien. Así. Vamos a hacerlo otra vez:
Tomar aire...
No, no preguntes, pero lo vamos a hacer con mucho gusto.
todo lo vamos a hacer de manera democrática, lo vamos a hacer
a través de la comunidad,
Aquí la oposición aparece clara. En los ejemplos (36)-(37) (sin SC)
el significado de la construcción es deóntico. El hablante propone una
actividad al oyente, pero el hablante mismo no participa de ella. El
Esta es una forma ya gramaticalizada, que ha seguido un camino análogo al de
let’s del inglés (Traugott 2003). La interpretación se origina en el significado deóntico de
propuesta que incluye tanto al hablante como al oyente (“hagamos esto tu y yo”), convir
Bermúdez • La “subida de clíticos”
103
componente modal del futuro (evaluación sobre la potencialidad del
evento) se desfocaliza y en su lugar se pone el foco en el evento mismo
descrito por el infinitivo. De esta manera, el hablante saca el foco del
“nosotros” del verbo y lo pone en el hacerlo (y en el oyente). La construcción funciona, pues, como un atenuador de la fuerza del enunciado. Es importante señalar que no hay ninguna ocurrencia en el corpus
de una construcción con SC (lo vamos a hacer) con este significado.
Por el contrario, en los ejemplos (38)-(39) con SC es justamente el
componente modal del futuro lo que se focaliza. Decíamos antes que
el hablante, al utilizar el futuro, afirma que el mundo actual tiene una
cierta disposición que favorece la realización del evento descrito por el
infinitivo. Al poner en foco justamente este carácter interpretativo (por
medio de la SC), lo que el hablante enfatiza es su propia participación
en tal interpretación del mundo, y, consecuentemente, señala que se
hace cargo de tal interpretación, y, por lo tanto, se dispone como origen de la interpretación y garante de la realización del evento. Esto
es, la vertiente modal de esta construcción con SC sería la de expresar
seguridad respecto de la realización del evento y garantizarla personalmente. El costado evidencial consistiría en que el hablante se describe
como la fuente de la información expresada. Estos ejemplos (38)-(39)
se interpretan prácticamente como una promesa. De hecho, resultaría totalmente natural agregarles a estos ejemplos “yo te lo garantizo”.
Nuevamente, no hay en el corpus ningún ejemplo de vamos a hacerlo
con esta interpretación.10
Vemos en todo el corpus que esta tendencia se sostiene: la estructura
con SC se utiliza como una estrategia de intensificación de la afirmación y focalización en el yo y la estructura sin SC, por el contrario, preferentemente como una estrategia de atenuación y de focalización en el
acto en sí y en el oyente. Veamos algunos datos.
El 65% de los casos con SC tienen el verbo ir en primera persona del
singular (te voy a decir, le voy a dar, etc.), lo que marca la tendencia a la
tiéndose luego en un mitigador con el que el hablante se sitúa empáticamente respecto
de las posibles objeciones del oyente a la actividad propuesta (“haz esto, ¿de acuerdo?”).
10
Hay un solo ejemplo que podría interpretarse en esta línea:
(i) Si de verdad quieren ustedes defender el vino, vamos a hacerlo, estamos a tiempo.
Sin embargo, es interesante que en este caso el nosotros es inclusivo del oyente. Más
adelante volveremos sobre esta discusión del nosotros inclusivo o exclusivo.
104
Lexis XXX.1
focalización del yo. En todos estos casos, el hablante explícitamente se
pone como sujeto de la interpretación del mundo y fuente de la información expresada en el enunciado.
Por otro lado, el 54% de la totalidad de los casos de ir a + Infinitivo
con SC se construye con verbos de lengua, mediante los cuales el hablante intensifica su aseveración y se pone como origen de la información y garante de su veracidad:
(40)
(41)
(42)
(43)
(44)
(45)
(46)
Bueno, te voy a decir que una de las últimas grandes figuras...
Están sencillamente porque no creen en el jurado y te voy a
decir más. Yo he hablado con delincuentes y dicen que...
...además los hacés a tu gusto. Te voy a decir que los elegiste
bien. Tienen un aire tan chic.
Te voy a decir que de labores de casa sé bastante, porque...
yo soy hija única y te voy a decir que los hijos únicos sufren
pero...
No, no, casi no, casi no. Yo te voy a decir inclusive, que... que
aquella actitud que yo tomé,
Ahora te voy a decir una cosa, patentar una... un invento... ya
no es...
De hecho, la construcción “te/le voy a decir” se ha gramaticalizado
como un marcador discursivo que justamente indica intensificación de
la aseveración y por lo tanto compromiso del hablante respecto de lo
afirmado:
(47)
(48)
(49)
(50)
La explotaban, te voy a decir, porque no le llegaron a pagar.
Sí, no... no había pensado en esto. Y te voy a decir, el... el trabajo no desmerece a nadie
Vos no fuiste una gran ayuda, te voy a decir.
Me gustaba... leer casi todo, te voy a decir, pero mucho eh...
Miguel Hernández, García Lorca...
Por el contrario, la construcción sin SC se utiliza, en general, de
diferentes maneras para sacar de foco la responsabilidad del hablante
al realizar un enunciado y atenuar su fuerza. Esto se ve claro en los siguientes ejemplos, en los que también se utilizan verbos de lengua pero
Bermúdez • La “subida de clíticos”
105
con un significado opuesto al señalado más arriba en relación con la
construcción con SC:
(51)
(52)
(53)
(54)
(55)
se lo ha quedado el Ayuntamiento, vamos a decirlo así, para la
exposición del noventa y dos ¿no?
...y que son doctos, vamos a decirlo así, en este tipo de escritura
que ése era e... el crecimiento, vamos a decirlo así, que había
tenido Caracas...
Yo creo que todo el paquete -vamos a llamarlo así- de medidas,
iniciativas, acciones, técnicas...
Uno de los puntos débiles, vamos a llamarlo así, que tiene la
atención médica privada es carecer...
El hablante utiliza el nosotros para incluir al oyente en la evaluación,
ya no del mundo, sino de la adecuación de la expresión utilizada. De esta
manera atenúa la aseveración realizada y toma en cuenta la imagen social
del oyente. Puede decirse entonces que este es un uso cortés del futuro.
De hecho, de la totalidad de los casos sin SC, el 54% se construye
en la primera persona del plural (frente al 9,7% de las construcciones
con SC), lo que marca la focalización hacia el oyente. Es importante
remarcar que en los casos con SC, el uso del nosotros es exclusivo, como
en (56)-(57), mientras que en los casos sin SC, el nosotros incluye (aunque sea retóricamente) al oyente, como se ve en (58)-(60). Esta es una
marca más de la orientación hacia el oyente de la construcción sin SC y
la orientación hacia el hablante de la construcción con SC:
(56)
(57)
(58)
(59)
(60)
un error que también, señor Moya, se lo vamos a hacer saber a
los ciudadanos de Castilla-La Mancha.
resulte en una mejoría para todos los ciudadanos. Lo vamos a
hacer con mucho cuidado, con mucha pulcritud, para que...
¿Está claro? Vamos a hacerlo con otro verbo : “Juan recibió a sus
padres”.
Eso ayudará a producir la tensión. Vamos a hacerlo. Tensiónala.
Muy bien. Un poco más.
Vamos a hacerlo con mucho entusiasmo, vamos a darle un
aplauso muy fuerte a...
106
Lexis XXX.1
Resumiendo, las construcciones con y sin SC de la perífrasis ir a +
Infinitivo muestran una diferencia de significado análoga a la ya señalada en el caso de deber: la construcción con SC focaliza el elemento modal del futuro perifrástico (evaluación de la potencialidad de un evento). Esto hace que esta construcción enfatice la responsabilidad del hablante en la evaluación del mundo, intensificando la afirmación hecha
y poniéndose como fuente y garante de la veracidad de lo expresado en
el enunciado. Por el contrario, la construcción sin SC tiende a interpretarse como una atenuación de la fuerza del enunciado y, crucialmente,
como una forma de tomar en cuenta las necesidades del oyente.
3.3. Estar + Gerundio
El significado del progresivo (estar + Gerundio) también es describible
como modal y aspectual al mismo tiempo. (61) es aspectual dado que
el evento se presenta desde una perspectiva interna o, dicho de otro
modo, como un evento “en proceso”:
(61)
Juan está cruzando la calle.
Sin embargo, (61) es también modal (Dowty 1977, Landman 1992,
Portner 1998), en el sentido de que el hablante, al usar el progresivo,
está indicando que, si nada inesperado ocurre, Juan terminará de cruzar la calle, lo cual es una referencia a mundos “verosímiles” en los que
el proceso en acción no es interrumpido (por ejemplo, por un coche
que lo atropella).
¿Cómo afectará, pues, la SC al progresivo? Nuevamente, la hipótesis
es que el componente modal ganará en saliencia, haciéndose, si fuera
posible, más subjetivo, más abstracto. La observación de Dowty (1977)
sobre el progresivo, señalada anteriormente, puede reinterpretarse en
estos términos. Dowty afirma que “Estoy haciéndolo sugiere algo hecho
de una sola vez”. Esto es, aquí es el costado aspectual (denotado por
el gerundio) el que está en foco. Estoy haciéndolo indica una acción en
proceso, “yo estoy en el proceso de hacer algo”. Eventualmente se terminará, pero lo que está en foco es el hecho de que estoy inmerso en el
proceso. Por el contrario, según Dowty, “Lo estoy haciendo sugiere algo
que puede ser interrumpido y luego retomado”. De hecho, yo puedo
no estar haciendo eso en el momento de la enunciación, es decir, puedo
Bermúdez • La “subida de clíticos”
107
encontrarme en una de esas “interrupciones” y sin embargo decir “lo
estoy haciendo”. Lo que está en foco en la construcción con SC es el costado modal, esto es, “voy a terminarlo”. Tal vez esté haciéndolo ahora
mismo, tal vez no, pero si nada extraordinario ocurre, lo terminaré.
El análisis extensivo del corpus confirma la hipótesis. Los ejemplos
sin SC tienden a interpretarse como aspectuales (X se encuentra en el
proceso de hacer algo), mientras que los casos con SC tienden a interpretarse como modales (X va a terminar de hacer algo). Los siguientes
ejemplos son muestras de este contraste:
(62)
(63)
(64)
(65)
(66)
–¿Con quién está haciendo la tesis?
–La estoy haciendo con el catedrático de Paleontología de Granada.
–Este mismo curso lo estoy haciendo en la Escuela de Educadoras de Párvulos, para un seminario de... de grado.
–...y decide arrastrarlo hacia la cama. Está haciéndolo cuando
aparece Teresa, en camisón y...
–No, yo todavía no daba clases. Estaba haciéndole una visita a
tía Merceditas y...
–Ni se sabe los años que estuvieron haciéndolo, fue un desastre
colosal de la construcción aquella sencilla ampliación.
En el caso de las construcciones con SC (62)-(63) se supone que, si
nada extraño ocurre, la tesis o el curso llegarán a su término, es decir, el
costado modal está en foco. Tal focalización salta a la vista al comparar
estos ejemplos con casos sin SC, donde el valor modal está completamente fuera de foco. En las construcciones sin SC (64)-(66) lo que está
en foco es el costado aspectual: el sujeto de la perífrasis se encuentra en
el proceso de arrastrar a alguien hacia la cama, de hacer una visita o de
ampliar un hospital, pero nada se supone respecto de la finalización del
evento; de hecho en (64) se supone que el proceso nunca fue completado a causa de la aparición de Teresa.
Además, una gran cantidad de casos de estar + Gerundio con SC se
interpretan con valor de intención, como en el ejemplo siguiente:
108
(67)
Lexis XXX.1
–No, lo que pasa es que no estamos preparados... eh... todavía,
como para poder nivelar las situaciones.
–No, no, no.
–Está la... está la mujer...
–Yo no te estoy hablando de nivelar. Digo, yo... yo te estoy diciendo que, desde el punto de vista intelectual, la mujer puede
tener tanto o más capacidad que el hombre.
–Ahá.
–Yo lo único que digo es... estoy hablando de naturaleza:
¿Quién tiene que cuidar a los niños?, ¿quién debe cuidar a los
hijos?, ¿quién, por su propio organismo en el nacimiento o en
la creación, tiene...
En (67) el hablante al decir “Yo no te estoy hablando de nivelar. Digo,
yo... yo te estoy diciendo que...” está comunicando “yo no quiero hablar
de nivelar, yo quiero decir que...” Este valor de intención no es en absoluto sorprendente. Como acabamos de decir, el progresivo tiene una
vertiente modal, que hace referencia a la potencialidad de que un evento se complete. Decíamos que la SC focaliza esta vertiente modal y que,
por lo tanto, se esperaría que la interpretación modal se intensificara,
se hiciera más abstracta o subjetiva. Esto es precisamente lo que ocurre
en la reinterpretación del progresivo como marcador de la intención
del hablante de completar el evento. Al intensificar el costado modal
de la construcción, es decir, al focalizar la eventual finalización o completamiento del evento, se generan significados de intención o meta
subjetiva: el hablante, al utilizar la construcción progresiva con SC, está
diciendo que es su intención que el evento se complete.
En la misma línea pueden explicarse los usos del progresivo con el
sentido cuasi futuro de inminencia:
(68)
Bueno, esa es la razón por la que le estamos mandando inmediatamente a su país para que sus... sus autoridades se encarguen de usted.
El significado de (68) es “esa es la razón por la que le vamos a mandar
inmediatamente a su país”. Este valor del progresivo se explica en relación
con el valor de intención ya señalado. El proceso de gramaticalización
Bermúdez • La “subida de clíticos”
109
es el siguiente: la construcción con SC, al focalizar el valor del auxiliar,
genera en contexto el valor de intención. Ulteriormente, la intención se
reinterpreta contextualmente como una característica fuerte que favorece la potencialidad de la realización del evento, tomando por lo tanto
un sentido análogo al futuro. Al mismo tiempo, el sentido de acción en
proceso, no focalizada pero de todos modos presente en el gerundio,
hace de este futuro un futuro inminente. Es necesario destacar que no
existen en el corpus ejemplos sin SC que tengan esta interpretación de
inminencia.
Otro hecho notable que apunta en la misma dirección es que todos
los casos del progresivo con el verbo ver sin SC se interpretan con el
significado concreto de “percibir con la vista” como en (69). Por el contrario, los casos con SC se interpretan metafóricamente como “darse
cuenta”, “entender”, “considerar”, como en (70)-(71):
(69)
Está muy contenta, las enfermeras y las auxiliares la rodean las
veinticuatro horas del día. Pueden estar viéndola continuamente, porque claro el plástico se de/ - es transparente y lo ve todo.
(70)
Entonces hay que estar preparado para poder recibirla. Desde
luego va a dar impresión. Yo no te digo que, ¡ay!... color de
rosa, un bebé, todo asi ¿no?... Da impresión. Pues tantas cosas
raras que uno por... por no ser médico, no las está viendo.
(71)
Yo no sé como el PRD tuvo oportunidad de contestar tan rápido, a qué hora le dieron el documento. A nosotros, a la Comisión de Acción Nacional le llegó por la tarde, están analizándola y la están viendo y saldrá una postura. Nosotros sólo
conocemos, yo en lo personal conozco el documento
La explicación de este fenómeno es directa: la construcción con SC,
decíamos, focaliza la potencialidad de completamiento del evento, implícita en el progresivo. De ahí que el verbo ver en la construcción con
SC se interprete preferentemente en el sentido metafórico de “darse
cuenta”, ya que en esta acepción ver es un verbo télico. Por el contrario,
la construcción sin SC se interpreta como verbo de estado, simplemente
de percibir con la vista.
110
Lexis XXX.1
4. Resumen y conclusiones
Creemos haber mostrado argumentos que apoyan dos afirmaciones hechas al comienzo de este trabajo:
1. Las construcciones con y sin subida de clíticos no son semánticamente equivalentes y su diferencia semántica no puede atribuirse
a “diferencias estilísticas”.
2. Las diferencias semánticas entre las construcciones con y sin subida de clíticos pueden describirse como resultado de la aplicación de un perfil diferente sobre la misma base conceptual.
Nos apoyamos en el análisis de las estructuras de elevación hecho
por Langacker (1999), según el cual los verbos modales y aspectuales
constituirían un caso más de “elevación de sujeto”. Adoptamos asimismo un análisis polisémico de la SC. Afirmamos que existen dos variantes de los verbos que permiten la subida de clíticos, que tienen los sentidos esquematizados en la Fig. 10.
a)
Construcción sin SC
b)
Construcción con SC
tr
tr
lm
za
za
Fig. 10 Construcciones con y sin SC
La flecha punteada simboliza la relación expresada por el verbo
“auxiliar” (deber, ir a, estar, etc.), sea ésta cual fuere. El contenido conceptual de ambas variantes es el mismo. La diferencia entre la construcción con y sin SC estriba en la prominencia focal del marco (landmark)
del proceso subordinado. En la construcción sin SC (Fig. 10a), el marco
Bermúdez • La “subida de clíticos”
111
del proceso subordinado no posee ninguna prominencia particular al
nivel del predicado de la cláusula principal, es sólo un participante en
un proceso conceptualmente subordinado. En la construcción con SC
(Fig. 10b), por el contrario, el marco del proceso subordinado adquiere prominencia focal al nivel de la cláusula principal, como marco del
predicado principal.
Al utilizar este acercamiento estamos afirmando que la subida de
clíticos sería analizable como un caso más de “elevación”, en este caso
como “elevación de objeto a objeto”, posibilidad no tomada en cuenta
por Langacker (1999).
La diferencia de prominencia del marco del proceso subordinado
tiene un efecto que puede rastrearse en las diferentes construcciones en
las que la SC es posible. En la configuración con SC el “verbo auxiliar”
toma al trayector y al marco del proceso subordinado como sus propios trayector y marco respectivamente, y esto ocasiona que el proceso
subordinado pierda prominencia y al mismo tiempo que el predicado
principal (elaborado por el auxiliar) la gane. El resultado es prototípicamente una lectura en la que el predicado principal (el verbo modal o
aspectual) recibe una interpretación más marcada o intensificada.
En el caso del verbo modal deber, la SC favorece una lectura epistémica mientras que la construcción sin SC se interpreta prototípicamente como deóntica. Este hecho encuentra su explicación en el análisis
de Sweetser (1982), según el cual la modalidad epistémica puede verse
como una extensión metafórica (más abstracta y subjetiva, aplicada sobre el mundo de los razonamientos) de la modalidad deóntica.
El así llamado futuro perifrástico (ir a + Infinitivo) es interpretado aquí como una evaluación de las disposiciones o características del
mundo actual que aumentan la potencialidad de un evento (Bermúdez
2005). De esta manera, la construcción con SC intensifica este rasgo de
evaluación del hablante (que suele construirse subjetivamente), haciendo que el significado de la construcción se haga por un lado evidencial
(el hablante se pone a sí mismo como fuente de la información) y, por
otro lado, que se acentúe la dimensión modal, dado que el hablante se
hace cargo de la evaluación realizada, lo que lo convierte en garante
de la realización del evento. El resultado global es que la construcción
pone el foco en el hablante e intensifica el acto mismo de la aseveración. Por el contrario, la construcción sin SC pone el foco en el evento
112
Lexis XXX.1
en sí y en el oyente, y funciona como un atenuador de la aseveración,
alejándola de la responsabilidad del hablante. Dos construcciones gramaticalizadas apoyan este análisis: la construcción vamos a + Infinitivo,
con un significado deóntico y que al mismo tiempo es un atenuador que
toma en cuenta las necesidades del oyente, análogo al let’s del inglés, se
construye en nuestro corpus sólo como construcción sin SC. En el otro
extremo del espectro, la forma te/le/les voy a decir con SC se ha convertido en un marcador discursivo de intensificación de la aseveración, que
al mismo tiempo sitúa la fuente de la información en el yo del hablante.
Ninguna de las ocurrencias de esta construcción sin SC (voy a decirte/le/
les) posee esta interpretación.
Por último, la construcción progresiva (estar + Gerundio) muestra un
patrón similar. Los casos sin SC tienden a interpretarse como meramente aspectuales: el sujeto se encuentra en el proceso o estado denotado
por el gerundio. Por el contrario, en los casos con SC, la interpretación
preferida es la modal, marcando la creencia del hablante de que si nada
inesperado ocurre, el proceso denotado por el gerundio llegará a completarse. Esta interpretación llega incluso a intensificarse y subjetificarse y convertirse en modal volitiva: el hablante, al utilizar el progresivo
con SC, expresa que el sujeto tiene la intención de lograr que el proceso
se lleve a cabo, lo cual es un rasgo que haría más probable el completamiento del evento. También en esta línea pueden interpretarse los
casos en los que la construcción progresiva se interpreta como aspectual
de inminencia: la intención del hablante se interpreta como una disposición del mundo actual que favorece la realización del evento, lo cual
es el significado prototípico del tiempo futuro. El rasgo de evento en
proceso, desfocalizado pero presente de todos modos en la construcción progresiva explica que este “futuro” sea un futuro inminente.
Por razones de espacio nos hemos limitado al análisis de estas tres perífrasis verbales; sin embargo, un patrón análogo puede rastrearse en las
demás construcciones que permiten la así llamada subida de clíticos.
Bermúdez • La “subida de clíticos”
113
BIBLIOGRAFÍA
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2004
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sujeto”. Boletín de Lingüística 22, 3-31.
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“Los tiempos verbales como marcadores evidenciales. El caso del
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2000
“La subida de clíticos en español medieval y en español moderno”.
En A. Anderle (ed.). Acta Hispánica, Tomo V. Szeged: Universidad
de Szeged, 83-95.
Bok-Bennema, Reineke y Brigitte Kampers-Manne
1984
“Transparency effects in Romance Languages”. En M. L. Mazzola
(ed.). Issues and theory in Romance Linguistics. Washington DC: Georgetown University Press, 199-217.
Bolinger, Dwight
1949
“Discontinuity of the Spanish Conjunctive Pronoun”. Language 25,
253-260.
Cinque, Guglielmo
1999
Adverbs and Functional Heads. A Cross-linguistic Perspective. Oxford:
Oxford University Press.
2000
“‘Restructuring’ and Functional Structure”. En L. Bruge (ed.), University of Venice Working Papers in Linguistics, Vol.11, 45 –127.
Coates, Jennifer
1983
The Semantics of the Modal Auxiliaries. London: Croom Helm.
Davies, Mark
1995
“Analyzing Syntactic Variation with Computer-Based Corpora: The
Case of Modern Spanish Clitic Climbing”. Hispania. 78, 370-380.
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1977
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Gábor, Kertes
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Subida de clíticos en corpus electrónicos. Szeged: Universidad de Szeged.
<http://tesina.galleus.com/tesina.pdf> [consultado el 20-3-2005]
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Kayne, Richard
1989
“Null Subjects and Clitic Climbing”. En O. Jaeggli y K. Safir (eds.).
The Null Subject Parameter. Dordrecht: Kluwer, 239-261.
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1977
“What ‘Must’ and ‘Can’ Must and Can Mean”. Linguistics and Philosophy 1, 337-355.
Landman, Fred
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Langacker, Ronald W.
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“Raising and Transparency”. Language 71, 1-62.
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Grammar and Conceptualization. New York: Mouton de Gruyter.
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1993
“La subida de clíticos y el modo en los complementos verbales del
español”. En O. Fernández Soriano (ed.). Los pronombres átonos. Madrid: Santillana, 235-281.
Perkins, Michael R.
1983
Modal Expressions in English. London: Frances Pinter.
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1998
“The Progressive in Modal Semantics”. Language 74, 760-787.
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Issues in Italian Syntax. Dordrecht: Foris Publications.
Roberts, Ian
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“Restructuring, Head Movement and Locality”. Linguistic Inquiry
28.3, 423-460.
Silva Corvalán, Carmen
1995
“Contextual conditions for the interpretation of poder and deber
in Spanish”. En J. Bybee y S. Fleischman (eds.). Modality in grammar
and discourse. Amsterdam: John Benjamin, 67-106.
Sweetser, Eve
1982
“Root and Epistemic Modals: Causality in two Worlds”. Proceedings
of the Eighth Annual Meeting of the Berkeley Linguistics Society. Berkeley:
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From ethymology to pragmatics. Cambridge: Cambridge University
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Talmy, Leonard
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49-100.
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“Clitic climbing from finite clauses and tense raising”. Probus 8,
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“Fuzzy sets”. Information and Control 8: 338-353.
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Frédéric Lordon: il lavoro tra desiderio e servitù
|
Studi e saggi
| 2,024
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cc-by
| 2,452
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Andrea Valzania, University of Siena, Italy, andrea.valzania@unisi.it, 0000-0003-3808-6826
Referee List (DOI 10.36253/fup_referee_list)
FUP Best Practice in Scholarly Publishing (DOI 10.36253/fup_best_practice)
Andrea Valzania, Frédéric Lordon: il lavoro tra desiderio e servitù, © Author(s), CC BY 4.0, DOI
10.36253/979-12-215-0319-7.119, in Giovanni Mari, Francesco Ammannati, Stefano Brogi, Tiziana Faitini,
Arianna Fermani, Francesco Seghezzi, Annalisa Tonarelli (edited by), Idee di lavoro e di ozio per la nostra
civiltà, pp. 1027-1032, 2024, published by Firenze University Press, ISBN 979-12-215-0319-7, DOI
10.36253/979-12-215-0319-7 1. Cenni biografici Frédéric Lordon, nato a Parigi nel 1962, è attualmente direttore di ricerca al
Centre National de la Recherche Scientifique. Sociologo, filosofo, economista,
difficilmente inquadrabile nell’ambito di una sola etichetta disciplinare, è noto
per il taglio spinoziano della sua critica al sistema economico capitalistico glo-
bale ed è tra gli artefici principali del recupero di emozioni, passioni e desideri
quali aspetti essenziali per comprendere il funzionamento delle società contem-
poranee. Autore di numerose pubblicazioni, tra le quali la più significativa resta
La société des affects: pour un structuralisme des passions (2013). In Italia sono stati
tradotti Capitalismo, desiderio e servitù (2015) e La condizione anarchica (2021)1. Frédéric Lordon: il lavoro tra desiderio e servitù Andrea Valzania 2. Spinoza e lo strutturalismo delle passioni Frédéric Lordon è noto per avere proposto un originale ritorno all’opera di
Spinoza nel dibattito sulle trasformazioni della società contemporanea, muo-
vendo dalla nota lezione di Deleuze risalente agli anni Settanta del Novecen-
to e ibridandola con altri contributi d’impianto prevalentemente strutturalista
(Lordon 2003; Lordon and Citton 2008). 1 Per ulteriori informazioni biografiche si rimanda al suo sito personale: <http://www.frede-
riclordon.fr>. Andrea Valzania Due sono, in estrema sintesi, gli obiettivi principali per le scienze sociali. In
primo luogo, riuscire a superare la contrapposizione tra approcci strutturalisti
e individualismo metodologico, trovando nel conatus una sintesi tra la neces-
saria presenza dell’agency e un’altrettanta necessaria presenza delle struttu-
re sociali: «le strutture sociali hanno il loro immaginario specifico in quanto
espressione di configurazione di desideri e affetti» (Lordon 2015, 71), ovvero
sono esterne e interne agli individui, oggettive ma anche soggettive, interioriz-
zate. Conatus e strutture, insomma, non sono affatto in conflitto tra loro ma in
un rapporto di continua e reciproca determinazione. In secondo luogo, torna-
re a mettere la dimensione non razionale dell’agire al centro della riflessione,
attualizzando il concetto spinoziano di affetto/passione e inserendosi – di fat-
to – nel dibattito che negli ultimi decenni è noto per avere valorizzato il ruolo
delle emozioni per l’analisi della società (Thoits, Hochschild, Flam, solo per
citare le studiose più note). p
Come sappiamo, Spinoza definisce il conatus come perseveranza nell’essere
(espressione di potenza in divenire) sostenendo come ciò che accade – «ciò che
ci accade» per dirla con Barthes – prescinda dalla volontà dell’individuo ma sia
piuttosto determinato dalla forza esterna ineluttabile degli affetti, a loro volta
definibili come gioiosi o tristi, a seconda delle situazioni. Un aspetto, questo,
che non solo lo separa da Marx (e anche da buona parte del pensiero sociologi-
co classico) ma sul quale, secondo Lordon, Spinoza oggi rappresenta un punto
di riferimento imprescindibile. Se infatti il comportamento umano è guidato
dalla centralità delle passioni – da logiche non razionali capaci di esercitare una
servitù passionale – risulta di conseguenza impossibile raggiungere la dimensio-
ne di soddisfacimento collettivo prefigurata da Marx con la sua idea di lavoro
e, più in generale, con l’avvento del comunismo. 2. Spinoza e lo strutturalismo delle passioni Al contrario, dato che siamo
continuamente immersi in rapporti di reciprocità fondati su interessi e passioni
che si manifestano come una più o meno prepotente forza esterna, si rende ne-
cessario rinunciare tanto al sogno di eliminare del tutto i rapporti di dipenden-
za quanto al raggiungimento di un Io perfettamente autonomo, e percorrere, al
contrario, la strada di un progressivo rafforzamento degli anticorpi nei confronti
del «desiderio-padrone», seppure in una ineliminabile dinamica di confronto. 3. Per una nuova teoria del valore Per questi motivi, lo strutturalismo delle passioni, in aperta rottura con il pa-
radigma marxiano dove è il tempo di lavoro astratto a produrre il valore di un
bene, sostiene come «l’operatore di ogni determinazione concreta nel mondo
storico-sociale» (Lordon 2018, 55) – il motore che muove i corpi e le menti –
sia invece l’affetto. Ovviamente non in termini di singolo conatus bensì come un
effetto prodotto dall’insieme dei conatus individuali. Saranno pertanto le mol-
titudini, altro concetto spinoziano che ha avuto una notevole fortuna nel dibat-
tito pubblico degli ultimi decenni (basta pensare agli scritti di Hardt e Negri),
con le loro mediazioni intersoggettive produttive di affetti, a determinare il va-
lore delle cose. Se sono le intensità affettive (la somma dei conatus individuali) 1028 Frédéric Lordon: il lavoro tra desiderio e servitù a produrre il valore delle cose, decade però, di fatto, il presupposto marxiano dei
rapporti di equivalenza quale base di costruzione del valore di un bene, pensato
come precedente allo scambio e direttamente implicato nella produzione: «in
una teoria socio-affettiva del valore, l’equivalenza è constatata ex post e per co-
sì dire de facto: essa assume quasi i tratti di una tautologia» (Lordon 2018, 56). Tutto avviene invece secondo la regola dell’immanenza, nella quale è lo scambio
tra gli individui a dare valore a una cosa a seconda dell’intensità delle passioni
che ne sono coinvolte e alla direzione che sprigionano. È pertanto il desiderio
del singolo, in forma aggregata di moltitudine, a definire il valore in itinere alla
stessa transazione economica. Lordon (2006) fa qui riferimento esplicito all’in-
formalità di buona parte di quelle che definiamo relazioni economiche: dagli
‘scambi di favori’ alla necessità di negoziare fino al ruolo della fiducia. E ci ri-
manda, seppur latamente, alle molteplici dimensioni non economiche dell’agire
economico che tanto spazio hanno avuto nelle scienze sociali contemporanee. 4. Il lavoro in epoca neoliberista Se la società funziona attraverso questa centralità delle passioni, che ne ca-
ratterizzano l’agire e le relazioni sociali, anche il lavoro non può esserne esente. Lordon si chiede come mai il capitalismo, con la sua logica di sfruttamento e
dominio, perduri nel tempo, e per quali motivi gli individui siano disponibili a
lavorare in condizioni, almeno apparentemente, spesso prive di senso. La spie-
gazione non può essere ricercata soltanto in negativo, come buona parte del-
la letteratura ha fatto: la violenza organizzativa del sistema, il fallimento della
lotta di classe, la crisi del movimento operaio. Il capitalismo di tipo fordista, ad
esempio, è stato in grado di arricchire «il complesso passionale del rapporto
salariale» (Lordon 2015, 47) attraverso l’invenzione e la diffusione del consu-
mo. In fondo, oltre a garantire la riproduzione materiale delle famiglie dei la-
voratori, che ha permesso la sostenibilità del modello, il lavoro salariato è stato
per lungo tempo nel Novecento anche generatore di desideri mediante i quali
soddisfare e controllare i lavoratori stessi. Con tutto il corollario necessario:
l’apparato ideologico, la pubblicità, il loisir, l’indebitamento al consumo ecc.t pp
g
p
La trasformazione post-fordista, tuttavia, non ha mutato soltanto il modello
di produzione e l’organizzazione del lavoro ma anche la funzione del «desiderio-
padrone». Il compito del sistema non è più, infatti, quello di incentivare desi-
deri ‘gioiosi’ nei lavoratori, anche se per lo più indotti attraverso il consumo, ma
quello di generare paura (di licenziamento, di restare senza protezione sociale,
di assenza delle condizioni primarie di sopravvivenza ecc.), sviluppando quin-
di la componente ‘triste’ del desiderio, per dirla con Benasayag. Si tratta di una
nuova fase storica nella quale le forze in campo sono talmente diseguali che la
partita appare già segnata in partenza: d’altronde, «l’egocentrismo del conatus,
quando gode di un’asimmetria di potenza favorevole, tende necessariamente
verso l’abuso» (Lordon 2015, 65). Di fronte alle trasformazioni del sistema ca-
pitalistico neoliberista, che consentono il licenziamento come modalità per ri-
durre i costi separandolo dal nodo etico che aveva nel fordismo e che rendono 1029 Andrea Valzania possibile delocalizzare una sede lavorativa senza che i sindacati possano avere
molta forza di opporsi, i lavoratori hanno solo la possibilità di adattarsi, «arruo-
landosi» (nell’allineamento perfetto prospettato dal capitale) alla volontà del
desiderio-padrone dominante. 4. Il lavoro in epoca neoliberista t In questo quadro, il neoliberismo, prendendo «direttamente in mano il la-
voro di ingegneria dei desideri e degli affetti» (Lordon 2015, 74), ha teorizzato
la necessità per i lavoratori di diventare imprenditori di sé stessi «pronti a met-
tersi in gioco nel processo permanente della concorrenza» (Dardot and Laval
2013, 235), trasferendo su di loro tutti i rischi sociali. Si tratta di un vero e pro-
prio cambio di paradigma nel quale è sempre più difficile separare il lavoro dal-
la vita (ovvero il tempo di lavoro dal tempo libero). Se infatti il vecchio modello
fordista incentivava necessità materiali o di evasione, costruendo appositi spazi
di fruizione e mantenendo la vita altrove, il neoliberismo incentiva, al contrario,
affetti intrinseci al solo lavoro. La grande novità è pertanto la simbiosi tra lavoro
e vita, così che la mobilitazione verso il desiderio-padrone sia totalizzante e le re-
sistenze, di qualunque natura esse siano, sempre più marginali. Il vero obiettivo
del neoliberismo è in ultima istanza produrre un allineamento dei salariati nei
confronti del desiderio-padrone improntato alla gioia: «l’assoggettato è felice
quando si vede proporre desideri che scambia per propri e che di fatto diventa-
no i suoi» (Lordon 2015, 84). 5. Riflessioni finali Sappiamo come una buona parte del Novecento sia stato contrassegnato an-
che dal dibattito sul processo di alienazione prodotto dal lavoro, che in seguito
ha vissuto una sorta di contrappasso storico finendo, forse troppo presto, nel
dimenticatoio. Nel 1964, epoca nella quale il tema aveva una grande rilevanza,
Goffredo Parise scrisse un romanzo – Il Padrone – nel quale un impiegato com-
piva, senza nessuna costrizione esterna ma al contrario ricercando un proprio
piacere personale, un vero e proprio processo di auto-reificazione di fronte al
proprio datore di lavoro. Il romanzo voleva essere allora una sorta di distopia
sarcastica, e allo stesso tempo polemica, nei confronti dell’egemonia pubblica
di una tematica e delle sue storture (per le quali, secondo alcune letture, tutto
finiva per essere alienante). Invece sembra che la distopia sia stata superata dal-
la realtà. Secondo Lordon, infatti, la finalità ultima del neoliberismo è proprio
quella che il lavoratore arrivi ad amare il padrone/imprenditore (o l’istituzio-
ne che permette di lavorare) «per farlo gioire e farsene amare», in una sorta di
totale identificazione simbolica, culturale e valoriale. Per raggiungere questo
obiettivo non è necessaria la costrizione, anzi, tutto ciò che è repressivo deve
essere accuratamente evitato e/o occultato. Il potere neoliberista, infatti, non
ricorre all’uso della forza ma vuole i propri salariati in una condizione di servitù
consensuale. Non esiste una «servitù volontaria» à la Boetiè, perché comunque
«al di là della costrizione fisica, non ci si potrà lasciare legare se non avendolo
più o meno voluto» (Lordon 2015, 33). Esiste al contrario una «servitù delle
passioni» che riguarda tutti: l’affezione crea il desiderio di una cosa e attraver- 1030 Frédéric Lordon: il lavoro tra desiderio e servitù so un processo di emulazione questo desiderio diventa collettivo (eteronomia
secondo la quale ci sottomettiamo volontariamente). Il processo emulativo raf-
forza l’asservimento ricercando nel comportamento dell’altro e nella sua appro-
vazione una conferma sociale. Il potere può agire per amore o per paura, come
insegna Spinoza, e il neoliberismo ha scelto questa seconda strada, imponendo
ai suoi membri una profonda assimilazione culturale al modello di riferimento. 5. Riflessioni finali Sono queste le basi sulle quali si fonda la sua capacità di trasformare, in manie-
ra irreversibile, le logiche ereditate dal fordismo, i cui effetti sono visibili oggi
nel funzionamento dei mercati del lavoro, a partire dai meccanismi di selezio-
ne improntati al mimetismo, alla somiglianza tra chi viene selezionato e il sele-
zionatore, fino ai processi di allineamento richiesti ai lavoratori, che assumono
una maggiore evidenza in situazioni segnate da precariato, in particolare di ti-
po cognitivo. Un meccanismo, quest’ultimo, noto anche come economia della
promessa, nel quale il lavoratore si trova ad accettare la situazione precaria nella
speranza si normalizzi (e perché comunque gli offre una seppur debole identi-
tà) mentre il datore di lavoro tende a procrastinarla non solo per mantenere la
disparità di potere tra lui e il lavoratore ma anche perché sa di poter contare su
una sorta di nuovo esercito di riserva da cui attingere. so un processo di emulazione questo desiderio diventa collettivo (eteronomia
secondo la quale ci sottomettiamo volontariamente). Il processo emulativo raf-
forza l’asservimento ricercando nel comportamento dell’altro e nella sua appro-
vazione una conferma sociale. Il potere può agire per amore o per paura, come
insegna Spinoza, e il neoliberismo ha scelto questa seconda strada, imponendo
ai suoi membri una profonda assimilazione culturale al modello di riferimento. Riferimenti bibliografici Dardot Pierre, and Christian Laval. 2010. La nouvelle raison du monde: Essai sur la société
néolibérale. Paris: Editions La Découverte (trad. it. La nuova ragione del mondo. Critica
della razionalità neoliberista. Roma: DeriveApprodi, 2013). pp
Lordon, Frédéric. 2003. “Revenir à Spinoza dans la conjuncture intellectuelle présente.”
L’Annèe de la Règulation 7: 147-66. g
Lordon, Frédéric. 2006. L’intérêt souverain. Essai d’anthropologie économique spinoziste. Paris: La Découverte.t Lordon, Frédéric, and Yves Citton. 2008. Spinoza et les sciences sociales. De l’économie
des affects à la puissance de la multitude. Paris: Éditions Amsterdam. f
Lordon, Frédéric. 2010. Capitalisme, désir et servitude. Marx et Spinoza. Paris: La Fabrique
éditions (trad.it. Capitalismo, desiderio e servitù. Antropologia delle passioni nel lavoro
contemporaneo. Roma: DeriveApprodi, 2015). p
pp
Lordon, Frédéric. 2013. La société des affects: pour un structuralisme des passions. Paris:
Seuil. Lordon, Frédéric. 2018. La condition anarchique. Paris: Seuil (trad.it. La condizione
anarchica. Vicenza: Neri Pozza, 2021).f Parise, Goffredo. 1964. Il padrone. Milano: Feltrinelli. Parise, Goffredo. 1964. Il padrone. Milano: Feltrinelli. Altri riferimenti bibliografici Altri riferimenti bibliografici Altri riferimenti bibliografici Bidet, Jacques, and Gèrard Dumenil. 2007. Altermarxisme. Un autre marxisme pour un
autre monde. PUF: Paris.fi Bove, Laurent. 1996. La Strategie Du Conatus: Affirmation Et Resistance Chez Spinoza:
Affirmation et résistance chez Spinoza. Paris: Librairie Philosophique J. Vrin.hh ffi
p
Klikauer, Thomas. 2016. “Spinoza and Marx on Desire and Management.” The Journal
of Labour & Society 19: 553-61. f
y
Lazzarato, Maurizio. 2004. Les Révolutions du capitalisme. Paris: Seuil. 1031 Andrea Valzania g
Marengo, Stefano. 2017. “Passioni e istituzioni. Frédéric Lordon, Spinoza e le scienz
sociali.” Quaderni materialisti 2014-2015, 13-14: 132-49. Lordon, Frédéric. 2015. Imperium. Structures et affects des corps politiques. Paris: La
Fabrique.f Andrea Valzania Lordon, Frédéric. 2015. Imperium. Structures et affects des corps politiques. Paris: La
Fabrique.f q
Lordon, Frédéric. 2016. Les affects de la politique. Paris: Éditions du Seuil. ff
p
q
Lordon, Frédéric. 2021. Figures du communism. Paris: La Fabrique. Marengo, Stefano. 2017. “Passioni e istituzioni. Frédéric Lordon, Spinoza e le scienze
sociali.” Quaderni materialisti 2014-2015, 13-14: 132-49. 1032
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Impact of Heat Shock Protein 90 Inhibition on the Proteomic Profile of Lung Adenocarcinoma as Measured by Two-Dimensional Electrophoresis Coupled with Mass Spectrometry
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Received: 24 June 2019; Accepted: 30 July 2019; Published: 31 July 2019 Abstract: Heat shock protein 90 (HSP90) is an important chaperone in lung adenocarcinoma, with
relevant protein drivers such as EGFR (epidermal growth factor receptor) and EML4-ALK (echinoderm
microtubule-associated protein-like protein4 fused to anaplastic lymphoma kinase) depending on it
for their correct function, therefore HSP90 inhibitors show promise as potential treatments for lung
adenocarcinoma. To study responses to its inhibition, HSP90 was pharmacologically interrupted
by geldanamycin and resorcinol derivatives or with combined inhibition of HSP90 plus HSP70
in lung adenocarcinoma cell lines. Two-dimensional electrophoresis was performed to identify
proteomic profiles associated with inhibition which will help to understand the biological basis for the
responses. HSP90 inhibition resulted in altered protein profiles that differed according the treatment
condition studied. Results revealed 254 differentially expressed proteins after treatments, among
which, eukaryotic translation initiation factor3 subunit I (eIF3i) and citrate synthase demonstrated
their potential role as response biomarkers. The differentially expressed proteins also enabled
signalling pathways involved in responses to be identified; these included apoptosis, serine-glycine
biosynthesis and tricarboxylic acid cycle. The proteomic profiles identified here contribute to an
improved understanding of HSP90 inhibition and open possibilities for the detection of potential
response biomarkers which will be essential to maximize treatment efficacy in lung adenocarcinoma. Keywords: lung cancer; proteomic; chaperones; HSP90 inhibitors cells cells Impact of Heat Shock Protein 90 Inhibition on the
Proteomic Profile of Lung Adenocarcinoma as
Measured by Two-Dimensional Electrophoresis
Coupled with Mass Spectrometry Ángela Marrugal 1
, Irene Ferrer 1,2, Maria Dolores Pastor 3, Laura Ojeda 1
,
Álvaro Quintanal-Villalonga 4, Amancio Carnero 2,3
, Sonia Molina-Pinelo 2,3,*,† and
Luis Paz-Ares 1,2,5,6,*,† Ángela Marrugal 1
, Irene Ferrer 1,2, Maria Dolores Pastor 3, Laura Ojeda 1
,
Álvaro Quintanal-Villalonga 4, Amancio Carnero 2,3
, Sonia Molina-Pinelo 2,3,*,† and
Luis Paz-Ares 1,2,5,6,*,† 1
H12O-CNIO Lung Cancer Clinical Research Unit, Instituto de Investigación Hospital 12 de Octubre &
Centro Nacional de Investigaciones Oncológicas (CNIO), 28029 Madrid, Spain
2
CIBERONC, 28029 Madrid, Spain
3
Instituto de Biomedicina de Sevilla (IBIS) (HUVR, CSIC, Universidad de Sevilla), 41013 Sevilla, Spain
4
Program in Molecular Pharmacology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
5
Medical Oncology Department, Hospital Universitario Doce de Octubre, 28041 Madrid, Spain
6
Medical School, Universidad Complutense, 28040 Madrid, Spain
*
Correspondence: smolina-ibis@us.es (S.M.-P.); lpazaresr@seom.org (L.P.-A.)
†
These authors contributed equally to this work. p
p
*
Correspondence: smolina-ibis@us.es (S.M.-P.); lpazaresr@seom.org (L.P.-A.) †
These authors contributed equally to this work. 1. Introduction Lung cancer is the leading cause of cancer-related death globally, with a 5-year relative survival
rate of only 18% on account of it being commonly diagnosed at advanced stages [1]. There are two
major types of lung cancer non-small-cell lung cancer (NSCLC), which accounts for 85% of lung
tumors, and small-cell lung cancer (SCLC) accounting for the rest. In turn, NSCLCs are histologically
classified according to three subtypes: adenocarcinoma, squamous cell carcinoma and large cell Cells 2019, 8, 806; doi:10.3390/cells8080806 www.mdpi.com/journal/cells www.mdpi.com/journal/cells 2 of 22 Cells 2019, 8, 806 carcinoma [2], with several molecular alterations underlying each histological subtype. This has
allowed therapies that target some of these molecular aberrations to be developed [3,4]. While such
targeted treatments have achieved improved responses and survival rates, acquired resistance to
these treatments is a problem. Moreover, not all NSCLC molecular subtypes have a specific targeted
therapy. There is consequently a clear need for novel and broader-spectrum therapies that improve
patient responses [5]. Focusing on adenocarcinoma, the main NSCLC subtype (50%), more than half of
the cases are driven by recognized oncogenic alterations, such as epidermal growth factor receptor
(EGFR), Kirsten rat sarcoma viral oncogene homolog (KRAS), Echinoderm Microtubule-associated
protein-like protein 4 fused to anaplastic lymphoma kinase (EML4-ALK), mesenchymal-epithelial
transition (MET) factor, serine/threonine-protein kinase B-Raf (BRAF) or human epidermal growth
factor 2 (HER2/ErbB2/neu) [6,7]. As most of these proteins are clients of 90kDa heat shock protein
(HSP90) [8], an elevated expression of HSP90 has consequently been correlated with a poorer clinical
prognosis [9,10] as well as with resistance to chemo- and radiotherapy [11–15]. p
g
[ ,
]
py [
]
HSP90 is one of the most abundant and evolutionarily conserved molecular chaperones. Besides representing 1–2% of all cellular proteins, this chaperone can increase its expression up to 10-fold
in response to physiological stress [16]. Dominant isoforms are the constitutively expressed HSP90β
and the stress inducible HSP90α which can be found in the cytoplasm, nucleus or even on the cell surface
and extracellular space (for HSP90α) [17]. Both isoforms are collectively named HSP90 unless specified,
and play a critical role in the maturation, stabilization and regulation of so-called “client” proteins
via an ATP-driven chaperone cycle regulated by co-chaperones such as HSP70 and p23 [18]. 2.2. siRNA Transfection Transient silencing of HSP90 was achieved through small interfering RNAs (siRNAs). For HSP90α
and HSP90β silencing, siRNAs were purchased from Origene (Rockville, MD USA) (SR302262 and
SR302264). In turn, control cells were transfected with scrambled siRNA (SR30002). LipofectamineTM
RNAiMAX (Invitrogen, Carlsbad, CA, USA), a cationic lipid transfection reagent, was used to transfect
all cell lines according to the manufacturer’s instructions. Simultaneous transfections were performed
to silence both genes in the same cell line. 2.3. Drug Treatment The HSP90 inhibitors used in this study included analogues of geldanamycin: tanespimycin
(17-N-allylamino-17-demethoxygeldanamycin, 17-AAG) (Selleckchem, Munich, Germany) and
retaspimycin hydrochloride ((4E,6Z,8S,9S,10E,12S,13R,14S,16R)-19-(allylamino)-13,20,22-trihydroxy-
8,14-dimethoxy-4,10,12,16-tetramethyl-3-oxo-2-azabicyclo[1 6.3.1]docosa-1(22),4,6,10,18,20-hexaen-9-
yl carbamate, IPI-504) (Eurodiagnóstico, Madrid, Spain) and radicicol derivatives: ganetespib
(3-(2,4-Dihydroxy-5-isopropylphenyl)-4-(1-methylindol-5-yl)-5-hydroxy-4H-1,2,4-triazole, STA-9090)
and
luminespib
(5-(2,4-dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4-(morpholinomethyl)phenyl)
isoxazole-3carboxamide, AUY-922) (Selleckchem). VER-155008 (5’-O-[(4-Cyanophenyl)methyl]-8-[[(3,4-
dichlorophenyl)methyl]amino]-adenosine) (ApexBio, Houston, TX, USA) was used as an HSP70
inhibitor. The chemical structure of all inhibitors used can be accessed on the website https:
//pubchem.ncbi.nlm.nih.gov/. All drugs were dissolved in dimethyl sulfoxide (DMSO) as stock
solutions according to manufacturers’ instructions. Cells were treated for 96 h with the different
inhibitors in concentrations ranging from 0.33 nM to 20 uM to calculate half-maximal inhibitory
concentrations (IC50). Thereafter, the concentration for each drug at which growth is reduced to 80%
(IC80) was calculated and applied to cell lines maintained in complete growth medium. Protein extracts
were collected at different times (10, 24 and/or 48 h) according to subsequent techniques to be used. 1. Introduction Many
of the approximately 300 client proteins (https://www.picard.ch/downloads/Hsp90interactors.pdf)
play key roles in oncogenic signalling and in different hallmarks of cancer such as proliferation,
evasion of apoptosis, immortalization, angiogenesis, invasion and metastasis. Due to of their strong
dependence on HSP90, inhibition of the latter leads to ubiquitin-mediated proteasomal degradation of
client proteins concluding with the downregulation of different oncogenic signalling pathways [19]. Since EGFR [20], BRAF [21], ERBB2 [22], MET [23,24] and the EML4-ALK translocation product [8] are
clients of HSP90, acting as oncodrivers in different clinico-pathological subsets of lung adenocarcinoma,
degradation of these oncoproteins through HSP90 inhibition leads to loss of tumor-cell viability [25–28]. Promising results have been shown in different clinical studies, especially in malignancies that possess
an HSP90 client as an oncodriver [29–31]. However, as not all lung adenocarcinomas respond equally
to HSP90 inhibitors [32,33], a better understanding of the cellular consequences of HSP90 inhibition
will therefore be key to improve clinical outcomes in this tumor type. Proteomic methods have been used widely to identify protein network alterations that may be
linked to drugs used to treat lung cancer in terms of sensitivity and resistance [34–37]. Specifically,
two-dimensional gel electrophoresis has been employed to analyze protein expression profiles and
identify novel diagnostic, prognostic or predictive biomarkers in these tumors [38–44]. Due to
the complex interactome of HSP90 and the enormous number of cellular processes in which it is
involved, the use of proteomic tools for the study of this chaperone and its clients is a common
approach [17,45–47]. However, additional information is needed to dissect responses to HSP90
inhibitors in lung cancer. For this reason, we analyzed proteome modulation by HSP90 inhibitors in
different lung adenocarcinoma cell lines using two-dimensional electrophoresis coupled with tandem
mass spectrometry. Comparisons of proteomic expression profiles in drug-treated and untreated cells
showed remarkable changes in both the number and expression levels of many proteins. Differentially
expressed proteins could help in the identification of plausible biomarkers and elucidate cellular
processes involved in responses arising from the inhibition of HSP90 in these tumors. 3 of 22 Cells 2019, 8, 806 2.1. Cell Cultures The human lung adenocarcinoma cell lines HCC827 (CRL-2868), A549 (CCL-185) and H1437
(CRL-5872) were obtained from American Type Culture Collection (ATCC), and the H3122 cell
line was kindly provided by Dr. Koivunen. Cell lines were propagated in RPMI-1640 medium
(Sigma-Aldrich, Saint Louis, MO, USA), with the exception of A549 which was cultured in DMEM
(Sigma-Aldrich). All cell lines were supplemented with 10% (v/v) fetal bovine serum (FBS, TICO
Europe, Amstelveen, The Netherlands), 1% (v/v) antibiotic-antimycotic solution (Sigma-Aldrich) and
1% (v/v) glutamine. Cells were maintained at 37 ◦C in a humidified incubator in a 5% CO2 and 95% air
atmosphere. Cells were authenticated and regularly checked for mycoplasma. 2.6. Two-Dimensional Electrophoresis and Gel Image Analysis In the experiment, 240 µg of total protein from each individual sample were diluted to up to
250 µL with DeStreak rehydration solution (GE Healthcare) and 0.5% 3–11 NL pH IPG buffer (GE
Healthcare). Each mixture was loaded onto 13 cm IGP DryStrips (GE Healthcare) with a 3–10 NL pH
gradient. The first-dimension separation—isoelectric focusing (IEF) —was then carried out on an Ettan
IPGphor II system (GE Healthcare). IPG DryStrips were equilibrated in a reducing agent followed by
an alkylating agent. The second dimension was performed by placing the strips on 8–16% CriterionTM
TGX stain-free acrylamide gels (Bio-Rad) to allow protein separation by electrophoresis in a Criterion
DodecaTM cell (Bio-Rad). The analytical gels were visualized with Typhoon 9400 (GE Healthcare) after
SYPRO® (Bio-Rad) staining. The digitalized 2-DE gel images were studied (protein spot detection,
spot matching and semi-quantitative statistical analysis) using PDQuest software (Bio-Rad). For each
studied condition, three different gel images were analyzed and a corresponding reference synthetic
image was obtained. To improve accuracy, detected spots and spot matches after in silico matching,
were manually edited. 2.4. Immunoblotting Total protein extracts from the studied cell lines were isolated with RIPA buffer (Sigma-Aldrich)
containing a protease inhibitor cocktail (cOmpleteTM Mini EDTA-free, Roche, Basel, Switzerland) and
a phosphatase inhibitor cocktail (PhosSTOP EASYpack, Roche). A standard western blot protocol
was employed with a miniProtean electrophoretic system (BioRad, Berkeley, CA, USA) and a wet
electroblotting system (BioRad). Primary antibodies against HSP90α (ab79849, Abcam, Cambridge, UK),
HSP90β (ab53497, Abcam), GRP94 (ab18055, Abcam), HSP70 (ab45133, Abcam), HSP27 (#2402, Cell
Signaling, Danvers, MA, USA) EGFR (#4267, Cell Signaling), pEGFR (#2234, Cell Signaling), ALK
(#3633, Cell Signaling), eIF3i (ab40745, Abcam), citrate synthase (#14309, Cell Signaling), α-Tubulin
(T9026, Sigma-Aldrich) or β-actin (A5316, Sigma-Aldrich) were used. Antibody-bound proteins were
detected by enhanced chemiluminescence (Clarity Western ECL Blotting Substrates (BioRad)) using
the ChemiDoc system (BioRad) after incubation with horseradish peroxidase-conjugated anti-mouse 4 of 22 Cells 2019, 8, 806 secondary antibody (#7076, Cell Signaling) or anti-rabbit secondary antibody (#7074, Cell Signaling). The ratios between signals from proteins of interest and α-tubulin or β-actin were calculated to
determine the relative protein expression values. No grouping of gels/blots cropped from different
parts of the same gel, or from different gels, fields or exposures was performed. 2.5. Proteomic Sample Preparation Cells were seeded into 10 cm diameter dishes with the relevant culture medium at the appropriate
density to reach 80 percent confluence 24 h post-seeding. On the following day, cells were treated
with the different HSP90 inhibitors or the HSP90 (17-AAG) plus HSP70 (VER-155008) inhibitors at
its IC80 while the negative control was treated with the same amount of vehicle DMSO used in the
inhibited cells. After 24 h, cells were collected, precipitated, washed with ice-cold PBS and finally each
pellet was shock-frozen in liquid nitrogen and stored at −80 ◦C until further use. Before use, pellets
were thawed on ice and then cells were resuspended in HEN buffer (50 mM HEPES (Sigma-Aldrich),
5 mM ethylenediaminetetraacetic acid (EDTA) (Invitrogen Ambion), 250 mM sodium chloride (NaCl)
(Panreac, Barcelona, Spain), 5 mM dithiothreitol (DTT) (GE Healthcare, Chicago, IL, USA), 1 mM
sodium orthovanadate (Sigma-Aldrich), 0.2% IGEPAL (Sigma-Aldrich) and 5 µL of protease inhibitor
cocktail (Sigma-Aldrich)) and lysed for 1 h on ice with intermittent vortexing. Protein samples from each cell line and condition were pre-treated with the 2-D Clean Up Kit (GE
Healthcare) and the final pellets were resuspended in 2-DE lysis buffer (8 M urea (GE Healthcare), 4%
CHAPS (GE Healthcare), 2% IPG buffer pH 3-11 nonlinear (NL) (GE Healthcare) and 40 mM DTT(GE
Healthcare)). Protein samples were maintained overnight at 4 ◦C with gentle rotation to ensure their
complete resolubilization. Protein concentrations in the 2-DE samples were determined by the 2-D
Quant-Kit (GE Healthcare). 2.7. MALDI-TOF/TOF Mass Spectrometry Analysis Spots that were present in only one of the conditions or displayed quantitative abundance changes
of more than 2-fold were selected for identification by MALDI-TOF/TOF. Protein spots of interest
were picked from the stained gel using the Investigator ProPic robotic workstation (Genomic Solution,
Huntingdon, UK) and were then washed and digested. The samples were mixed with a matrix
solution CCA (α-cyano-4-hydroxycinnamic acid), spotted on a MALDI plate (Applied Biosystems,
Foster City, CA, USA) and allowed to air-dry. To obtain a peptide mass fingerprint (PMF), lists of
peak intensities and mass-to-charge (m/z) values were analyzed with a 4800 Proteomics Analyzer
MALDI-TOF/TOF Mass Spectrometer (Applied Biosystems). 5 of 22 Cells 2019, 8, 806 2.9. Protein Functional Analysis The gene ontology enrichment analysis of identified proteins was performed using the PANTHER
(Protein ANalysis THrough Evolutionary Relationships) online database (http://pantherdb.org/) [48,49]. Pathway analyses were carried out for differentially expressed proteins from the cell lines studied in
response to different inhibitors. This database was also used to categorize identified proteins according
to their biological processes and molecular functions. Only results whose adjusted p-value was lower
than 0.05 were considered statistically significant and used in the study. 2.8. Protein Identification High-resolution tandem mass spectra MS + (MS/MS) data, peptide matching and protein
identification were determined with web-based MASCOT software (http://www.matrixscience.com/)
using SWISS-PROT as the protein sequence database with a mass tolerance of ± 50 ppm. Candidate
proteins were selected from each spot, using the number of matched peptides, sequence coverage,
molecular mass and isoelectric point, among other variables, as criteria for accepting the identification. 3.1. Characterization of Lung Adenocarcinoma Cell Lines Lung adenocarcinoma cell lines from different backgrounds were characterized based on protein
expression levels measured for HSP90, other related HSPs and HSP90 client proteins such as EGFR
and EML4-ALK (Figure 1). The EGFR-mutant cell line (HCC827) showed highest level of EGFR
expression (followed by the KRAS-mutant A549 cells) and was the only cell line showing EGFR
activation. The presence of EML4-ALK fusion protein was detected exclusively in the H3122 cell line. Focusing on HSPs, three of the four cell lines studied showed similar levels of cytosolic HSP90 (α and
β) protein expression, while the H1437 cell line (triple-negative; EGFR, ALK and KRAS wild-type)
exhibited a higher expression of HSP90α and a lower expression of HSP90β than the other cell lines
studied. Similarly, GRP94 (the endoplasmic reticulum HSP90 isoform) expression was higher in the
H1437 cell line than the other cell lines. On the other hand, protein levels of HSP70 were similar in all
cell lines although slightly higher in the A549 and H1437 cell lines. Furthermore, the HCC827 and
A549 cell lines showed high HSP27 protein expression levels compared to almost undetectable levels
in the other cell lines. Figure 1. Characterization of a panel of adenocarcinoma cell lines. Western blot analysis of
adenocarcinoma cell lines whose oncodrivers are either directly (epidermal growth factor receptor
(EGFR) in HCC827 and EML4-ALK in H3122 cell lines) or indirectly Kirsten rat sarcoma (KRAS) viral
oncogene homolog) in the A549 cell line) related to HSP90, as well as an EGFR, anaplastic lymphoma
kinase (ALK) and KRAS wild-type cell line (H1437). This technique was employed to study protein
expression levels of constitutive and inducible HSP90, other related heat shock proteins (HSPs) (GRP94,
HSP70 and HSP27) as well as the HSP90 clients EGFR, its phosphorylated form pEGFR, and EML4-ALK. For Western blots, a representative image is shown. Densitometry analysis may be found in Figure S1. Figure 1. Characterization of a panel of adenocarcinoma cell lines. Western blot analysis of
adenocarcinoma cell lines whose oncodrivers are either directly (epidermal growth factor receptor
(EGFR) in HCC827 and EML4-ALK in H3122 cell lines) or indirectly Kirsten rat sarcoma (KRAS) viral
oncogene homolog) in the A549 cell line) related to HSP90, as well as an EGFR, anaplastic lymphoma
kinase (ALK) and KRAS wild-type cell line (H1437). 3.2. Effectiveness of HSP90 Inhibitors in Lung Adenocarcinoma Cell Lines In relation to HSP90 inhibition,
IC50 values ranged from 2814 to 30,733 µM, with the radicicol derivatives (STA-9090 and AUY-922)
generally having lower IC50 values particularly in the EGFR-mutant and EML4-ALK translocated
cell lines. Subsequently, western blot analyses were used to evaluate responses to HSP90 inhibition at
different time points based on IC80 values (Figure 2). We identified that HSP90 inhibitors induced
compensatory expression of HSP70 and HSP90, along with significant reductions in the HSP90 clients
EGFR and EML4-ALK since the 10 h time-point in analyzed treatments. Moreover, EGFR degradation
was faster and more pronounced in the EGFR-mutant cell line HCC827 than in the KRAS-mutant cell
line A549 despite the high initial expression of EGFR. It is also worth noting that HSP90 inhibition in
the H3122 cell line dramatically inhibited expression of its oncodriver EML4-ALK. nM = nanomolar; µM = micromolar. The studied cell lines (especially the cell line harboring EML4-ALK translocation, H3122) were
more sensitive to HSP90 inhibitors than to the HSP70 inhibitor used. In relation to HSP90 inhibition,
IC50 values ranged from 2,814 to 30,733 µM, with the radicicol derivatives (STA-9090 and AUY-922)
generally having lower IC50 values particularly in the EGFR-mutant and EML4-ALK translocated
cell lines. Subsequently, western blot analyses were used to evaluate responses to HSP90 inhibition
at different time points based on IC80 values (Figure 2). We identified that HSP90 inhibitors induced
compensatory expression of HSP70 and HSP90, along with significant reductions in the HSP90
clients EGFR and EML4-ALK since the 10 h time-point in analyzed treatments. Moreover, EGFR
degradation was faster and more pronounced in the EGFR-mutant cell line HCC827 than in the
KRAS-mutant cell line A549 despite the high initial expression of EGFR. It is also worth noting that
HSP90 inhibition in the H3122 cell line dramatically inhibited expression of its oncodriver
EML4 ALK HCC827
EGFR
Tubulin
HSP70
HSP90α
17-AAG
IPI-504
STA-9090
AUY-922
A549
EGFR
Tubulin
HSP70
HSP90α
17-AAG
IPI-504
STA-9090
AUY-922
H1437
HSP90α
Tubulin
HSP70
17-AAG
IPI-504
STA-9090
AUY-922
H3122
ALK
Tubulin
HSP70
HSP90α
17-AAG
IPI-504
STA-9090
AUY-922
A
C
B
D
HSP90α
EGFR
HSP70
Tubulin
HCC827
VER-155008+17-AAG
E
Figure 2. Analysis of sensitivity of adenocarcinoma cell lines to HSP90 inhibitors. Western blot
determination of the effect of 17-AAG, IPI-504, STA-9090 and AUY-922 inhibitors on
adenocarcinoma cell lines characterized by (A) mutated EGFR, (B) EML4-ALK translocation, (C)
mutated KRAS and (D) EGFR, ALK and KRAS wild-type. 3.2. Effectiveness of HSP90 Inhibitors in Lung Adenocarcinoma Cell Lines 3.2. Effectiveness of HSP90 Inhibitors in Lung Adenocarcinoma Cell Lines Cells were treated with different doses of HSP90 (17-AAG, IPI-504, STA-9090 and AUY-922) and
HSP70 (VER-155008) inhibitors with a view to calculating IC50 values and examining the efficacy of
each drug in each cell line (Table 1). Cells 2019, 8, x FOR PEER REVIEW
6 of 22 Cells were treated with different doses of HSP90 (17-AAG, IPI-504, STA-9090 and AUY-922) and
HSP70 (VER-155008) inhibitors with a view to calculating IC50 values and examining the efficacy of
each drug in each cell line (Table 1). Cells 2019, 8, x FOR PEER REVIEW
6 of 22 Table 1. IC50 values of different HSP90 and HSP70 inhibitors in the studied cell lines. Cell
Line
HSP90 Inhibitors
HSP70
Inhibitor
EGFR
ALK
KRAS
IC50
17-AAG
nM
IC50
IPI-504
nM
IC50
STA-9090
nM
IC50
AUY-922
nM
IC50
VER-155008
µM
HCC827
26.255
17.145
5.138
4.167
2.081
E746-A750
del
WT
WT
H3122
26.165
28.371
7.991
9.11
33.898
WT
EML4-ALK
v1
WT
A549
16.296
19.492
6.31
30.733
24.487
WT
WT
p.G12S
H1437
3.708
3.473
6.794
2.814
24.811
WT
WT
WT
nM = nanomolar; µM = micromolar. 3.2. Effectiveness of HSP90 Inhibitors in Lung Adenocarcinoma Cell Lines
Cells were treated with different doses of HSP90 (17-AAG, IPI-504, STA-9090 and AUY-922)
and HSP70 (VER-155008) inhibitors with a view to calculating IC50 values and examining the
efficacy of each drug in each cell line (Table 1). Table 1. IC50 values of different HSP90 and HSP70 inhibitors in the studied cell lines. Cell
Line
HSP90 Inhibitors
HSP70
Inhibitor
EGFR
ALK
KRAS
IC50
17-AAG
nM
IC50
IPI-504
nM
IC50
STA-9090
nM
IC50
AUY-922
nM
IC50
VER-155008
µM
HCC827
26.255
17.145
5.138
4.167
2.081
E746-A750
del
WT
WT
H3122
26.165
28.371
7.991
9.11
33.898
WT
EML4-ALK
v1
WT
A549
16.296
19.492
6.31
30.733
24.487
WT
WT
p.G12S
H1437
3 708
3 473
6 794
2 814
24 811
WT
WT
WT Table 1. IC50 values of different HSP90 and HSP70 inhibitors in the studied cell lines. Effectiveness of HSP90 Inhibitors in Lung Adenocarcinoma Cell Lines
Cells were treated with different doses of HSP90 (17-AAG IPI-504 STA-9090 and AUY-9 The studied cell lines (especially the cell line harboring EML4-ALK translocation, H3122) were
more sensitive to HSP90 inhibitors than to the HSP70 inhibitor used. 3.1. Characterization of Lung Adenocarcinoma Cell Lines This technique was employed to study protein
expression levels of constitutive and inducible HSP90, other related heat shock proteins (HSPs) (GRP94,
HSP70 and HSP27) as well as the HSP90 clients EGFR, its phosphorylated form pEGFR, and EML4-ALK. For Western blots, a representative image is shown. Densitometry analysis may be found in Figure S1. Figure 1. Characterization of a panel of adenocarcinoma cell lines. Western blot analysis of
adenocarcinoma cell lines whose oncodrivers are either directly (epidermal growth factor receptor
(EGFR) in HCC827 and EML4-ALK in H3122 cell lines) or indirectly Kirsten rat sarcoma (KRAS) viral
oncogene homolog) in the A549 cell line) related to HSP90, as well as an EGFR, anaplastic lymphoma
kinase (ALK) and KRAS wild-type cell line (H1437). This technique was employed to study protein
expression levels of constitutive and inducible HSP90, other related heat shock proteins (HSPs) (GRP94,
HSP70 and HSP27) as well as the HSP90 clients EGFR, its phosphorylated form pEGFR, and EML4-ALK. For Western blots, a representative image is shown. Densitometry analysis may be found in Figure S1. Cells 2019, 8, 806 6 of 22 3.2. Effectiveness of HSP90 Inhibitors in Lung Adenocarcinoma Cell Lines (E) Study of combined treatment of HSP70
and HSP90 inhibitors in the HCC827 cell line. HSP90α and HSP70 expression was determined in
Figure 2. Analysis of sensitivity of adenocarcinoma cell lines to HSP90 inhibitors. Western blot
determination of the effect of 17-AAG, IPI-504, STA-9090 and AUY-922 inhibitors on adenocarcinoma
cell lines characterized by (A) mutated EGFR, (B) EML4-ALK translocation, (C) mutated KRAS and
(D) EGFR, ALK and KRAS wild-type. (E) Study of combined treatment of HSP70 and HSP90 inhibitors
in the HCC827 cell line. HSP90α and HSP70 expression was determined in each of the cell lines along
with the HSP90 clients EGFR and EML4-ALK. For Western blots, a representative image is shown. Densitometry analysis may be found in Figure S2. H3122
ALK
Tubulin
HSP70
HSP90α
17-AAG
IPI-504
STA-9090
AUY-922
B HCC827
EGFR
Tubulin
HSP70
HSP90α
17-AAG
IPI-504
STA-9090
AUY-922
A
C B A HSP90α
EGFR
HSP70
Tubulin
HCC827
VER-155008+17-AAG
E E D H1437
HSP90α
Tubulin
HSP70
17-AAG
IPI-504
STA-9090
AUY-922
D C A549
EGFR
Tubulin
HSP70
HSP90α
17-AAG
IPI-504
STA-9090
AUY-922
C Figure 2. Analysis of sensitivity of adenocarcinoma cell lines to HSP90 inhibitors. Western blot
determination of the effect of 17-AAG, IPI-504, STA-9090 and AUY-922 inhibitors on
adenocarcinoma cell lines characterized by (A) mutated EGFR, (B) EML4-ALK translocation, (C)
mutated KRAS and (D) EGFR, ALK and KRAS wild-type. (E) Study of combined treatment of HSP70
and HSP90 inhibitors in the HCC827 cell line. HSP90α and HSP70 expression was determined in
Figure 2. Analysis of sensitivity of adenocarcinoma cell lines to HSP90 inhibitors. Western blot
determination of the effect of 17-AAG, IPI-504, STA-9090 and AUY-922 inhibitors on adenocarcinoma
cell lines characterized by (A) mutated EGFR, (B) EML4-ALK translocation, (C) mutated KRAS and
(D) EGFR, ALK and KRAS wild-type. (E) Study of combined treatment of HSP70 and HSP90 inhibitors
in the HCC827 cell line. HSP90α and HSP70 expression was determined in each of the cell lines along
with the HSP90 clients EGFR and EML4-ALK. For Western blots, a representative image is shown. Densitometry analysis may be found in Figure S2. Cells 2019, 8, 806 7 of 22 3.3. Effects of HSP90 Inhibition on Protein Expression Patterns in Adenocarcinoma Cell Lines 3.3. 3.2. Effectiveness of HSP90 Inhibitors in Lung Adenocarcinoma Cell Lines Effects of HSP90 Inhibition on Protein Expression Patterns in Adenocarcinoma Cell Lines To identify proteins correlated with HSP90 inhibition, we used 2-DE to analyze the proteome
from each cell line treated with the four selected HSP90 inhibitors, the HSP90 (17-AAG) plus HSP70
(VER-155008) inhibitors combination, or vehicle treatment for 24 h. To eliminate gel to gel variation,
isoelectric focusing and the second dimension of all samples for each cell line were performed at the
one time in a single assay. Different proteome profiles of treated and control groups in the studied cell
lines could be visualized following image analysis by PDQuest 2-D software. Representative 2-DE
gels for inhibited and untreated HCC827, H3122, A549 and H1437 cells are shown in Figures S3–S7
where the two most over- and down-expressed protein spots for each condition and cell line are
given. An average of 473, 201, 257 and 263 protein spots were detected, quantified, normalized and
inter-gel-matched in the EGFR, EML4-ALK, KRAS, and triple-negative cell lines, respectively (Table 2). Table 2. Number of matched protein spots in the considered cell lines under the different
treatment conditions. Condition
Cell Line
HCC827
H3122
A549
H1437
Control
492
199
263
253
17-AAG
488
200
259
264
IPI-504
480
202
266
262
STA-9090
499
200
235
265
AUY-922
507
197
259
261
VER-155008+17AAG
374
209
257
275 Protein spots included in the analyses were those with significant quantitative differences of at
least 2-fold expression variation between treatments and control or those qualitative spots present only
in some of the treatments or the control. The results, after mass spectrometry analysis, of identified
protein variations after treatments are shown in Table 3 and Tables S1–S5. Table 3. Number of differentially expressed proteins identified in the studied cell lines for the
different treatments. Table 3. Number of differentially expressed proteins identified in the studied cell lines for the
different treatments. Cells 2019, 8, 806 Cells 2019, 8, 806 8 of 22 Regarding differentially expressed proteins in the EGFR cell line, HSP70, enolase and mutL-like 1
(MLH1) protein were among the most overexpressed proteins, while HSP90AA1 (HSP90α), sumo3 and
HSC71 were the most down-expressed. In the EML4-ALK cell line, anterior gradient protein 2 homolog
(AGR2) was the most overexpressed protein while eukaryotic translation initiation factor 3 subunit I
(eIF3i) was the most down-expressed protein after inhibition. MHC class I antigen, citrate synthase (CS),
transketolase and elongation factor 1-alpha were some of the most overexpressed proteins in the KRAS
cell line, while pyruvate kinase and ATP synthase subunit beta were among the most down-expressed. Lastly, in the triple-negative cell line, transketolase, mitochondrial aldehyde dehydrogenase 2 variant
(ALDH2) and phenylalanine tRNA ligase beta subunit were among the most overexpressed proteins,
whereas 3-hydroxyacyl-CoA dehydrogenase type-2, alternative protein fibroblast growth factor 1
(FGF1) and creatine kinase U type were commonly detected as down expressed after treatments F1) and creatine kinase U-type were commonly detected as down-expressed after treatments. Concerning HSP90 inhibitors, the radicicol derivative STA-9090 was the treatment in response
to which more proteins were deregulated and the only one that showed a higher number of
down-expressed than overexpressed proteins. On the other hand, the geldanamycin derivative
IPI-504 showed the second highest dysregulation of the HSP90 inhibitors. It was also the treatment
which produced the largest number of overexpressed proteins, which was mainly due to the response
of the A549 and H1437 cell lines exposed to this inhibitor. Finally, the HSP90 (17-AAG) plus HSP70
(VER-155008) inhibitor combination led to greater protein dysregulation than that seen with the HSP90
inhibitors studied alone. Like under HSP90 inhibition, the highest number of deregulated proteins
was detected in the EGFR cell line. Besides, this along with the KRAS cell line were the only cell lines
where the number of down-expressed proteins was greater that than of overexpressed proteins in
response to the combined inhibition treatment. Following an in-depth analysis of the geldanamycin-derivative inhibition, we identified 65
differentially expressed proteins in the 17-AAG treated cell lines, four of which were deregulated in
several of the studied cell lines. eIF3 subunit I was down-expressed in the HCC827 (EGFR-mutated)
and H3122 (EML4-ALK rearrangement) cell lines. MHC class 1 antigen was down-expressed after
treatment of HCC827 cells but overexpressed in A549 (KRAS-mutated) cells. 3.2. Effectiveness of HSP90 Inhibitors in Lung Adenocarcinoma Cell Lines Treatment
Protein Deregulation
Cell Line
HCC827
H3122
A549
H1437
17-AAG
Up
23
4
9
10
Down
15
3
2
3
IPI-504
Up
26
3
11
18
Down
16
2
6
3
STA-9090
Up
25
14
6
9
Down
18
4
32
3
AUY-922
Up
23
5
6
9
Down
8
4
11
5
VER-155008 + 17AAG
Up
30
10
12
15
Down
61
6
15
3 The EGFR (HCC827) cell line showed the highest number of deregulated proteins after
treatment with the different HSP90 inhibitors studied without major differences between treatments. The triple-negative cell line (H1437) had a higher number of differentially expressed proteins following
treatment with geldanamycin derivatives whereas more deregulated proteins were detected in the
EML4-ALK (H3122) and KRAS (A549) cell lines after treatment with radicicol derivatives. The KRAS
cell line was the only line where the number of down-expressed proteins was higher than that of
overexpressed proteins, concretely after treatment with radicicol derivatives. Cells 2019, 8, 806 Ras-related protein
Rab-37 and transketolase were also identified in the A549 as well as in the H1437 (triple-negative;
EGFR, ALK and KRAS wild-type) cell lines. Rab-37 was overexpressed after treatment of A549 cells
but down-expressed in H1437 cells. Transketolase on the other hand was overexpressed in both cell
lines. In the case of the IPI-504 treatment, 79 proteins were identified. Five of them were deregulated
in two or more of the cell lines tested. HSP90α was down-expressed in the EGFR-mutated cell line
(HCC827) and overexpressed in the ALK translocation (H3122) cell line. HSP70 protein in its 1A and
1B variants was overexpressed in HCC827 and A549 cell lines in response to treatment. Transketolase
was detected again, this time in three different cell lines, where its expression was decreased in H3122
and increased in A549 and H1437 cells after inhibition with IPI-504. Finally, Heat shock cognate 71 kDa
protein (HSC71), also known as heat shock 70 kDa protein 8 (HSPA8), was down-expressed in HCC827
and overexpressed in H1437 cells. p
With respect to the radicicol derivatives used, inhibition with STA-9090 led to 107 different
proteins being identified, four of which were found in two or more cell lines. HSP90α as well as the
mitochondrial 60kDa heat shock protein, were down-expressed in HCC827 cells but overexpressed
in H3122 cells. Rab-37 was again identified as being down-expressed following treatment of the
HCC827 and A549 cell lines. Finally, the MHC class II antigen was overexpressed in H3122 cells and
down-expressed in A549 cells. On the other hand, sixty-nine differentially expressed proteins were
detected after cells were treated with AUY-922. Only two proteins were identified in more than one
cell line. HSP70 was overexpressed in HCC827 and A549 cells while transketolase was overexpressed
in A549 and H1437 cells. Combination of the HSP90 inhibitor 17-AAG plus the HSP70 inhibitor VER-155008 gave rise to
143 deregulated proteins, 9 of which were common to two cell lines. Six proteins were deregulated in
HCC827 and A549 cells. Retinoic acid receptor RXR-beta, lipoamide acyltransferase component of 9 of 22 Cells 2019, 8, 806 branched-chain alpha-keto acid dehydrogenase complex mitochondrial and cysteine and glycine-rich
protein 2 were down-expressed while HSP70 1A was overexpressed in both cell lines. SAM and
SH3 domain-containing protein 1 were overexpressed in HCC827 and down-expressed in A549 cells. In contrast, MHC class I antigen expression was decreased in HCC827 cells and increased in A549 cells. Cells 2019, 8, 806 Heat shock 70 kDa protein 6 was overexpressed in the H3122 and A549 cell lines, while 60S acidic
ribosomal protein P0 was one of the more highly expressed proteins following treatment of A549 and
H1437 cells. The double inhibition treatment resulted in transketolase overexpression in HCC827 and
H1437 cells. With the purpose of searching a common protein signature after HSP90 inhibition and taking
together results for the most deregulated proteins and those detected in two or more cell lines for
the same inhibitor, six shared proteins (HSP70, HSP90α, HSC71, eIF3i, MHC class 1 antigen and
transketolase) were identified to be involved in the response to HSP90 inhibitors. It is important to note
that adenocarcinoma is a molecularly diverse entity, in which many different molecular alterations have
been identified as drivers of different subsets of patients in this context, each of them showing different
prognosis and response to therapy. This fact makes each cell line harboring a different molecular driver
unique. Nevertheless, a protein dysregulation pattern common for all cell lines tested was identified
in this study, where 90% of the dysregulated proteins were found among the first ten proteins with
average ratios greater than 5, which indicates the strength of our results. It is also remarkable that not
only the expected HSP70, but also eIF3i, were detected unexpressed and down-expressed, respectively,
in different cell lines. 3.4. Functional Annotation Analysis of Differentially Expressed Proteins iGluRs pathway = ionotropic
glutamate receptor pathway, PPP= pentose phosphate pathway, TCA cycle = tricarboxylic acid cycle,
5-HT degradation = 5-hydroxytryptamine or serotonin degradation, PDGF pathway =
platelet-derived growth factor signaling pathway, EGFR pathway = epidermal growth factor
receptor pathway, IL pathway = interleukin signaling pathway, SER GLY biosynthesis = serine and
glycine biosynthesis, VEGF pathway = vascular endothelial growth factor signaling pathway, FRU
GAL metabolism = fructose and galactose metabolism, insulin/IGF-PKB cascade = insulin/
insulin-like growth factor pathway-protein kinase B signaling cascade, PI3K pathway =
phosphoinositide-3 kinase pathway, PA cascade = plasminogen activating cascade, PLP biosynthesis
= pyridoxal-5-phosphate biosynthesis, TGFβ pathway = transforming growth factor beta signaling
pathway, FGF pathway = Fibroblast Growth Factor signaling pathway, adrenaline and NA synth. =
adrenaline and noradrenaline biosynthesis, EGF pathway = epidermal growth factor pathway,
heterotrimeric G-protein signaling pathway = heterotrimeric G-protein signalling pathway Gi alpha-
and Gs alpha-mediated pathway, 5-HT1 type receptor pathway = serotonin 1A receptor-mediated
Figure 3. Canonical pathway analysis of proteins identified by two-dimensional electrophoresis
and mass spectrometry (2-DE/MS) in response to HSP90 inhibition. Functional analysis of the
differentially expressed proteins in response to (A) 17-AAG, (B) IPI-504, (C) STA-9090, (D) AUY-922
and (E) VER-155008 + 17-AAG inhibitors in the adenocarcinoma cell lines HCC827, H3122, A549
and H1437. Enriched pathways were determined from the PANTHER database. The number
of proteins involved in each identified pathway is represented on the y axis. iGluRs pathway =
ionotropic glutamate receptor pathway, PPP= pentose phosphate pathway, TCA cycle = tricarboxylic
acid cycle, 5-HT degradation = 5-hydroxytryptamine or serotonin degradation, PDGF pathway =
platelet-derived growth factor signaling pathway, EGFR pathway = epidermal growth factor receptor
pathway, IL pathway = interleukin signaling pathway, SER GLY biosynthesis = serine and glycine
biosynthesis, VEGF pathway = vascular endothelial growth factor signaling pathway, FRU GAL
metabolism = fructose and galactose metabolism, insulin/IGF-PKB cascade = insulin/ insulin-like
growth factor pathway-protein kinase B signaling cascade, PI3K pathway = phosphoinositide-3 kinase
pathway, PA cascade = plasminogen activating cascade, PLP biosynthesis = pyridoxal-5-phosphate
biosynthesis, TGFβ pathway = transforming growth factor beta signaling pathway, FGF pathway =
Fibroblast Growth Factor signaling pathway, adrenaline and NA synth. = adrenaline and noradrenaline
biosynthesis, EGF pathway = epidermal growth factor pathway, heterotrimeric G-protein signaling
pathway = heterotrimeric G-protein signalling pathway Gi alpha- and Gs alpha-mediated pathway,
5-HT1 type receptor pathway = serotonin 1A receptor-mediated signalling pathway, MAN metabolism
= mannose metabolism. gure 3. 3.4. Functional Annotation Analysis of Differentially Expressed Proteins Canonical pathway analysis of proteins identified by two-dimensional electrophoresis and
ass spectrometry (2-DE/MS) in response to HSP90 inhibition. Functional analysis of the
fferentially expressed proteins in response to (A) 17-AAG, (B) IPI-504, (C) STA-9090, (D) AUY-922
d (E) VER-155008 + 17-AAG inhibitors in the adenocarcinoma cell lines HCC827, H3122, A549 and
1437. Enriched pathways were determined from the PANTHER database. The number of proteins
volved in each identified pathway is represented on the y axis. iGluRs pathway = ionotropic
utamate receptor pathway, PPP= pentose phosphate pathway, TCA cycle = tricarboxylic acid cycle,
HT degradation = 5-hydroxytryptamine or serotonin degradation, PDGF pathway =
atelet-derived growth factor signaling pathway, EGFR pathway = epidermal growth factor
ceptor pathway, IL pathway = interleukin signaling pathway, SER GLY biosynthesis = serine and
ycine biosynthesis, VEGF pathway = vascular endothelial growth factor signaling pathway, FRU
AL metabolism = fructose and galactose metabolism, insulin/IGF-PKB cascade = insulin/
sulin-like growth factor pathway-protein kinase B signaling cascade, PI3K pathway =
hosphoinositide-3 kinase pathway, PA cascade = plasminogen activating cascade, PLP biosynthesis
pyridoxal-5-phosphate biosynthesis, TGFβ pathway = transforming growth factor beta signaling
thway, FGF pathway = Fibroblast Growth Factor signaling pathway, adrenaline and NA synth. =
renaline and noradrenaline biosynthesis, EGF pathway = epidermal growth factor pathway,
terotrimeric G-protein signaling pathway = heterotrimeric G-protein signalling pathway Gi alpha-
d Gs alpha-mediated pathway, 5-HT1 type receptor pathway = serotonin 1A receptor-mediated
Figure 3. Canonical pathway analysis of proteins identified by two-dimensional electrophoresis
and mass spectrometry (2-DE/MS) in response to HSP90 inhibition. Functional analysis of the
differentially expressed proteins in response to (A) 17-AAG, (B) IPI-504, (C) STA-9090, (D) AUY-922
and (E) VER-155008 + 17-AAG inhibitors in the adenocarcinoma cell lines HCC827, H3122, A549
and H1437. Enriched pathways were determined from the PANTHER database. The number
of proteins involved in each identified pathway is represented on the y axis. 3.4. Functional Annotation Analysis of Differentially Expressed Proteins Proteins that were differentially expressed in response to the different HSP inhibitors were
analyzed using PANTHER software for mapping of the molecular pathways that were possibly
involved (Figure 3). Results showed that the deregulated proteins identified in response to 17-AAG
inhibition participated in nine pathways, including the pentose phosphate pathway (PPP), which was
the only pathway enriched in two different cell lines (A549 (KRAS) and H1437 (TN)) (Figure 3A). In the
case of the other geldanamycin-derivative inhibitor, IPI-504 19, enrichment pathways were identified
that included the platelet-derived growth factor (PDGF) and interleukin pathways which were shared
by the EGFR (HCC827) and the EML4-ALK (H3122) cell lines. Two other pathways also coincided,
consisting of apoptosis in HCC827, A549 and H1437 cells, and PPP in H3122, A549 and H1437 cells
(Figure 3B). On the other hand, for radicicol derivatives, the set of STA-9090-associated proteins were
assigned to 16 pathways, three of which were shared by different cell lines: serine glycine synthesis
(detected in the EGFR and EML4-ALK cell lines), deregulated glycolysis (in the EML4-ALK and KRAS
cell lines) and ATP synthesis (in the KRAS and triple-negative cell lines) (Figure 3C). AUY-922 was
the HSP90 inhibitor with more shared pathways among the cell lines studied. These included serine
glycine synthesis (again detected in the EGFR and the EML4-ALK cell lines), glycolysis (enriched in
the EGFR, KRAS and triple-negative cell lines), and deregulated apoptosis (in the EGFR and KRAS cell
lines). The A549 (KRAS) and H1437 (TN) cell lines also showed enrichment in ATP synthesis, PPP and
pyruvate metabolism (Figure 3D). 10 of 22
of 22 10 of 22
of 22 Cells 2019, 8, 806
lls 2019, 8, x FOR PE Figure 3. Canonical pathway analysis of proteins identified by two-dimensional electrophoresis and
mass spectrometry (2-DE/MS) in response to HSP90 inhibition. Functional analysis of the
differentially expressed proteins in response to (A) 17-AAG, (B) IPI-504, (C) STA-9090, (D) AUY-922
and (E) VER-155008 + 17-AAG inhibitors in the adenocarcinoma cell lines HCC827, H3122, A549 and
H1437. Enriched pathways were determined from the PANTHER database. The number of proteins
involved in each identified pathway is represented on the y axis. 3.4. Functional Annotation Analysis of Differentially Expressed Proteins iGluRs pathway =
ionotropic glutamate receptor pathway, PPP= pentose phosphate pathway, TCA cycle = tricarboxylic
acid cycle, 5-HT degradation = 5-hydroxytryptamine or serotonin degradation, PDGF pathway =
platelet-derived growth factor signaling pathway, EGFR pathway = epidermal growth factor receptor
pathway, IL pathway = interleukin signaling pathway, SER GLY biosynthesis = serine and glycine
biosynthesis, VEGF pathway = vascular endothelial growth factor signaling pathway, FRU GAL
metabolism = fructose and galactose metabolism, insulin/IGF-PKB cascade = insulin/ insulin-like
growth factor pathway-protein kinase B signaling cascade, PI3K pathway = phosphoinositide-3 kinase
pathway, PA cascade = plasminogen activating cascade, PLP biosynthesis = pyridoxal-5-phosphate
biosynthesis, TGFβ pathway = transforming growth factor beta signaling pathway, FGF pathway =
Fibroblast Growth Factor signaling pathway, adrenaline and NA synth. = adrenaline and noradrenaline
biosynthesis, EGF pathway = epidermal growth factor pathway, heterotrimeric G-protein signaling
pathway = heterotrimeric G-protein signalling pathway Gi alpha- and Gs alpha-mediated pathway,
5-HT1 type receptor pathway = serotonin 1A receptor-mediated signalling pathway, MAN metabolism
= mannose metabolism. signalling pathway, MAN metabolism = mannose metabolism. It is noteworthy that the largest number of pathways was identified when a HSP90 inhibitor
as used in combination with the HSP70 inhibitor (VER155008). Four of 21 pathways were shared
y cell lines. Glycolysis was detected in the EGFR, EML4-ALK and triple-negative cell lines, while
rine glycine synthesis was found in the EGFR and KRAS cell lines. Moreover, ATP synthesis was
nriched in the EML4-ALK and triple-negative cell lines such as 5-hydroxytryptamine (5-HT)
It is noteworthy that the largest number of pathways was identified when a HSP90 inhibitor was
used in combination with the HSP70 inhibitor (VER155008). Four of 21 pathways were shared by cell
lines. Glycolysis was detected in the EGFR, EML4-ALK and triple-negative cell lines, while serine
glycine synthesis was found in the EGFR and KRAS cell lines. Moreover, ATP synthesis was enriched
in the EML4-ALK and triple-negative cell lines such as 5-hydroxytryptamine (5-HT) degradation was
found in the KRAS and triple-negative cell lines (Figure 3E). egradation was found in the KRAS and triple-negative cell lines (Figure 3E). 5 Protein Expression Profiles and Signalling Pathways Associated with the HSP90 Inhibitor Family in Lung
3.5. Protein Expression Profiles and Signalling Pathways Associated with the HSP90 Inhibitor Family in Lung
Adenocarcinoma Cell Lines 5. 3.4. Functional Annotation Analysis of Differentially Expressed Proteins Protein Expression Profiles and Signalling Pathways Associated with the HSP90 Inhibitor Family in Lung
denocarcinoma Cell Lines
From the deregulated proteins under HSP90 inhibition in the four studied cell lines, Venn
agrams were constructed to illustrate the overlap of altered proteins within each HSP90 inhibitor
mily. Among the differentially expressed proteins after treatment with the geldanamycin
From the deregulated proteins under HSP90 inhibition in the four studied cell lines, Venn
diagrams were constructed to illustrate the overlap of altered proteins within each HSP90 inhibitor
family. Among the differentially expressed proteins after treatment with the geldanamycin derivatives,
33 were specific for 17-AAG and 47 for IPI-504, with 32 proteins (Table S6) common to both inhibitors 11 of 22 Cells 2019, 8, 806 (Figure 4A). Among the proteins common to both inhibitors, the overexpression of HSP70, CS, AGR2,
enolase and sumo3 are of note. On the other hand, only V-ATPase 116 kDa isoform a1, anamorsin,
calcium-independent phospholipase A2-gamma (iPLA2-2) and 3-hydroxyacyl-CoA dehydrogenase
type-2 were exclusively down-expressed under geldanamycin derivatives. Four proteins (HSC71,
MHC class I antigen, HSP90α and transketolase) showed different expression patterns depending on
the cell line under consideration. both inhibitors (Figure 4A). Among the proteins common to both inhibitors, the overexpression of
HSP70, CS, AGR2, enolase and sumo3 are of note. On the other hand, only V-ATPase 116 kDa
isoform
a1,
anamorsin,
calcium-independent
phospholipase
A2-gamma
(iPLA2-2)
and
3-hydroxyacyl-CoA dehydrogenase type-2 were exclusively down-expressed under geldanamycin
derivatives. Four proteins (HSC71, MHC class I antigen, HSP90α and transketolase) showed
different expression patterns depending on the cell line under consideration. p
p
p
g
Figure 4. Study of cell line response to HSP90 inhibitors according to inhibitor family. Venn
diagrams showing overlap of differentially expressed proteins as well as those specific to inhibition
with (A) geldanamycin or (B) radicicol derivatives. The analysis of shared proteins following (C)
geldanamycin or (D) radicicol treatments was performed with PANTHER software which identified
deregulated pathways associated with each HSP90 inhibitor family. The number of proteins
involved in each identified pathway is represented on the y axis. PPP= pentose phosphate pathway,
TCA cycle = tricarboxylic acid cycle, 5-HT degradation = 5-hydroxytryptamine or serotonin
degradation, PDGF pathway = platelet-derived growth factor signaling pathway, SER GLY
biosynthesis = serine and glycine biosynthesis, PLP biosynthesis = pyridoxal-5-phosphate
bi
h
i
VEGF
h
l
d
h li l
h f
i
li
h
Figure 4. 3.4. Functional Annotation Analysis of Differentially Expressed Proteins The number of proteins
involved in each identified pathway is represented on the y axis. PPP= pentose phosphate pathway,
TCA cycle = tricarboxylic acid cycle, 5-HT degradation = 5-hydroxytryptamine or serotonin
degradation, PDGF pathway = platelet-derived growth factor signaling pathway, SER GLY
biosynthesis = serine and glycine biosynthesis, PLP biosynthesis = pyridoxal-5-phosphate
g
p
y
y
p
in each identified pathway is represented on the y axis. PPP= pentose phosphate pathway, TCA
cycle = tricarboxylic acid cycle, 5-HT degradation = 5-hydroxytryptamine or serotonin degradation,
PDGF pathway = platelet-derived growth factor signaling pathway, SER GLY biosynthesis = serine
and glycine biosynthesis, PLP biosynthesis = pyridoxal-5-phosphate biosynthesis, VEGF pathway =
vascular endothelial growth factor signaling pathway. biosynthesis, VEGF pathway = vascular endothelial growth factor signaling pathway. The radicicol derivatives group shared 52 deregulated proteins (Table S7). Fifty-five proteins
were exclusively related to STA-9090 treatment while 18 were exclusive to AUY-922 inhibition
(Figure 4B). Among the 30 shared proteins that were overexpressed, HSP70, AGR2 and enolase were
again present in addition to new ones such as transketolase, vinculin or radixin. A smaller number of
proteins were down-expressed such as V-ATPase 116 kDa isoform a1, iPLA2-2, 3-hydroxyacyl-CoA
dehydrogenase type-2, ATP synthase subunit beta, HSC71, eIF3i and sumo3. MHC class I antigen,
HSP90α, pyruvate kinase (PKM) and glucose-6-phosphate 1-dehydrogenase (G6PD) showed
diff
i
i
di
h
ll li
i
i
The radicicol derivatives group shared 52 deregulated proteins (Table S7). Fifty-five proteins were
exclusively related to STA-9090 treatment while 18 were exclusive to AUY-922 inhibition (Figure 4B). Among the 30 shared proteins that were overexpressed, HSP70, AGR2 and enolase were again present
in addition to new ones such as transketolase, vinculin or radixin. A smaller number of proteins were
down-expressed such as V-ATPase 116 kDa isoform a1, iPLA2-2, 3-hydroxyacyl-CoA dehydrogenase
type-2, ATP synthase subunit beta, HSC71, eIF3i and sumo3. MHC class I antigen, HSP90α, pyruvate
kinase (PKM) and glucose-6-phosphate 1-dehydrogenase (G6PD) showed different protein expression
patterns according to the cell lines in question. different protein expression patterns according to the cell lines in question. We identified that proteins that were deregulated following treatment with geldanamycin
derivatives were involved in six pathways, while those detected after radicicol derivative treatments
participated in 11 pathways. Three pathways (apoptosis, TCA cycle and 5-HT degradation) were
exclusively enriched in response to treatment with geldanamycin derivatives (Figure 4C). 3.4. Functional Annotation Analysis of Differentially Expressed Proteins Study of cell line response to HSP90 inhibitors according to inhibitor family. Venn
diagrams showing overlap of differentially expressed proteins as well as those specific to inhibition
with (A) geldanamycin or (B) radicicol derivatives. The analysis of shared proteins following
(C) geldanamycin or (D) radicicol treatments was performed with PANTHER software which identified
deregulated pathways associated with each HSP90 inhibitor family. The number of proteins involved
in each identified pathway is represented on the y axis. PPP= pentose phosphate pathway, TCA
cycle = tricarboxylic acid cycle, 5-HT degradation = 5-hydroxytryptamine or serotonin degradation,
PDGF pathway = platelet-derived growth factor signaling pathway, SER GLY biosynthesis = serine
and glycine biosynthesis, PLP biosynthesis = pyridoxal-5-phosphate biosynthesis, VEGF pathway =
vascular endothelial growth factor signaling pathway. Figure 4. Study of cell line response to HSP90 inhibitors according to inhibitor family. Venn Figure 4. Study of cell line response to HSP90 inhibitors according to inhibitor family. Venn
diagrams showing overlap of differentially expressed proteins as well as those specific to inhibition
with (A) geldanamycin or (B) radicicol derivatives. The analysis of shared proteins following (C)
geldanamycin or (D) radicicol treatments was performed with PANTHER software which identified
deregulated pathways associated with each HSP90 inhibitor family. The number of proteins
involved in each identified pathway is represented on the y axis. PPP= pentose phosphate pathway,
TCA cycle = tricarboxylic acid cycle, 5-HT degradation = 5-hydroxytryptamine or serotonin
degradation, PDGF pathway = platelet-derived growth factor signaling pathway, SER GLY
biosynthesis = serine and glycine biosynthesis, PLP biosynthesis = pyridoxal-5-phosphate
Figure 4. Study of cell line response to HSP90 inhibitors according to inhibitor family. Venn
diagrams showing overlap of differentially expressed proteins as well as those specific to inhibition
with (A) geldanamycin or (B) radicicol derivatives. The analysis of shared proteins following
(C) geldanamycin or (D) radicicol treatments was performed with PANTHER software which identified
deregulated pathways associated with each HSP90 inhibitor family. The number of proteins involved
in each identified pathway is represented on the y axis. PPP= pentose phosphate pathway, TCA
cycle = tricarboxylic acid cycle, 5-HT degradation = 5-hydroxytryptamine or serotonin degradation,
PDGF pathway = platelet-derived growth factor signaling pathway, SER GLY biosynthesis = serine
and glycine biosynthesis, PLP biosynthesis = pyridoxal-5-phosphate biosynthesis, VEGF pathway =
vascular endothelial growth factor signaling pathway. g
y
(
)
p
deregulated pathways associated with each HSP90 inhibitor family. 3.6. Validation of the Differential Expression of Proteins Involved in the Response to Chaperone Inhibitors
geldanamycin derivatives and to PKM for radicicol derivatives. Two proteins (eIF3i and CS) were evaluated by western blotting owing to their important changes
in expression in different cell lines in response to inhibitors as well as to their relationship with the
deregulated pathways. The first of these, eIF3i, was the most down-expressed protein in response to
treatment with 17-AAG, STA-9090, and VER-155008 + 17-AAG, and the second one under AUY-922
in the EML4-ALK cell line H3122. The down-expression of this important initiation factor was also
detected in HCC827, the EGFR cell line, under the inhibitor 17-AAG. Western blotting of eIF3i and the
corresponding internal control of the four studied cell lines are shown in Figure 5A. Since this technique
is not as sensitive as 2-DE, the expression trend was observed more clearly in genetic silencing of HSP90
(α and β) than under drug inhibition of HSP90 in the EGFR and EML4-ALK cell lines. At the same
time, these cell lines presented a noticeable eIF3i degradation after treatment with VER-155008, the
HSP70 inhibitor, alone or in combination with 17-AAG. In contrast, the triple-negative cell line (H1437),
showed an increased expression after genetic silencing of HSP90 and most of the studied conditions. In fact, H1437 cells showed an exclusive, small decreased expression of eIF3i after inhibition with
17-AGG in monotherapy or in conjunction with VER-155008. Finally, the KRAS cell line showed an
intermediate expression pattern with increased expression of eIF3i after HSP70 inhibitor in conjunction
with 17-AAG, as well as a decreased expression of this protein under genetic silencing or inhibition
of HSP90. 3.6. Validation of the Differential Expression of Proteins Involved in the Response to Chaperone Inhibitors
Two proteins (eIF3i and CS) were evaluated by western blotting owing to their important
changes in expression in different cell lines in response to inhibitors as well as to their relationship
with the deregulated pathways. The first of these, eIF3i, was the most down-expressed protein in
response to treatment with 17-AAG, STA-9090, and VER-155008 + 17-AAG, and the second one
under AUY-922 in the EML4-ALK cell line H3122. The down-expression of this important initiation
factor was also detected in HCC827, the EGFR cell line, under the inhibitor 17-AAG. Western
blotting of eIF3i and the corresponding internal control of the four studied cell lines are shown in
Figure 5A. 3.4. Functional Annotation Analysis of Differentially Expressed Proteins The
differentially expressed proteins related to these pathways were the different isoforms of HSP70 and
HSC71 (for apoptosis), CS (for TCA cycle) and aldehyde dehydrogenase (for 5-HT degradation). In
turn, the integrin signalling pathway, angiogenesis, the PDGF pathway, serine glycine biosynthesis,
ATP synthesis pyridoxal 5 phosphate (PLP) biosynthesis the VEGF pathway and vitamin B6
We identified that proteins that were deregulated following treatment with geldanamycin
derivatives were involved in six pathways, while those detected after radicicol derivative treatments
participated in 11 pathways. Three pathways (apoptosis, TCA cycle and 5-HT degradation)
were exclusively enriched in response to treatment with geldanamycin derivatives (Figure 4C). The differentially expressed proteins related to these pathways were the different isoforms of HSP70 and
HSC71 (for apoptosis), CS (for TCA cycle) and aldehyde dehydrogenase (for 5-HT degradation). In turn,
the integrin signalling pathway, angiogenesis, the PDGF pathway, serine glycine biosynthesis, ATP
synthesis, pyridoxal-5-phosphate (PLP) biosynthesis, the VEGF pathway and vitamin B6 metabolism 12 of 22 Cells 2019, 8, 806 12 of 22 were specific to radicicol derivative treatment (Figure 4D). In this case, the proteins related to the
enriched pathways (in parentheses) were BRAF and vinculin (integrin signalling pathway), BRAF
and FGF1 (angiogenesis), BRAF (PDGF pathway), phosphoserine aminotransferase (serine glycine
biosynthesis), ATP synthase subunit α and β (ATP synthesis), phosphoserine aminotransferase (PLP
biosynthesis), BRAF (VEGF pathway) and phosphoserine aminotransferase (vitamin B6 metabolism). Cells 2019, 8, x FOR PEER REVIEW
12 of 22
related to the enriched pathways (in parentheses) were BRAF and vinculin (integrin signalling
pathway), BRAF and FGF1 (angiogenesis), BRAF (PDGF pathway), phosphoserine aminotransferase
(serine glycine biosynthesis)
ATP synthase subunit α and β (ATP synthesis)
phosphoserine y
p
y
p
p
Only three signalling pathways (PPP, glycolysis, and pyruvate metabolism) were detected across
the two HSP90 inhibitor families. Transketolase was the protein whose dysregulation was involved in
the enrichment of PPP in both inhibitor families. Enolase was the common protein related to glycolysis
dysregulation, but PKM also took part in inhibition by radicicol family derivatives. The final common
pathway, pyruvate metabolism, was related to CS for inhibition by geldanamycin derivatives and to
PKM for radicicol derivatives. (serine glycine biosynthesis), ATP synthase subunit α and β (ATP synthesis), phosphoserine
aminotransferase (PLP biosynthesis), BRAF (VEGF pathway) and phosphoserine aminotransferase
(vitamin B6 metabolism). Only three signalling pathways (PPP, glycolysis, and pyruvate metabolism) were detected
across the two HSP90 inhibitor families. 3.6. Validation of the Differential Expression of Proteins Involved in the Response to Chaperone Inhibitors
geldanamycin derivatives and to PKM for radicicol derivatives. Since this technique is not as sensitive as 2-DE, the expression trend was observed more
clearly in genetic silencing of HSP90 (α and β) than under drug inhibition of HSP90 in the EGFR and
EML4-ALK cell lines. At the same time, these cell lines presented a noticeable eIF3i degradation after
treatment with VER-155008, the HSP70 inhibitor, alone or in combination with 17-AAG. In contrast,
the triple-negative cell line (H1437), showed an increased expression after genetic silencing of HSP90
and most of the studied conditions. In fact, H1437 cells showed an exclusive, small decreased
expression of eIF3i after inhibition with 17-AGG in monotherapy or in conjunction with VER-155008. Finally, the KRAS cell line showed an intermediate expression pattern with increased expression of
eIF3i after HSP70 inhibitor in conjunction with 17-AAG, as well as a decreased expression of this
protein under genetic silencing or inhibition of HSP90. Figure 5. Western blotting validation of proteins identified as differentially expressed in the
2-DE/MS analysis. Protein lysates from HCC827, H3122, A549 and H1437 cells without treatment or
inhibited with 17-AAG, IPI-504, STA-9090, AUY-922 VER-155008 + 17AAG and VER-155008 as well
as the genetic silencing of HSP90 were immunoblotted. Expression study of (A) eIF3i and (B) CS after
Figure 5. Western blotting validation of proteins identified as differentially expressed in the 2-DE/MS
analysis. Protein lysates from HCC827, H3122, A549 and H1437 cells without treatment or inhibited
with 17-AAG, IPI-504, STA-9090, AUY-922 VER-155008 + 17AAG and VER-155008 as well as the
genetic silencing of HSP90 were immunoblotted. Expression study of (A) eIF3i and (B) CS after HSP90
inhibition as well as its loading control are shown. eIF3i = eukaryotic translation initiation factor 3
subunit I, CS = citrate synthase. For Western blots, a representative image is shown. Densitometry
analysis may be found in Figure S8. Figure 5. Western blotting validation of proteins identified as differentially expressed in the
2-DE/MS analysis. Protein lysates from HCC827, H3122, A549 and H1437 cells without treatment or
inhibited with 17-AAG, IPI-504, STA-9090, AUY-922 VER-155008 + 17AAG and VER-155008 as well
as the genetic silencing of HSP90 were immunoblotted. Expression study of (A) eIF3i and (B) CS after
Figure 5. Western blotting validation of proteins identified as differentially expressed in the 2-DE/MS
analysis. 3.4. Functional Annotation Analysis of Differentially Expressed Proteins Transketolase was the protein whose dysregulation was
involved in the enrichment of PPP in both inhibitor families. Enolase was the common protein
related to glycolysis dysregulation, but PKM also took part in inhibition by radicicol family
derivatives The final common pathway pyruvate metabolism was related to CS for inhibition by 4. Discussion In this study we used 2-DE coupled with mass spectrometry to analyze differences that take
place in the proteome of lung adenocarcinoma cell lines in response to HSP90 inhibition. For each
lung adenocarcinoma cell line studied, we identified clear alterations in the protein profiles in treated
compared to untreated control cell lines. These differences were notable for individual HSP90 inhibitors
and in combination with an HSP70 inhibitor, with responses always being more effective for radicicol
derivative treatments given the lower IC50 values and higher proteomic dysregulation in the studied
cell lines treated with these inhibitors. Treatment with HSP90 inhibitors of lung adenocarcinoma cell lines, as expected, showed
remarkable changes in the homeostasis of HSPs, leading to deregulation of HSP90, HSP70, HSP60,
HSC71, HSP70 protein 6 (HSPA6), and mitochondrial HSP70 (mitHSP70). It has been reported that
HSP90 inhibitors usually result in induction of the HSR leading to HSP70 overexpression mediated
by heat shock factor 1 (HSF1) [50]. HSP70 upregulation has thus been related to the confirmation of
HSP90 inhibition [51,52]. Besides HSP70, HSP90 overexpression occurs after HSP90 inhibition due
to the HSR after inhibition as well as dissociation of HSF1 from HSP90 [53]. In this way, we have
identified compensatory responses specific for each HSP90 inhibitor used, alone or in combination
with a HSP70 inhibitor. On the other hand, we found that HSP60 was down-expressed in the EGFR
mutant cell line after treatment with STA-9090 and 17-AAG plus VER-155008 combination; however, it
was overexpressed in the EML4-ALK cell line in response to STA-9090 inhibition. This protein could
be related to STA-9090 sensitivity, and if its inhibition in the EML4-ALK cell line was successful, this
could indicate that the HSR may not be as important here as in other cell lines. A cytoprotective role of
mitochondrial HSP60 in the apoptotic process was previously described [54,55]. Concretely in lung
cancer, low expression of this chaperone has been proposed as a good prognostic factor of disease-free
survival [56,57]. HSC71 was detected to be down-regulated after treatment with geldanamycin and
radicicol derivatives, which could be related to an optimal response in the EGFR-mutated cell line. However, the expression of this chaperone was increased in response to STA-9090 treatment in the
EML4-ALK cell line as well as in the triple negative cell line following IPI-504 treatment. 3.6. Validation of the Differential Expression of Proteins Involved in the Response to Chaperone Inhibitors
geldanamycin derivatives and to PKM for radicicol derivatives. Protein lysates from HCC827, H3122, A549 and H1437 cells without treatment or inhibited
with 17-AAG, IPI-504, STA-9090, AUY-922 VER-155008 + 17AAG and VER-155008 as well as the
genetic silencing of HSP90 were immunoblotted. Expression study of (A) eIF3i and (B) CS after HSP90
inhibition as well as its loading control are shown. eIF3i = eukaryotic translation initiation factor 3
subunit I, CS = citrate synthase. For Western blots, a representative image is shown. Densitometry
analysis may be found in Figure S8. Cells 2019, 8, 806 13 of 22 We also validated CS, which was differentially expressed across cell lines. This protein is an enzyme
of the TCA cycle whose regulation is related to ATP synthesis and other metabolic pathways such as
glycolysis, which were detected as deregulated pathways after HSP90 inhibition in the enrichment
analysis. CS showed the largest increase of expression in the A549 cell line after treatments, confirming
the 2-DE results, where approximately 7-fold expression changes were detected after 17-AAG, IPI-504
and VER-155008 + 17-AAG treatment. It should be pointed out that the EGFR cell line HCC827 was
the only line where a decrease in expression of this protein after treatments was detected (Figure 5B). 4. Discussion This result is
consistent with other studies on different tumor types where HSC71 plays a regulatory role in the HSR
and homeostasis [47,58]. Another HSP70 family member, inducible HSPA6, was overexpressed in the
H3122 and A549 cell lines following the combined inhibition of HSP90 plus HSP70. The upregulation of
this protein, which is only minimally expressed under normal conditions, could be induced through the
HSE/HSF1 system [59] and is related to cell survival in response to extracellular stress such as toxicity
exposure [60,61]. However, in a recent study, HSPA6 expression was correlated with the inhibition
of proliferation, migration and invasion, thereby enhancing the anti-tumor effect of garlic extract in
bladder cancer [62]. This could be the reason why we observed a greater number of deregulated
pathways in the combined inhibition protocol. Other member of the HSP70 family, mitHSP70, also
known as mortalin, has been shown to promote and contribute to the process of carcinogenesis in
numerous ways, including the merging and inactivation of tumor suppressor protein p53, which plays
a key role in anti-apoptosis pathways, in regulation of the RAS RAF MEK pathway and activation
of EMT signalling [63,64]. As mortalin is enriched in many types of cancers its overexpression has
been used as a predictor of poor outcome in lung cancer [65,66]. In the present study, mortalin was
down-expressed in the EGFR cell line after treatment with the inhibitor 17-AAG and overexpressed 14 of 22 14 of 22 Cells 2019, 8, 806 in the EML4-ALK cell line with STA-9090. In cancer cells, mortalin responds to stress, including
chemicals, and sequesters p53 in the cytoplasm to thereby avoid apoptosis [67]. Since cell death
induced by inhibition of HSP90 partly depends on the p53 pathway [33], the mutational status of the
tumor suppressor could be in charge of the differential response since in our study the EGFR cell line
(HCC827) expresses wild-type (wt) p53 [68] while the EML4-ALK cell line (H3122) expresses mutated
p53 [69]. Since wt and mutant p53 as well as Akt, whose action suppresses p53 activity, have been
described as HSP90 clients, the inhibition of this chaperone would drive the activation of wt p53 or
down-regulation of mutant p53 [70,71]. 4. Discussion This fact could be the cause of differential expression in the
HCC827 and H3122 cell lines, with degradation of mortalin after nuclear translocation of wt p53 in the
EGFR cell line and mortalin accumulation after breaking of the HSP90-mutated p53 complex in the
EML4-ALK cell line. Furthermore, the different mutational state of p53 could be related to a larger HSP
response in the EML4-ALK cell line than in the EGFR cell line as it was seen with increased HSP90α,
HSP60 and HSC71 expression, which could modulate the response to inhibition [72]. In this way, in
lung adenocarcinoma cells presenting mutated p53, such as H3122 cells, sensitivity to HSP90 inhibition
could be increased by the restoration of wt p53. Other response biomarkers to HSP90 inhibition were eIF3i, Rab-37, MLH1, AGR2 or transketolase. Deregulation of eIF3, an extremely complex multiprotein assembly with a key role in translation
initiation and termination as in ribosomal recycling [73], has been correlated with the onset and
progression of cancer [74–76]. Concretely, the I subunit of the eIF3 complex (eIF3i) was found to
be overexpressed in different tumor types including colon adenocarcinoma and adenoma, head
and neck squamous cell carcinomas, hepatocellular carcinoma, breast cancer, cervical cancer and
metastatic melanoma [77–81]. Consistent with this, it was reported that eIF3i overexpression leads
to malignant phenotype of cells [82,83] and could promote tumor angiogenesis [84,85]. Therefore,
the down-expression of eIF3i observed in HCC827 and H3122 cell lines after treatment with HSP90
inhibitors could be a good prognostic factor for this therapeutic strategy in lung adenocarcinoma. Rab-37
is a small Rab-GTPase which regulates both exocytic and endocytic pathways and signal transduction
pathways involving cell proliferation, migration, nutrition, innate immunity and fragmentation
of compartments [86–88]. In lung cancer, low hRAB-37 mRNA expression has been associated
with tumor metastasis [89]. It was demonstrated that Rab-37 suppresses lung cancer metastasis by
mediating TIMP1 (tissue inhibitor of metalloproteinase) exocytosis, which inactivates extracellular
matrix metallopeptidase 9 (MMP9) and suppresses cell invasion signalling [90,91]. However, MMP9 is
an HSP90 client, so this protein should be degraded in response to HSP90 inhibition independently
of Rab-37. For this reason, expression of this GTPase differed in response to treatments used on the
different cell lines studied. However, while the MMP9 was not detected, it should be considered in
future experiments for validation of its use as a possible prognostic factor of the response to inhibition. 4. Discussion However, it was only
down-expressed in H3122 cells after inhibition with IPI-504 while it was overexpressed in the A549 and
H1437 cell lines under all treatments. These findings suggest that transketolase could have a key role in
metabolic activity of lung cancer as well as its downregulation could be also a good prognostic factor. y
g
g
g
p
g
These results showed a strong intervention of HSP90 inhibition on cell metabolism. Keeping
in mind that a hallmark of lung cancer metabolism is the hyperactivity of glycolysis independent
of oxygen availability [106,107], we looked for proteins related to this metabolic pathway in our
assay. Furthermore, the study of protein expression in cell lines in response to treatment with specific
inhibitor families led to the identification of six deregulated pathways following treatment with
geldanamycin derivatives and 11 pathways after treatment with radicicol derivatives. Only three
of these pathways (glycolysis, PPP and pyruvate metabolism) were shared between families, thus
confirming our previous results of the importance of metabolism in HSP90 inhibition. One of the three
geldanamycin-specific pathways was the TCA cycle. We showed here that CS, a key enzyme in the
TCA cycle, was overexpressed in the KRAS-mutant and triple-negative cell lines after inhibition with
17-AAG and IPI-504. This deregulation could be related to a possible resistance mechanism as was
previously reported in ovarian carcinoma where, after chemotherapy, an increase of CS expression
levels was related to protection from apoptosis [108,109]. Concerning the 11 specific pathways detected
with radicicol derivative treatment, the biosynthesis pathway for serine and glycine was among the
most remarkable, since this metabolic pathway, besides leading to the production of amino acids,
purines and pyrimidines, is linked to the NADH production needed to maintain redox balance [110]. The upregulation of serine and glycine synthesis has thus been associated with different tumor
types [111,112], including lung cancer, where expression of the SHMT1 enzyme has been correlated
with good prognosis and expression of SMHT2 with poor prognosis [113]. Interestingly, we found that
SMHT2 was downregulated after treatment with STA-9090 in the EGFR cell line, which could be a
good signal of treatment efficacy. In summary, a classical proteomic approach was carried out to detect proteomic changes after
HSP90 inhibition in lung adenocarcinoma cell lines. The protein profiles, which were based on 2-DE
and mass spectrometry, were identified in response to five different inhibitors. 4. Discussion On the other hand, in the study of responses according to inhibitor family, approximately 50% of
the differentially expressed proteins were common to inhibitors of the same family. In addition
to previously mentioned proteins such as HSP70 and HSP90α, other interesting proteins such as
MLH1 and AGR-2 were detected for both HSP90-inhibitor families studied. MLH1 is one of the most
important members of the DNA mismatch repair (MMR) system [92], and hence its downregulation
was related to tumor progression and chemoresistance in different cancer types, among which is
lung cancer [93–97]. In our study, the EGFR cell line showed increased MLH1 expression following
treatment with all HSP90 inhibitors. We suggest that inhibition in this context could be restoring the
expression of this tumor suppressor and consequently help drug resistance tumors become sensitive to
other therapies. For its part, AGR2, which belongs to the protein disulfide isomerase (PDI) family,
acts as a chaperone in periods of physiological stress [98]. This protein has been detected to be highly
expressed in different tumor types, thus giving rise to the hypothesis that AGR2 acts as an oncogene
due to its important role in the activation of survival and metastasis pathways [99,100]. However,
other studies showed a down-expression of AGR2 in different cancer types [101–103], which raises
serious doubts about its oncogenic role. In our study, AGR2 was overexpressed in the EML4-ALK cell Cells 2019, 8, 806 15 of 22 line (which has mutated p53) in response to all inhibitor treatments. These findings suggest that AGR2
could serve as a good prognostic factor of HSP90 inhibition, which is in agreement with a recent report
where tumor aggressiveness and poorer disease-free survival were related to the downregulation of
AGR2, p21 and cyclin D1 in the presence of mutated p53 in ovarian carcinoma [104]. Functional annotation analysis in our study showed that the differentially expressed proteins
were involved in 40 different pathways. Six of these pathways (PPP, apoptosis, pyruvate metabolism,
glycolysis, 5-HT degradation and ATP synthesis) were shared between all studied HSP inhibitors. In connection with this, the elevated expression of transketolase, an enzyme in the PPP, has been
detected in lung cancer [105], confirming an increase in metabolic activity in this tumor type. We
found that transketolase was deregulated in all of tested HSP90 inhibitors. 5. Conclusions We analyzed the proteomics associated with HSP90 inhibition in lung adenocarcinoma using a
combined approach of two-dimensional electrophoresis and mass spectrometry analysis. A total of
254 deregulated proteins were found after treatment with HSP90 inhibitors. Functional annotation
analysis of differentially expressed proteins revealed different pathways as serine and glycine synthesis
or TCA cycle which could help improve our understanding of the biology behind HSP90 inhibition. eIF3i, HSC71, MHC class I antigen, transketolase, citrate synthase, Rab-37, MLH1 and AGR2 might
be putative biomarkers in HSP90 inhibition response in lung adenocarcinoma. However, a thorough
in vivo validation is required to future clinical uses in lung adenocarcinoma. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4409/8/8/806/s1,
Figure S1: Graphical summary of densitometry results obtained from the Western blots of (A) pEGFR, (B) EGFR,
(C) EML4-ALK, (D) GRP94, (E) HSP90α, (F) HSP90β, (G) HSP70 and (H) HSP27 of Figure S1. All experiments
were independently reproduced three times in the laboratory.; Figure S2: Graphical summary of densitometry
results obtained from the Western blots of (A) EGFR, (B) HSP90α and (C) HSP70 of the cell line HCC827, Western
blots of (D) EML4-ALK, (E) HSP90α and (F) HSP70 of the cell line H3122, Western blots of (G) EGFR, (H) HSP90α
and (I) HSP70 of the cell line A549, and Western blots of (J) HSP90α and (K) HSP70 of the cell line H1437; such as
Western blots of (L) EGFR, (M) HSP90α, (N) HSP70 after HSP70 and HSP90 inhibition in the cell line HCC827. All
of quantified Western blots may be observed in the Figure S2. VER = VER-1555008.; Figure S3: Representative
two-dimensional (2D) gel images for the cell lines (A) HCC827, (B) H3122, (C) A549 and (D) H1437 untreated versus
treated with the HSP90 inhibitor 17-AAG. For each cell line, the two spots that were most up- and down-expressed
proteins are highlighted by circles, with the accompanying numbers matching those listed in Table S1.; Figure S4:
Representative two-dimensional (2D) gel images for the cell lines (A) HCC827, (B) H3122, (C) A549 and (D) H1437
untreated versus treated with the HSP90 inhibitor IPI-504. 5. Conclusions For each cell line, the two spots that were most up- and
down-expressed proteins are highlighted by circles, with the accompanying numbers matching those listed in
Table S2.; Figure S5: Representative two-dimensional (2D) gel images for the cell lines (A) HCC827, (B) H3122,
(C) A549 and (D) H1437 untreated versus treated with the HSP90 inhibitor STA-9090. For each cell line, the two
spots that were most up- and down-expressed proteins are highlighted by circles, with the accompanying numbers
matching those listed in Table S3.; Figure S6: Representative two-dimensional (2D) gel images for the cell lines
(A) HCC827, (B) H3122, (C) A549 and (D) H1437 untreated versus treated with the HSP90 inhibitor AUY-922. For each cell line, the two spots that were most up- and down-expressed proteins are highlighted by circles,
with the accompanying numbers matching those listed in Table S4.; Figure S7: Representative two-dimensional
(2D) gel images for the cell lines (A) HCC827, (B) H3122, (C) A549 and (D) H1437 untreated versus treated with
the combination of the HSP70 inhibitor VER-155008 plus the HSP90 inhibitor 17-AAG. For each cell line, the
two spots that were most up- and down-expressed proteins are highlighted by circles, with the accompanying
numbers matching those listed in Table S5.; Figure S8: Graphical summary of densitometry results obtained from
the Western blots of the eukaryotic translation initiation factor3 subunit I (eIF3) in the cell lines (A) HCC827,
(B) H3122, (C) A549 and (D) H1437, such as Western blots of citrate synthase (CS) in the cell lines (E) HCC827,
(F) H3122, (G) A549, (H) H1437 of Figure 5. All experiments were independently reproduced three times in the
laboratory. 4. Discussion Taken together, we
identified a panel of 254 deregulated proteins after HSP90 inhibition. Different proteins detected
in our study, such as eIF3i, may provide useful information concerning the specific mode of action
of inhibitors. Bioinformatic analyses of the altered proteins suggested that HSP90 inhibitors could
affect many pathways, most of which are related to energy production, metabolism and apoptosis. Our results showed that HSP90 interacts with many intracellular pathways which were deregulated
after its inhibition. The effects of some of the identified proteins to alter these pathways, such as CS
in the TCA cycle, could be related to a possible mechanism that gives rise to an undesired form of
HSP90 inhibition. However, further research including models that take tumor microenvironment into
account, such as mouse models, as well as clinical investigation, are required to provide context to the
significance of these possible biomarkers of response to HSP90 inhibition. Despite the limitations in
the models used in this study, the differential protein profiles presented here, such as the pathways
identified, contribute to our expanding knowledge of the mechanism of action underlying HSP90
inhibition and the potential development of novel response biomarkers or therapeutic targets. Cells 2019, 8, 806 16 of 22 16 of 22 Author Contributions: Conceptualization A.M., I.F., A.C., S.M.-P., and L.P.-A.; methodology, A.M., M.D.P.,
I.F., S.M.-P., L.O., and A.Q.-V.; investigation, A.M., L.P.-A. and S.M.-P.; validation, A.M., and M.D.P.; formal
analysis, A.M., and S.M.-P.; writing—original draft preparation, A.M., S.M.-P. and L.P.-A.; writing—review and
editing, A.M., I.F., A.C., M.D.P., L.O., A.Q.-V., S.M.-P. and L.P.-A.; supervision, S.M.-P., and L.P.-A.; funding
acquisition L.P.-A. References 1. Siegel, R.L.; Miller, K.D.; Jemal, A. Cancer statistics. Cancer J. Clin. 2018, 68, 7–30. [CrossRef] [PubMed] 1. Siegel, R.L.; Miller, K.D.; Jemal, A. Cancer statistics. Cancer J. Clin. 2018, 68, 7–30. [CrossRef] [PubMed]
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after 17-AAG treatment of the HCC827, H3122, A549 and H1437 cell lines.; Table S2: Differentially expressed
proteins identified by mass spectrometry after IPI-504 treatment of the HCC827, H3122, A549 and H1437 cell
lines.; Table S3: Differentially expressed proteins identified by mass spectrometry after STA-9090 treatment of
the HCC827, H3122, A549 and H1437 cell lines.; Table S4: Differentially expressed proteins identified by mass
spectrometry after AUY-922 treatment of the HCC827, H3122, A549 and H1437 cell lines.; Table S5: Differentially
expressed proteins identified by mass spectrometry after combined treatment with VER-155008 and 17-AAG of
the HCC827, H3122, A549 and H1437 cell lines.; Table S6: List of 32 shared differentially expressed proteins under
geldanamycin derivatives inhibitors.; Table S7: List of 52 common deregulated proteins after treatments with
radicicol derivatives Author Contributions: Conceptualization A.M., I.F., A.C., S.M.-P., and L.P.-A.; methodology, A.M., M.D.P.,
I.F., S.M.-P., L.O., and A.Q.-V.; investigation, A.M., L.P.-A. and S.M.-P.; validation, A.M., and M.D.P.; formal
analysis, A.M., and S.M.-P.; writing—original draft preparation, A.M., S.M.-P. and L.P.-A.; writing—review and
editing, A.M., I.F., A.C., M.D.P., L.O., A.Q.-V., S.M.-P. and L.P.-A.; supervision, S.M.-P., and L.P.-A.; funding
acquisition L.P.-A. Funding: L.P.A. was funded by the Comunidad de Madrid, CAM, (B2017/BMD3884), ISCIII (PIE15/00076,
PI17/00778 and DTS17/00089) and CIBERONC (CB16/12/00442), and co-funded by FEDER from Regional
Development European Funds (European Union). S.M.P. is funded by the Fundación Mutua Madrileña (2014)
Ministry of Health and Social Welfare of Junta de Andalucía (PI-0046-2012, Nicolas Monardes Program C-0040-2016),
ISCIII (PI17/00033), and co-funded by FEDER from Regional Development European Funds (European Union). I.F. is funded by the AECC (AIO2015) and ISCIII (PI16/01311), and co-funded by FEDER from Regional Development
European Funds (European Union). AC was funded by grants from the Spanish Ministry of Economy and
Competitiveness Plan Estatal de I+D+I 2018 co-funded by FEDER: RTI2018-097455-B-I00; CIBER de Cáncer 17 of 22 Cells 2019, 8, 806 17 of 22 (CB16/12/00275), co-funded by FEDER from Regional Development European Funds. Especial thanks to the
Fundación AECC. L.O. is funded by the Ministerio de Educación, Cultura y Deporte (FPU13/02595). (CB16/12/00275), co-funded by FEDER from Regional Development European Funds. Especial thanks to the
Fundación AECC. L.O. is funded by the Ministerio de Educación, Cultura y Deporte (FPU13/02595). Conflicts of Interest: The authors declare that they have no competing interests. Conflicts of Interest: The authors declare that they have no competing interests. References Park, K.-S.; Oh, B.; Lee, M.-H.; Nam, K.-Y.; Jin, H.R.; Yang, H.; Choi, J.; Kim, S.-W.; Lee, D.H. The HSP90
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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A One-Stage Ensemble Framework based on Convolutional Autoencoder for Remaining Useful Life Estimation
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Noname manuscript No.
(will be inserted by the editor) Noname manuscript No. (will be inserted by the editor) Noname manuscript No. (will be inserted by the editor) Y-K. Park, M-K Kim and J. Um
Kyung Hee University, 1732 Deogyeong-daero, Yongin-si, Republic of Korea
Tel.: +82-31-201-3695
Fax: +82-31-203-4004
E-mail: jayum@khu.ac.kr Received: date / Accepted: date Received: date / Accepted: date Received: date / Accepted: date Abstract The research on predictive maintenance of rotating machines, the
most important element in manufacturing facilities, has been very active. The
widespread availability of smart factory solutions has led to improved data
collection from machines and processes and is able to provide key information. For our purpose, the collected information enables the maintenance system to
predict the remaining useful life using deep learning models. The introduction
of multi-layer perceptron of signal processing originating from bearings, in
time series data, has been discussed in many publications. However, estimat-
ing accuracy for the remaining useful life is determined by the selection of the
feature domain and the concatenation network model. Herein, we introduce a
convolutional Autoencoder based on multi-domain ensemble learning in order
to include various feature domains and a concatenation network operated by
latent space into a single neural network. The performance of the proposed
model is evaluated by using a simple health indicator and a PRONOSTIA
dataset and compared with a simple concatenation model, 2-stage Autoen-
coder, and a recurrent neural network. Keywords Remaining useful life · Convolutional auto-encoder · Ensemble
learning · Prognostics health management An One-Stage Ensemble Framework based on
Convolutional Autoencoder for Remaining Useful
Life Estimation Yong-Keun Park · Min-Kyung Kim ·
Jumyung Um* Yong-Keun Park · Min-Kyung Kim ·
Jumyung Um* 1.1 prognostics and health management 1.1 prognostics and health management At the PHM stage, data is collected using sensors, and after determining the
health of the system based on the collected data, an abnormal situation is diag-
nosed and RUL (Remaining Useful Life) is predicted. In addition, HI (Health
Indicator) extraction, which is an indicator of health for evaluating machine
health, is an indispensable process. Finally, the time of machine failure can
be predicted via HI. It may result in the completion of PdM technology that
can be serviced only when needed. In addition, based on this, the optimum
maintenance cycle and range for formulating a maintenance strategy can be
determined, so there is a cost reduction effect[5]. For predictive maintenance of machine, bearing is the main component of
analysis. There are many rotating part in machine. Bearings account for about
40% of machine failures[6] and are the most breakdown component in failure. For these reasons, among the various component of machine, bearing re-
maining useful life prediction is used in the main predictive maintenance
method. Bearing remaining useful life prediction is mainly analyzed via vi-
bration signals and sound[7], and various signal processing technologies such
as noise reduction and fault frequency measurement are used. There are several methods to extract features in various areas; however,
the use of a single domain is problematic. For example, if only the time do-
main model is used, a prediction model skips learning the crack signal occurred
in the frequency spectrum; thus, the model can miss the information that the
features have in each domain, and even though some models and machines per-
form well, the problem resides in that the pre-processing method depends on
the data specific to each domain. Therefore, many studies have suggested fea-
tures created from various domains of time, frequency, and time-frequency in
order to complement the shortcomings of aforementioned methods consisting
of extracting a single domain feature. However, despite these attempts, there
exist drawbacks, since the features of the three domains are lumped into one
data set, i.e simple concatenate, that is used as the input of the model. This
method is insufficient to efficiently extract the information included in each
domain, and to conduct as a solution for multi-domain features extraction. Selecting the best feature data set or using latent space of AE (Auto En-
coder) were the typical methods to achieve multi-domain feature extraction. Both methods, however, have some drawbacks. 1 introduction The field of PHM(Prognostics and Health Management) was initiated from
electronics area in 2006 [1] and extended to the are of rotary machinery in
2014 [2]. It is intended to provide the increase of productivity in machine 2 Yong-Keun Park et al. maintenance. Since 2011, Industry 4.0 widely spreads and was initiative to
build the infrastructure able to collect data from device, machine and whole
system. And an increasing amount of literature has been published in PHM
over a diverse methods from machine learning to deep learning. 1.1 prognostics and health management Using a particular feature
makes a learning model rely on specific metrics. It diminishes the benefits
to build a data set from multiple domains. For example, it is worthwhile to
add a specific feature capturing the critical moments of the breakage, even
though giving the model a negative influence in the most period during the 3 Title Suppressed Due to Excessive Length learning process. Feature selection ignores features showing the correlation in
only a certain moment. learning process. Feature selection ignores features showing the correlation in
only a certain moment. To eliminate the drawbacks of feature selection, several studies have adopted
latent space to reduce the dimension using deep learning models, in particular
AE , in feature extraction. However, feature extraction through AE has a com-
plexity problem arising from the need to learn two different prediction models:
the first AE for learning and the second latent space and RUL for predicting
HI. Learning the two models has the disadvantage of increasing the complexity
of the model structure, resulting in that efficient learning of the latent space
is not performed. The reason for this is that after learning AE, features are
extracted using the latent space. The extracted features are then used to train
the RUL prediction model. The disadvantage of this method is that HI, which
is the final output value of the RUL prediction model, is learned without any
interaction with the features extracted through the latent space. In this paper, the authors suggest the method using an all-domain data set
in a one-stage learning model including latent spaces of AE and RUL model to
predict HI. The remainder of this paper is structured as follows: section 2 sur-
veys the state-of-the-art of relevant research. section 3 describes the proposed
model architecture. Section 4 compares the proposed model with relevant mod-
els of HI prediction. Section 5 provides the discussion and conclusion. 2 Literature survey Sensor data driven approach is required for pre-processing of sensor signal. Signal processing techniques are used to analyze vibration signals. Prepro-
cessing is performed on 3 main domain. There are 3 types: (1) time domain
feature, (2) frequency domain feature, and (3) time-frequency domain feature. (1) In the case of time domain features Various statistics have been widely
used to efficiently capture bearing failure signals. Typically, RMS (Root Mean
Square), Kurtosis, etc. have been used. These statistics describe the overall fail-
ure process of bearing. However, there is a drawback that it does not describe
the detailed failure steps. (2) Frequency domain features are mainly analyzed
by transforming time domain signals such as FT (Fourier Transformation)
and Hilbert-Huang transformation to the frequency domain. The advantage of
these features is that the technology is known to efficiently perform the steps
of initial failure and final failure. However, it has the disadvantage of not de-
scribing the procedure for intermediate failures. (3) Time-Frequency domain
features are often STFT (Short Time Fourier Transform) and WT (Wavelet
Transformation). These transformation features are known as have few errors
without loss of information[11]. After the preprocessing is done, selecting meaningful features is performed. Such a method is called feature selection. Feature selection select more efficient
feature for obtaining bearing failure sign and removing unwanted features. in
this process, various metrics are used, that are monotonicity, trendability, ro-
bustness, etc[12]. Features are selected based on these metrics. The process for 4 Yong-Keun Park et al. selecting sensitive features includes not only feature selection, but also feature
extraction. Feature extraction is a method of creating new features by us-
ing existing features. PCA(Principle Component Analysis), ICA(Independent
Component Analysis), Partial Least Square, SOM(Self-Organizing Map), etc. are used[13]. In addition to this, a feature extraction method through deep
learning is also used, using a method in which a model or neural network
extracts its own function using CNN(Convolutional Neural Network), or us-
ing AE(AutoEncoder). AE is one of the feature extraction methods that has
attracted a great deal of attention by utilizing the latent space. RUL prediction has been extensively studied, but two general methods
have been widely used. The first method is to predict the change of breakage
through a physical model[8], and the second is to predict the degradation based
on sensor data[9]. The physical model is practically difficult to implement a
complex actual facility operating environment. 2 Literature survey In other words, expressing all
situations with few failure mechanisms is bound to be limited. For this reason,
machine learning techniques that learn from sensor data based on a black box
model are widely applied[10]. 2.2 Feature selection and Feature extraction After collecting a feature in each area, both feature selection and feature ex-
traction remove unnecessary signals and remain only useful information. Vari-
ous metrics such as monotonicity, trendability, and robustness are used for fea-
ture selection. In [15], only trendability among features is applied to machine
learning. In other machine learning techniques, [26] selected three metrics,
trendability, monotonicity, and the average of the two metrics, while [18] used
Mahalanobis distance of features. [31] highlighted that there have also been
studies to select features by using information theory metric. These studies
are to use statistical techniques as feature selection. Sensitive features using
the chi-square test[32] and F-test[33] are applied to feature selection. Following feature selection, there are many studies in which AE is per-
formed as a feature extraction. Lin and Tao extracted features using a latent
space after ensembles a number of AEs, and used it as an input to a model
that predicts RUL [34]. Similar studies using three domains as feature sets
conducted, But Ren et al. used latent space and the remaining set of domain
features as inputs to the RUL prediction model after feature extraction via
AE only in the time domain [11]. Xu et al also applied AE model to only
prediction of final output value of HI [21]. AE is a deep learning model with
the same input and output, but there is a difference in that the input data
is not restored. Similarly, there is a PHM study that does not restore input
data [35], and the difference is that a network is designed for bearing failure
classification from the final output value. Some studies tried to conduct both
feature selection and feature extraction simultaneously. Hu et al. and She et al. performed feature extraction through RBM and then selected features through
HI metric [30][25]. 2.1 Domain Selection In many studies, to analyze the bearing vibration signal, various features have
been used in 3 domain which are time domain, frequency domain, and time-
frequency domains. In many studies, to analyze the bearing vibration signal, various features have
been used in 3 domain which are time domain, frequency domain, and time-
frequency domains. q
y
Using time domain as input features is to apply a set of statistics fac-
tors extracting from time-series signal data. Fundamentally RMS (Root Mean
Square) have been used as bearing diagnosis and HI [16]. Adding the window
size in RMS is suggested to depict the changes along the time [17]. Among the
statistics used as features in the time domain[15], a statistic called waveform
entropy was presented. In [18], after decomposing signals into specific levels
using Wavelet Packet Decomposition (WPD), 14 statistics were extracted for
each decomposed signal level. In addition, many studies have been conducted
using the original signal[19]. The window size is adjusted and the original
signal is used as an input. In addition, there have been attempts to find a
meaningful information only the original signal through CNN[20]. [21] constructed input data by using the frequency signal applying a moving
average after applying FT. In addition, [22] and [15] research using FT as input
data. Finally, there are studies using the time-frequency domain. There are also
studies in which an input shape similar to an image in a two-dimensional
format was created using STFT (Short Time Fourier Transformation) and
learned through CNN[23]. In addition, [24] imaged each signal using WT and
then extracted HI using a CNN network. There are studies using a multi domain input. [25], [11], [26] make features
using statistics as a time domain feature, FT as a frequency domain feature, 5 Title Suppressed Due to Excessive Length and WT as a time frequency domain feature. [27] also make feature in 3 do-
mains, reduces the dimension through RBM (Restricted Boltzmann machine),
and predicts HI through GRU (Gated Recurrent Units). In addition, [28] that
do not use all 3 domains and use statistics and raw signals in some cases,
and [29] use an RVM (Relevance vector machine) to construct a model. [30]
predicted HI using statistics and frequency domain feature. 2.4 Summary and Opportunities 2.4 Summary and Opportunities As the survey of previous researches, many studies have used specific domain
to extract features. There are cases where a single domain is used as a feature,
and other studies used two or more domain as a feature. However, A common
point of both cases is that feature extraction was simply made into one data
set and used as an input to the model. The problem of using only one domain
as a feature is missing the characteristics of remaining features. This issue
arises even when more than one domain is used as a feature. It is simply
concatenate of one data set and used to input the model. Simple concatenation
is insufficient to learn all domains efficiently. From previous literature, AE secures the better performance of feature ex-
traction and of feature selection. There have been various researches on feature
selection and extraction, but accuracy of learning models fluctuated, because
feature selection goes through a process of selecting features based on specific
metrics. Feature extraction via AE shows the high accuracy than using other
dimension reduction methods. However, learning two models at the same time,
feature extraction via AE and RUL prediction, increase model complexity un-
necessarily. As a solution to this, there have also been studies predicting HI
directly through AE. Even in these cases, the difference between inputs and
outputs appear a problem that the latent space cannot be trained efficiently. the study of constructing the two models into one model are insufficient. In
this work, we present a model consisting of one model, along with latent spa-
tial layer learning of AE. The conclusions we can draw after reviewing the
literature are: 1. Feature sources need to be rich and diverse. Estimating remaining useful
lifetime require for multiple aspects of bearing sensor signals. As existing
literature describe, time-series data, frequency data, and time-frequency
data are employed for feature extraction because bearing signals usually
are vibration, current, and acoustic noise. 2. Feature selection is required for reducing abnormal effects from whole
dataset. The disturbance of machine learning is caused by the series of
data not to follow the learning direction of whole dataset. It is noise or
event happened by very specific accident. Selecting features is critical de-
cision to reduce the effect of noise like this. It is inevitable to ignore useful
data too. 2.3 Deep learning model of remaining useful life After extracting the feature for bearing failure from the vibration signal in
various domain, the remaining useful life is predicted via a deep learning model. Deep running models typcially used MLP(Multi Layer Perceptron), and uses
RNN(Recurrent Neural Network) and CNN [14]. The remaining useful life
was efficiently predicted through the features extracted in the above. After
demonstrating the efficiency of the latent space using AE, AE was used to
reduce the dimensions and perform feature extraction. The features extracted 6 Yong-Keun Park et al. in this way were used as inputs to the model for predicting the remaining useful
life, and created a bearing prediction model with even higher performance [9]. in this way were used as inputs to the model for predicting the remaining useful
life, and created a bearing prediction model with even higher performance [9]. 2.4 Summary and Opportunities The abandon of certain data need to logical reason in order to
keep consistence of estimating RUL. 3. Feature synthesis improves the possibility of finding hidden correlation of
raw data. Both mathematical approach and data driven approach are used
for evaluating correlations and reducing data dimension. Title Suppressed Due to Excessive Length 7 From these conclusions it appear that there is opportunity to motivate new
predictive maintenance algorithm of rotary parts. 3 Feature Ensemble AutoEncoder Architecture Motivated by the opportunities mentioned in previous section, then, the im-
provement of feature extraction, and selection are viewed as the way of en-
hancing the performance of RUL prediction. In this paper, it is proposed for
comprehensive data flow turned into an architecture incorporating ensemble
learning with FEAE(Feature Ensemble AutoEncoder) and is depicted in Fig-
ure 1. The proposed model framework is design for covering with following
aspects 1. Wavelet Packet Decomposition to distinguish low and high frequency in
time-frequency domain. 2. Applying AutoEncoder to Multi-domain approach 2. Applying AutoEncoder to Multi-domain approach 3. Reducing dimensions of multi-domain by using latent space generated by
AutoEncoder 4. 1-stage model with combined loss function is applied to enhance the learn-
ing rate of whole model. Signal processing technique is used to remove noise and redundant informa-
tion of signal. Then after applying signal processing technique, extract various
domain features that time, frequency, and raw signal source, it is used as input
data of the FEAE model. The FEAE model consists of two parts. First, FEAE
learn latent space through each AE. At the same time, each latent space is
coming through input of the RUL model, and finally HI is predicted. Fig. 1 Procedure of proposed framework for estimating RUL Fig. 1 Procedure of proposed framework for estimating RUL Yong-Keun Park et al. 8 3.1 Signal processing of Wavelet Packet Decomposition Since bearing vibration signal has rich information such as trouble, faulty of
machine, accurate vibration analysis is essential process. First, Direct Compo-
nent(DC) value remove from the signal purpose of right transformation such as
fourier and wavelet. Second, to remove noise in signal apply window function
because of edge of signal has many uncertainty. Since bearing vibration signal has rich information such as trouble, faulty of
machine, accurate vibration analysis is essential process. First, Direct Compo-
nent(DC) value remove from the signal purpose of right transformation such as
fourier and wavelet. Second, to remove noise in signal apply window function
because of edge of signal has many uncertainty. Wavelet Packet Decomposition(WPD) is a time-frequency analysis method
that is widely used in the field of signal processing. We apply WPD to pre-
processing of proposed procedure in order to improve the resolution of complex
signal data. In the case of WPD, the signal is decomposed into coefficients (low
frequency components) and details (high frequency components) at the first
level. 3 Feature Ensemble AutoEncoder Architecture This can be thought of as the low-frequency and high-frequency pass
filter components of the signal[36]. Zhang, Z., Wang, Y., Wang, K. (2013). Fault diagnosis and prognosis using
wavelet packet decomposition, Fourier transform and artificial neural network. Journal of Intelligent Manufacturing, 24(6), 1213-1227. The main difference between the WT(Wavelet Transform) and WPD is
the way the signal is more decomposed after the first level. Simply put, WPD
are WT that pass more filters in signal processing. WT decomposes only
low-frequency components at subsequent levels, while WPD decomposes low-
frequency and high-frequency components at each level[37]. Therefore, WPD
provides better resolution in various areas where the signal contains high fre-
quency information. The equation W (p, q) presented below is an equation for WT. x (t) is the
signal to apply WT, ψ∗(t) means a certain wavelet (mother wavelet), and
ψ∗means complex conjugate. To do. Further, p plays a role of reducing or
increasing a certain wavelet as a scaling parameter, and q means a variable that
moves a certain wavelet as a movement parameter. The formula DWT(Discrete
Wavelet Transformation) (i, j) can be obtained from the dioxide of W (p, q). p and q are replaced by 2i, 2j. Repeated decomposition can be used to obtain
low frequency (cA1) and high frequency (cD1) band signals figure 2 Fig. 2 Procedure of Discrete Wavelet Transformation Fig. 2 Procedure of Discrete Wavelet Transformation 9 Title Suppressed Due to Excessive Length 9 When the original signal is decomposed into level 2 via the WPD process,
it can be decomposed into a total of 4 signals as shown in figure 3. WT is said
to be a nonstationary signal that works as a frequency filter and is effective
in reducing noise [38], [39]. The decomposed signal contains low-frequency to
high-frequency features. Next, in order to execute the preprocessing of the
input data, the four decomposition signals and the original signal are created
as one data set, and then the preprocessing is executed. Fig. 3 Decomposition result of WPD up to Level 2 Fig. 3 Decomposition result of WPD up to Level 2 In particular, the resolution of WPD helps to detect progressive increase
of specific vibration caused by the growth of cracks in rotating components. Figure 4 shows the advantage of WPD in estimating RUL. 3 Feature Ensemble AutoEncoder Architecture The input signal
data is to made by combining narrow low-frequency signal, decreasing signal,
increasing signal, wide high-frequency, and random noise signal in all different
frequencies. The result of WPD keeps the trends of each signal even though
the noise signal is mixed. The accuracy of WPD resolution is evaluated with
the result of FFT filter and DC removing filter. Orange peaks cover with blue
peaks of original signal. 3.2 Preprocessing of Multi-domain dataset In this section, we will turn our focus to the way to extract feature sets. From
raw signal, pre-processing is required to extract bearing degradation signal ef-
ficiently. Typically feature sets of bearing data can be categorized into Signal,
Statistics, Frequency. In the case of time-domain basis feature set, several tra-
ditional features were introduced for predicting the failures of bearings such 10 Yong-Keun Park et al. Fig. 4 Resolution result of different mixed signals by using Level 2 WPD Fig. 4 Resolution result of different mixed signals by using Level 2 WPD as RMS, kurtosis, skewness, etc. In the case of frequency-domain basis feature
set, Fourier transform that transforms time domain vibration data into a fre-
quency spectrum is used to extract frequency domain information. In the case
of time-frequency domain basis feature set, wavelet analysis is used to analyze
signal as different resolution. In this paper uses 3 domain features that are
below discussed 1. Signal: Original signal & wavelet signal are used to signal domain feature
S
S
f
& 1. Signal: Original signal & wavelet signal are used to signal domain feature 2. Statistics: Statistics of original signal & wavelet signal are used to statistics
domain feature 3. Frequency: Unlike above mentioned features, Frequency feature only work
original signal. 3. Frequency: Unlike above mentioned features, Frequency feature only work
original signal. 3.2.2 Statistics feature set 3.2.1 Signal feature set The basic signal is an input type that is widely used in various studies applying
deep learning. This has the advantage of not requiring preprocessing. Also, in
the case of basic signals, it is used in the most common form to which the basic
signal processing algorithms and WPD described in Section 3.2 are applied. As shown in Figure 5, in the case of the final data set, it is used as one data
set to which the original signal and the wavelet are applied. 3.2.2 Statistics feature set Extract the statistic of the converted signal and the original signal using WPD Extract the statistic of the converted signal and the original signal using WPD. Statistics often used in RUL prediction are RMS, Kurtosis, Peak-to-peak, Mar- Extract the statistic of the converted signal and the original signal using WPD. Statistics often used in RUL prediction are RMS, Kurtosis, Peak-to-peak, Mar- Statistics often used in RUL prediction are RMS, Kurtosis, Peak-to-peak, Mar- Title Suppressed Due to Excessive Length 11 Fig. 5 Pre-processing of each signal source Fig. 5 Pre-processing of each signal source gin factor, Mean absolute deviation, Skewness, Kurtosis, and Waveform en-
tropy. A total of 8 statistics are used. Since eight statistics of the wavelet-
transformed signal and the original signal are extracted, a total of 40 types of
statistics are extracted. Therefore, it is possible to efficiently extract various
information possessed by each frequency signal. The overall procedure for the
Statistics feature set is shown in Figure 5. The proposed model uses the easy-to-obtain frequency domain signals as
the input of the deep learning model basically. Time-domain features are cor-
related with the overall trend for the bearing faulty and are the fundamental
inputs of deep learning model for machine degradation. But not only that, but
also because of the nature of raw data that is not found with frequency filters,
primary statistics of raw data is selected as first input of proposed model. It is
also includes statistical figures directly obtained from raw data and a method
for collecting raw data characteristics, unlike in the frequency domain. 3.3 Ensemble learning of latent space generated by AutoEncoders 3.3 Ensemble learning of latent space generated by AutoEncoders In statistics and machine learning, ensemble methods use multiple learning al-
gorithms to obtain better predictive performance than could be obtained from
any of the constituent learning algorithms alone. Unlike a statistical ensemble
in statistical mechanics, which is usually infinite, a machine learning ensemble
consists of only a concrete finite set of alternative models, but typically allows
for much more flexible structure to exist among those alternatives . An ensem-
ble model method is a machine learning process to obtain better prediction
performance by strategically combining multiple learning algorithms . There
are three primary advantages brought by ensemble methods . 1) The first one
is called statistical reason which is related to lack of sufficient data to prop-
erly represent the data distribution. Without sufficient data, many hypotheses
which give the same training accuracy may be chosen as the learning algorithm. Ensemble methods can thus reduce the risk of selecting the wrong model by
aggregating all these candidate models. 2) The second is computational rea-
son. Many learning algorithms, such as decision tree and neural network, work
by performing some form of local search. These methods can frequently result
in locally optimal solutions. Ensemble methods show their advantages in this
scenario by running many local search from different starting points. 3) The
last reason is representational. In most cases, the true function f cannot be
represented by any single hypothesis H. However, the function can be better
approximated by a weighted sum of several hypotheses. 3.2.3 Frequency feature set FT has the advantage of being able to move signals in the time domain to the
frequency domain. In addition, various frequencies of the signal can be decom-
posed by frequency via a periodic function to confirm the characteristics in an
intuitive understanding of FT. The following equation is a Fourier transform
equation that decomposes a signal via a sine periodic function. In the case of
Fourier transform, it plays a role of converting the signal in the time domain
into the frequency domain. However, since the signal obtained via WPD is
already a signal decomposed for each frequency, the same information will be
obtained when FT is applied. For these reasons, as shown in Figure 5, WPD
was not applied to Frequency, which is a frequency domain feature set, and
only FT was applied to the original signal. Yong-Keun Park et al. 12 3.4 1-stage learning model for RUL prediction Finally, all the methods described in the methodology are used to provide a
new model, FEAE. First, proceed with the ensemble of input data. In order
to efficiently compress and extract the information that each input data has,
the structure shown in figure 6 is used. This structure is a model based on
three AEs, and each model uses a feature set of three regions as input values. Each feature set is the three input data of Signal, Statistics, and Frequency
described above. Also, the latent space of each AE is connected to the RUL
model that predicts HI. The AE and RUL models consist of one stage, that
is, one model. There are two main models proposed, and the first is AE that learns latent
space. The second part is a model that predicts HI using the latent space as an
input to the RUL model. FEAE will eventually use the feature set of the three
areas Signal, Statistics, and Frequency as input. In addition, the final output
has a total of 4 outputs with 3 AE restoration results for each feature set and 1
RUL model. The advantage of the structure of these models is that the latent
space of AE is efficiently trained by two elements. First, the latent space is
trained through the restoration loss function of AE. Second, each latent space
is used as an input to the RUL model and predicts HI. There is an advantage Title Suppressed Due to Excessive Length 13 that the latent space can be learned via MSE (Mean Square Error), which is
the loss function of this RUL model. Fig. 6 Network model of 1-stage feature ensemble autoencoder for estimating RUL Fig. 6 Network model of 1-stage feature ensemble autoencoder for estimating RUL 3.5 Health indicator of remaining useful life There are many ways in which HI can be designed. However, HI, which pre-
dicts the failure time of another machine, is generally highly volatile, making
it difficult to set a failure threshold. In general, even in the case of bearings,
the threshold value is determined experimentally because the HI value is dif-
ferent for each work and data. To dispel these ambiguities, this paper also uses
common HI design methods. The HI value in the initial state is set to 0%, and
when a complete failure occurs, the HI value is set to 100% to present the HI
value that can be applied in any situation. For example, if the total drive time
is 2800s and the current time is 1400s, the current HI is set to 0.5 fiture 7. (xt, yt), where t ∈T, xt ∈F, (F = Feature set)
(1) (1) (xt, yt), where t ∈T, xt ∈F, (F = Feature set) Yong-Keun Park et al. 14 14
Yong-Keun Park et al. Fig. 7 HI Construction
4 Experiment
4.1 Dataset
The data used in the experiment to validate the methodology is the IEEE PHM
challenge PRONOSTIA dataset in 2012 [40]. The PRONOSTIA data consists
of 3 working conditions, and the number of bearings in each experimental
condition is 7, 7, and 3. In addition, the sampling frequency of each bearing
is 25.6kHz, and accelerometers are attached to the x and y axes, and the
vibration of the two axes can be obtained. Finally, it consists of a run-to-failure
dataset that collected from the start of initial operation until the occurrence
of corruption. The summary of data is shown in Table 2. In addition, 3 data are used for the test data required for methodological
verification. Each dataset has a tendency as shown in Fig. 11, and a detailed
explanation is given in Table 3. It has individual working condition. Bear- 14
Yong Keun Park et al. Fig. 7 HI Construction
4 Experiment Fig 7 HI Construction Fig. 7 HI Construction 4.1 Dataset The data used in the experiment to validate the methodology is the IEEE PHM
challenge PRONOSTIA dataset in 2012 [40]. The PRONOSTIA data consists
of 3 working conditions, and the number of bearings in each experimental
condition is 7, 7, and 3. In addition, the sampling frequency of each bearing
is 25.6kHz, and accelerometers are attached to the x and y axes, and the
vibration of the two axes can be obtained. Finally, it consists of a run-to-failure
dataset that collected from the start of initial operation until the occurrence
of corruption. The summary of data is shown in Table 2. In addition, 3 data are used for the test data required for methodological
verification. Each dataset has a tendency as shown in Fig. 11, and a detailed
explanation is given in Table 3. It has individual working condition. Bear- Title Suppressed Due to Excessive Length 15 Table 1 Experiment condition
Experiment condition
number of bearing
notation
Experiment info
1800RPM & 4000N
7
Bearing1_1
bearing 1_7
sampling period interval: 10s
1800RPM & 4000N
7
Bearing1_1
bearing 1_7
sampling frequency: 25.6KHz
1800RPM & 4000N
7
Bearing1_1
bearing 1_7
0.1s snapshot, x,y axis accelerometer
Table 2 Experiment condition
Bearing
Description
Bearing1_1
general failure pattern, monotonic increasing amplitude
Bearing2_1
general failure pattern, sudden increasing amplitude
Bearing3_1
general failure pattern, sudden increasing amplitude, outlier Table 1 Experiment condition
Experiment condition
number of bearing
notation
Experiment info
1800RPM & 4000N
7
Bearing1_1
bearing 1_7
sampling period interval: 10s
1800RPM & 4000N
7
Bearing1_1
bearing 1_7
sampling frequency: 25.6KHz
1800RPM & 4000N
7
Bearing1_1
bearing 1_7
0.1s snapshot, x,y axis accelerometer
Table 2 Experiment condition
Bearing
Description
Bearing1_1
general failure pattern, monotonic increasing amplitude
Bearing2_1
general failure pattern, sudden increasing amplitude
Bearing3_1
general failure pattern, sudden increasing amplitude, outlier ing1_1, Bearing2_1, and Bearing3_1, in that order, have more and more
complex failure patterns with common failure patterns. Fig. 8 Model Framework(a) Test Bearing1_1, (b) Test Bearing2_1, (C) Test Bearing3_1 Fig. 8 Model Framework(a) Test Bearing1_1, (b) Test Bearing2_1, (C) Test Bearing3_1 4.2 Model structure 4.2 Model structure 4.3 comparision 4.3 comparision 4.3.1 model structure 4.3.1 model structure In this section we compare the FEAE model with a single input model. As
shown in Figure 12, a comparison is made between using a single input model
and using all input data. This is an experiment to evaluate the performance
of the model structure. 1. Signal AutoEncoder 2. Statistics AutoEncoder 3. Frequency AutoEncoder Bearing1_1: g _
This is the case of the simplest bearing failure pattern with continuous am-
plitude increase. All models predict HI almost accurately, and Statistics AE
predicts the HI closest to the label in a single input model. However, in the Yong-Keun Park et al. 16 Fig. 9 Experiment of model structure compare to FEAE
(a) Test Bearing1_1, (b) Test Bearing2_1, (c) Test Bearing3_1 Fig. 9 Experiment of model structure compare to FEAE
(a) Test Bearing1_1, (b) Test Bearing2_1, (c) Test Bearing3_1 case of the Frequency AE model, there is a rapid increase and some differ-
ences from other models can be seen. Finally, it can be seen that the Signal
AE model looks similar to the proposed FEAE model, but after the second
half point, HI decreases again and shows unstable prediction. Bearing2_1: g _
Amplitude in the initial state of these, it is a case where it continues until
the latter half and has a failure pattern after repeating momentary changes in
amplitude. In the initial state, Statistics AE and Signal AE tend to increase
in close proximity to label, while Frequency AE and FEAE models tend to
increase rapidly. In the middle state, Signal AE tends to decrease suddenly,
although the tendency is almost similar. In the latter half, in the case of Statis-
tics AE and Frequency AE, there is a pattern in which HI decreases again in
the last part. Due to the characteristics of RUL It is a bad tendency to de-
crease again. In addition, the proposed FEAE and Signal AE models tend to Title Suppressed Due to Excessive Length 17 increase to the end. In the final RUL part, the FEAE model shows the HI
closest to the label and tends to increase to the end. increase to the end. In the final RUL part, the FEAE model shows the HI
closest to the label and tends to increase to the end. Bearing3_1: This data have a pattern in which a value equal to or greater than the oc-
currence of a momentary change in amplitude occurs after maintaining the
amplitude in the initial state. Statistics AE and Signal AE show a downtrend
in the last part, showing bad predictions. The Frequency AE and FEAE mod-
els tend to be closest to the label, showing a steadily increasing trend. ?? shows the calculated metric that evaluate HI for the 3 models. Mon(X) =
1
N −1
No.of
d
dx > 0 −No.of
d
dx < 0
(2)
Corr(X, T) =
PN
i=1(Xi
0 −¯X)(T i −¯T)
qPN
i=1(Xi
0 −¯X) PN
i=1(T i −¯T)
(3)
Cri = Corr + Mon
2
(4) (2) (4) Fig. 10 metric In case of Monotonicity, K is the number of data in the entire life cycle. Monotonicity = 1 mean that there is a complete monotonic, in other cases
it means that HI oscillates. There is an irreversible relationship between the
actual machine failure tendencies. Failure do not recover spontaneously with-
out human intervention. Appropriate HI Monotonicity tends to increase or
decrease monotonically, which is usually the case. Trendability shows the linear correlation between operating time and HI. At this time, K is the number of data in the entire life cycle, x means HI,
and t means the operating time. As the operating time increases, the faulty
is gradually failing. Therefore, the trendability of the general situation, that
is, the time and HI linear correlation, a value close to 1 will be a meaningful
metric. 18 Yong-Keun Park et al. Criteria means the average value of the both metric. Rather than relying
on only one of the two indicators, using the average of both values provides a
more reliable indicator. Summarizing the experiments on the 3 datasets, Statistics AE is perform-
ing well on average. And in certain situations, it shows good performance
when using a certrain feature set. However, case of the FEAE model, you can
see that it maintains the stable tendency in any situation and has strong ro-
bustness. These characteristics are the result of efficiently learning the unique
information of the vibration signal that only each time, frequency, and signal
has. 4.3.2 input structure 4.3.2 input structure The FEAE model uses all feature set combinations, and at the same time, the
model is trained through individual AEs that Signal, Statistics, and Frequency
each feature sets that have the different meaning. We also use the same dataset
to compare with the basic simple concat model in Figure 16. The comparison
of both models uses the same data, but you can see a comparison of whether
the performance of the models changes depending on how you train them. Fig. 11 Network models of 1-stage FEAE(left) and Simple concat(right) Fig. 11 Network models of 1-stage FEAE(left) and Simple concat(right) Bearing1_1:
There is no big difference between the FEEA and Simple concat model Bearing2_1:
Bearing2_1 is The same aspect is shown in the case of Bearing1_1. but there
are a few differences. The Simple concat model shows a increase in the initial Title Suppressed Due to Excessive Length 19 Fig. 12 (a) Average reconstruction error as a function of the number of groups. (b) Average
reconstruction errors of ten scenarios. Fig. 12 (a) Average reconstruction error as a function of the number of groups. (b) Average
reconstruction errors of ten scenarios. state, whereas the FEAE model does not. It can also be seen that the FEAE
model shows even better predictive power in the final HI predictive value. Bearing3_1: Bearing3_1: There is a big difference in performance from the previous test bearing. Bear-
ing3_1 is data with outliers, and there is a big difference between the two
models in terms of performance. Both models also show a steady rise, but the
FEAE model can be seen to perform better. The difference between the two models performance change depending on
how the input data is trained. The Simple concat model and the FEAE model
learned the same data. The FEAE model train the features of statistics, signals,
and frequency domains through different AEs for each domain. In contrast,
the Simple concat model concatenates three domains into a single dataset and
uses the merged dataset as the training data. The difference between these 20 Yong-Keun Park et al. learning methods appears in the performance of models, and the method of
learning features as one dataset as in existing research is to inefficiently extract
the unique information of each domain that each source has. 4.3.3 1stage and 2stage 4.3.3 1stage and 2stage FEAE model(1stage model) and two model(2stage model) that includes for
feature extraction through AE and prediction of HI are compared in this sec-
tion. The difference between the 1stage model(FEAE) and the 2stage model
is that learning is performed at the same time, or features are extracted using
the AE that has been trained, and the RUL model is trained. This experiment
is a comparative experiment of how the latnet space can be learned efficiently
by the two loss functions. Fig. 13 Network models of 1-stage FEAE(left) and 2-stage FEAE(right) Fig. 13 Network models of 1-stage FEAE(left) and 2-stage FEAE(right) Bearing1_1:
It looks similar until the initial state, but as time goes on, the 2stage model
shows that HI is no longer increasing. Bearing2_1 & Bearing3_1: Two experiments can see that the initial HI
start is non-zero. This can be seen as a result of the vibration signals of the
two bearings being generated with the initial amplitude maintained. In ad-
dition, the 2stage model does not show a large increase with the passage of
time. These features indicate that the latent space of the trained AE does not
act as an efficient input feature, and discussions related to training the RUL
prediction model apply. be able to. Learning the RUL prediction model using the latent space of trained AE
as an input feature is common to both models (1stage, 2stage). However, the
difference between both models is that using a trained AE, it trains the AE as Title Suppressed Due to Excessive Length 21 Fig. 14 (a) Average reconstruction error as a function of the number of groups. (b) Average
reconstruction errors of ten scenarios. Fig. 14 (a) Average reconstruction error as a function of the number of groups. (b) Average
reconstruction errors of ten scenarios. well as the RUL prediction model. In the case of 1stage model (FEAE), the
latent space is trained by two loss function that are the loss function of AE
and RUL prediction model. The two loss functions efficiently learn the latent
space of each AE through the backpropagation method, which is a learning
method of deep learning, and at the same time, the final output of the model
has better prediction performance of HI. 4.3.3 1stage and 2stage However, in the case of the 2stage
model, the models that predict AE and HI that perform feature extraction
have to show poor performance because they are trained separately. 4.3.4 RNN type model Finally, a comparison was made between the AE-based model and the RNN-
based model. RNN-based models have been used to handle time-series data
traditionally and are one of the used classic methods. This experiment de- 22 Yong-Keun Park et al. scribes the difference between a model that uses the latent space of AE and a
model that predicts HI using an existing RNN-based model. Fig. 15 Network models of 1-stage FEAE(left) and RNN(right) Fig. 15 Network models of 1-stage FEAE(left) and RNN(right) Fig. 16 (a) Average reconstruction error as a function of the number of groups. (b) Average
reconstruction errors of ten scenarios. Fig. 16 (a) Average reconstruction error as a function of the number of groups. (b) Average
reconstruction errors of ten scenarios. Bearing1_1:
In the case of the RNN model, there is continuous oscillation compared to the
FEAE model. There is a continuous increasing trend until the second half. However, in the end, there is a tendency for it to decrease as it progresses, and
then to increase again. Bearing1_1:
In the case of the RNN model, there is continuous oscillation compared to the
FEAE model. There is a continuous increasing trend until the second half. However, in the end, there is a tendency for it to decrease as it progresses, and
then to increase again. Bearing3_1: In the initial state, the RNN model and the FEAE model have the same ten-
dency, but it can be seen that the RNN model does not rise over time as it goes
beyond the middle state. And finally the RNN model can be seen to increase
a little at the end. In the case of the FEAE model and the RNN model, the difference is that
they are the AE-based model and the RNN model. A model using the latent
space of AE can show higher performance than an RNN model using the data
of each domain as a feature without performing feature extraction. It can also
be seen that the FEAE model increases more stably than the RNN model. Bearing1_1: Title Suppressed Due to Excessive Length 23 Bearing2_1:
The RNN model starts at 0.2 and can be seen to look similar to FEAE until
the second half. However, it can be seen that the FEAE model increases in
the part that predicts the final HI, but the RNN model does not increase, but
predicts a constant HI. 5 Conclusion In this study, we proposed a methodology for solving the problems related to
the selection and extraction of input data and features that should be con-
sidered in bearing life prediction. As a method to solve the problem of input
data, the signal processing technique classically used for vibration signal anal-
ysis was applied. In addition, vibration signals divided each frequency band
via WPD, and the each domain was pre-processed to propose method in vari-
ous studies was used. We applied feature extraction through AE to solve the
problem of feature selection. By integrating the 2stage model, which is a draw-
back of the model using AE, into 1stage, we were able to solve the complicated
problem displayed when using the two models, and obtain a model with high
performance. Experiments were performed using the PRONOSTIA dataset to
verify this methodology. Through the FEAE model presented in this study,
it was confirmed that higher performance was obtained than when a single
AE was used. And the FEAE model made it possible to learn the meaning
of the feature more efficiently than when the functions of each area were cre-
ated as one data set. In addition, feature extraction through AE was efficient. By converting the 2stage model to the 1stage model, which is a drawback of
the model using these structures, we were able to obtain a model with even
better performance. There are various facilities and working conditions at the
site. However, there are many difficulties in applying one algorithm to each
facility. If there is an area that best describes each facility, and if the working
conditions change, the above problems can occur. However, when applied to
equipment via the proposed method, it will be possible to create models that
are even stronger or have better performance through ensembles in various
areas. When predicting the remaining life based on these advantages, field 24 Yong-Keun Park et al. workers can obtain more reliable results. Research for predictive maintenance
of bearings is the main field of prediction of remaining life. However, the di-
rection of research to be pursued now will be important to analyze the value
of HI calculated by any factor through explainable artificial intelligence. As a
first step for that, it will be necessary to analyze the latent space of AE. 6 Acknowledgements This research was supported by the Ministry of Trade, Industry Energy(MOTIE),
Korea Institute for Advancement of Technology(KIAT) through the project of
Development of Customized Smart HMI Systems (No.20012807) 5 Conclusion Then,
more information can be obtained from the data by analyzing how each input
function is related to the latent space and how the HI value is interpreted. PHM . . . . . . . . . . . . Prognostics and Health Management
PdM . . . . . . . . . . . . Predictive Maintenance
RUL . . . . . . . . . . . . . Remaining Useful Life
HI . . . . . . . . . . . . . . . Health Indicator
WT . . . . . . . . . . . . . . Wavelet Transformation
DWT . . . . . . . . . . . . Discrete Wavelet Transformation
FT . . . . . . . . . . . . . . . Fourier Transformation
WPD . . . . . . . . . . . . Wavelet Packet Decomposition
AE . . . . . . . . . . . . . . AutoEncoder
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How do people use and view infographics that summarise health and medical research? A cross-sectional survey
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© 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. Abstract Background: Understanding how people use infographics and their opinion on them has important implications
for the design of infographics but has not been investigated. The aim of this study was to describe people’s use of
and opinions about infographics summarising health and medical research, preferences for information to include in
infographics, and barriers to reading full-text articles. Methods: We conducted an online cross-sectional survey of consumers of infographics that summarise health or
medical research. Demographic and outcome data were collected and summarised using descriptive statistics. A
sensitivity analysis explored whether being a researcher/academic influenced the findings. Results: Two hundred fifty-four participants completed the survey (88% completion rate). Participants included
health professionals (66%), researchers (34%), academics (24%), and patients/the public (13%). Most used Twit‑
ter (67%) and smartphones (89%) to access and view infographics, and thought infographics were useful tools to
communicate research (92%) and increase the attention research receives (95%). Although most participants were
somewhat/extremely likely (76%) to read the full-text article after viewing an infographic, some used infographics as
a substitute for the full text at least half of the time (41%), thought infographics should be detailed enough so they
do not have to read the full text (55%), and viewed infographics as tools to reduce the time burden of reading the full
text (64%). Researchers/academics were less likely to report behaviours/beliefs suggesting infographics can reduce
the need to read the full-text article. Conclusions: Given many people use infographics as a substitute for reading the full-text article and want infograph‑
ics to be detailed enough so they don’t have to read the full text, a checklist to facilitate clear, transparent, and suf‑
ficiently detailed infographics summarising some types of health and medical research may be useful. Keywords: Infographics, Visual abstract, Graphical abstract, Health, Medicine, Cross-sectional, Survey Joshua R. Zadro1*, Giovanni E. Ferreira1, Mary O’Keeffe1, Will Stahl‑Timmins2, Mark R. Elkins3 and
Christopher G. Maher1 Joshua R. Zadro1*, Giovanni E. Ferreira1, Mary O’Keeffe1, Will Stahl‑Timmins2, Mark R. Elkins3 and
Christopher G. Maher1 *Correspondence: joshua.zadro@sydney.edu.au 1 Institute for Musculoskeletal Health, The University of Sydney and Sydney
Local Health District, PO Box M179, Level 10 North, King George V Building,
Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050,
Australia Full list of author information is available at the end of the article Introduction ‘Infographic’ is an abbreviated term for an information
graphic [1]. Infographics generally combine text, images
and data visualisations to present information visually,
increase the attention it receives, and to improve com-
prehension and recall [1–3]. Infographics are becom-
ing increasingly popular as a method for summarising
research findings [3–5], although they often have other Zadro et al. BMC Medical Education (2022) 22:677
https://doi.org/10.1186/s12909-022-03744-6 Zadro et al. BMC Medical Education (2022) 22:677
https://doi.org/10.1186/s12909-022-03744-6 Open Access Study design, participants, and recruitment Study design, participants, and recruitment
We conducted an online cross-sectional survey, with
data collected between August and December 2021. We
posted a link advertising our study on Twitter and Face-
book to recruit people who self-identified as consumers
of infographics summarising health or medical research
(e.g. health professionals, researchers, patients or mem-
bers of the public). People had to be 18 years or older
to participate and could be living in any country. Study
advertisements were posted from the Institute for Mus-
culoskeletal Health Twitter (@msk_health; 4500 fol-
lowers) and Facebook (@IMHSydney1; 150 followers)
accounts, which reach a mix of researchers, academics,
health professionals, and members of the public. These
accounts mostly post, retweet or share the findings of
interesting musculoskeletal research. They were not post-
ing infographics produced by the Institute for Muscu-
loskeletal Health at the time this survey was conducted,
although infographics from journals are occasionally
retweeted or shared. gy
J
p
)
Infographics appear to be increasing in popularity [1]
and evidence suggests some infographics increase the
attention an article receives on social media [6–8]. How-
ever, it is unknown whether people use different types of
infographics more commonly as stand-alone resources to
interpret research findings or to decide whether to seek
more information about a study. Either approach could
explain why some infographics decrease or have no effect
on the attention full-text articles receive in some cases [3,
8–10]. There is evidence of harmful misuse of research
[11] when clinicians only read the abstract of an arti-
cle. For example, the President of Global Strategies for
HIV Prevention (Arthur Amman) tells a story of a phy-
sician engaged in perinatal HIV prevention in southern
Africa who started delivering a less effective preventative
treatment because they only had access to the abstract
of an article, which spun the study’s results. The physi-
cian’s decision to alter their prevention approach based
on the article’s abstract may have harmed many people
[11]. We are concerned a similar scenario could occur if
health professionals, researchers, or patients view info-
graphics as stand-alone resources. These concerns are
compounded by our recent work showing that most info-
graphics from health and medical journals do not report
enough information to allow readers to adequately inter-
pret and apply study findings (e.g. key study characteris-
tics, limitations, effect sizes) [1]. © 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. Zadro et al. BMC Medical Education (2022) 22:677 Zadro et al. BMC Medical Education (2022) 22:677 Page 2 of 9 use infographics that summarise health and medical
research, their opinions about these infographics, their
preferences for what information to include in these info-
graphics, and barriers to reading full-text articles that
might lead them to solely rely on an infographic. uses in research (e.g. present information more visu-
ally or interactively, highlight certain information from
a research article). Many health and medical journals
now use infographics, such as visual abstracts, graphi-
cal abstracts and interactive graphics, to boost the vis-
ibility and dissemination of the research they publish [6]
(e.g. New England Journal of Medicine, The BMJ, JAMA
Oncology, British Journal of Sports Medicine). Study design, participants, and recruitment Conversely, if research is
not accessible because it takes too long to read for over-
whelmingly busy clinicians or is too challenging for many
patients to understand, valuable health innovations could
be under-utilised. Data collection
Th
T itt
d The Twitter and Facebook post included a link to com-
plete an online survey hosted in Qualtrics survey soft-
ware. The first page of the survey briefly described the
study, provided a link to read the Participant Informa-
tion Statement (which defined infographics as per the
opening sentences of this manuscript), and included a
Participant Consent Form. There was no financial incen-
tive for participants. All recruitment and data collection
procedures were approved by the University of Sydney
Human Research Ethics Committee (Reference number:
2021/542). All methods were carried out in accordance
with relevant guidelines and regulations. Before starting
the survey, all participants provided consent by check-
ing a box that confirmed they had read the Participant
Information Statement and Consent Form and agreed to
participate. Participants’ rights were protected. Informed
consent was obtained from each participant. Understanding people’s use of and opinions about info-
graphics has important implications for their design but
has not been investigated. Infographics won’t ever be able
to replace the full-text article. However, if most people
use infographics as stand-alone resources or substitutes
for reading the full-text article, there may be a need to
ensure information presented in infographics is more
comprehensive (i.e. through enhanced visuals or text)
and reflects important features of the full-text article. People may also have different opinions about the func-
tions of infographics, the type of information they expect
to see, and experience barriers to reading full-text articles
which should be considered when designing infograph-
ics. The aims of this study were to describe how people Participants were asked to provide demographic data
such as age, gender, educational attainment, employment
status, and background (researcher, health professional,
academic, patient or member of the public, other). Par-
ticipants were asked: “When did you last come across an
infographic that summarised research you were inter-
ested in (e.g. on social media, in a journal)?” Response Zadro et al. BMC Medical Education (2022) 22:677 Page 3 of 9 outcome data for participants involved (vs. not involved)
in research and/or academia since these groups may
use infographics for different reasons (e.g. due to a lack
of access or time to read full-text articles). We used a
Chi2 test to investigate differences in outcomes between
these groups and conducted analyses using Stata BE Ver-
sion 17.1 (StataCorp LLC, College Station, Texas, USA). Preferences for information to include in infographics Preferences for information to include in infographics
Participants were asked to indicate what information
from the full-text article should be included in an info-
graphic (e.g. ‘conclusion or ‘take away’ message’, ‘descrip-
tion of the population, intervention(s), comparison(s)
and outcome(s)’, ‘sample size’). Sample characteristics 88% of those who commenced the survey completed
it (n = 254/289). Table 1 reports the characteristics of
participants who completed the survey (n = 254). These
participants completed the survey in a median time of
7 minutes (IQR 5 to 10). Use of infographicsh This included questions on how likely participants are to
find and read the full text article after viewing an info-
graphic (5-point Likert scale from ‘Extremely unlikely’
to ‘Extremely likely’), how often they use infographics as
a substitute for reading the full text article (5-point Lik-
ert scale from ‘Never’ to ‘Always’), and how participants
access (e.g. social media, journals) and view infographics
(e.g. smart phone, laptop). Barriers to reading full‑text articles Participants were asked to indicate what barriers they
experience when trying to read full-text articles.hi The survey finished with a free-text response question
where participants could add any other comments that
would help the researchers understand how they use or
view infographics (Additional file 2). Data collection
Th
T itt
d Responses to the free-text question at the end of the sur-
vey were grouped into themes (Additional file 2). options included: “In the past week”; “In the past month”;
“In the past 6 months”; “In the past 12 months”; “I have
never come across an infographic that summarised
research I was interested in”. Participants who selected
the last option were excluded from the study. The full
survey is in Additional file 1. Use of infographics Most participants were somewhat/extremely likely to
find and read the full-text article after viewing an info-
graphic (76%). Some used infographics as a substitute for Participants were asked whether they think infograph-
ics should be detailed enough so readers can translate
the findings to their context without having to read the
full-text article (5-point Likert scale from ‘Definitely not’
to ‘Definitely yes’), whether they think infographics are
useful tools for communicating research and increasing
the attention research receives (5-point Likert scale from
‘Definitely not’ to ‘Definitely yes’), and what they think
the functions of an infographic should be. This last ques-
tion included pre-specified options (e.g. ‘reduce the time
burden of reading the full-text article’, ‘entice people to
read the full-text article’) and a free-text box for partici-
pants to list other functions. Table 1 Demographics of the participants who completed the
survey (N = 254)
n number of participants satisfying the item, N number of participants with data,
SD standard deviation
a Percentages do not add to 100% because participants could select multiple
options
Demographics
Descriptive statistics
Female, n (%)
116 (46%)
Age (years), mean (SD)
37 (12)
Education, n (%)
High school (completed)
12 (5%)
Non-university tertiary education
6 (2%)
University
236 (93%)
Employment, n (%)
Employed
217 (85%)
Student
32 (13%)
Unemployed or retired
5 (2%)
Background, n (%)a
Researcher
85 (33%)
Academic
62 (24%)
Health professional
174 (69%)
Patient or member of the public
25 (10%)
Researcher or academic
116 (46%)
Not involved in research or academia
138 (54%)
Last come across an infographic, n (%)
In the past week
193 (76%)
In the past month
47 (19%)
In the past 6 months
13 (5%)
In the past 12 months
1 (< 1%) Table 1 Demographics of the participants who completed the
survey (N = 254) Data analysis All data were summarised descriptively using counts
and percentages, means and standard deviations (SD),
and median and interquartile ranges (IQR), as appropri-
ate. We performed a sensitivity analysis summarising Zadro et al. BMC Medical Education (2022) 22:677 Zadro et al. BMC Medical Education (2022) 22:677 Page 4 of 9 to determine study quality (26%), interpret the meth-
ods (23%), and interpret the results (24%). Participants
not involved (vs. involved) in research or academia
were more likely to report lack of access (83% vs. 56%,
p < 0.001) and being unsure how to determine study qual-
ity as barriers (35% vs. 16%, p = 0.001) (Table 2). reading the full-text article at least half of the time (41%),
and this was more common among those not involved
(vs. involved) in research or academia (53% vs. 29%;
p < 0.001). Most access infographics via Twitter (67%)
and view infographics on a smartphone (89%). Partici-
pants not involved (vs. involved) in research or academia
were less likely to access infographics via Twitter (58%
vs. 78%, p = 0.001), more likely to access infographics via
Instagram (57% vs. 29%, p < 0.001), and less likely to use
a laptop to view infographics (47% vs. 62%, p = 0.017)
(Table 2). Other comments about use or views on infographics
The most common themes from free-text responses
about use of or views about infographics were that they
help communicate research in user-friendly way, inform
people and support decision making, and are not all
equal in quality (Additional file 2). Strengths and weaknesses of the study We recruited participants from a range of backgrounds
(e.g. researchers, health professionals, patients, mem-
bers of the public) and had a high completion rate (88%). The main limitation of this study is that data on people’s
use of infographics is self-reported and may be different
in real life. Another limitation is only recruiting through
Twitter and Facebook, which may have led to a biased
sample. For example, the high proportion of health pro-
fessionals, academics, and researchers likely reflects the
composition of people who follow the Institute for Mus-
culoskeletal Health Twitter and Facebook accounts, and
the type of people who were liking, re-tweeting, or shar-
ing our study advertisement. Future research should con-
sider ways to reach a broader audience in the medical and
research community. Summary of main findings Although most participants were somewhat/extremely
likely to read the full-text article after viewing an info-
graphic, some used infographics as a substitute for read-
ing the full-text article at least half of the time, thought
infographics should be detailed enough so they don’t
have to read the full-text article, and viewed infographics
as tools to reduce the time burden of reading the full-text
article. Most of these behaviours and beliefs were more
common among those who were not involved in research
or academia. Most participants used Twitter and smart-
phones to access and view infographics, and thought
infographics were useful tools to communicate research
and increase the attention research receives. A large
majority expected to see the conclusion or ‘take away’
message and the four PICO elements in infographics. Lack of time was the most common barrier to reading
full-text articles in the total sample, while lack of access
was the most common barrier for those not involved in
research or academia. Preferences for information to include in infographics
Most participants expected to see a conclusion
or ‘take away’ message in an infographic (95%), a
description of the four PICO elements – population
(81%), intervention(s) (92%), outcome(s) (87%) and
comparison(s) (74%) – the sample size (65%), and sta-
tistics summarising the effects of an intervention (58%). Fewer participants expected to see study limitations
(39%) and conflicts of interest (26%). Participants not
involved (vs. involved) in research or academia were less
likely to want a description of the population in an info-
graphic (76% vs .87%, p = 0.026) (Table 2). Opinions about infographics Many participants thought infographics should probably/
definitely be detailed enough so readers can translate the
findings to their context without having to read the full-
text article (55%), and this was more common among
those not involved (vs. involved) in research or academia
(65% vs. 43%; p = 0.007). Most thought infographics
probably/definitely were useful tools for communicating
research (92%), and this was more common among those
not involved (vs. involved) in research or academia (96%
vs. 87%, p = 0.022). Most thought infographics prob-
ably/definitely were useful tools for increasing the atten-
tion research receives (95%) and viewed infographics as
a way to communicate research in a more user-friendly
way (89%). Most viewed infographics as a way to reduce
the time burden of reading the full-text article (64%), and
this was more common among those not involved (vs. involved) in research or academia (76% vs. 49%, p < 0.001)
(Table 2). Other functions of infographics included deliv-
ering evidence-based, accessible information to patients
and the public (including those with low health literacy),
reaching new audiences, and using infographics as a
recap after reading the full-text article. Barriers to reading full text articlesh The most common barriers to reading full-text arti-
cles were lack of time (77%) and access (71%). Less fre-
quently reported barriers included being unsure how Page 5 of 9 Zadro et al. Barriers to reading full text articlesh BMC Medical Education (2022) 22:677 Page 6 of 9 Table 2 (continued) Table 2 (continued)
Total sample
Not involved in
research/ academia
Involved in
research/ academia
Chi2, p-value**
Reduce the time burden of reading the full text
162 (64%)
105 (76%)
57 (49%)
19.8, p < 0.001*
Help readers quickly decide whether to read the full text
161 (63%)
89 (65%)
72 (62%)
0.2, p = 0.690
Entice readers to read the full text
146 (58%)
72 (52%)
74 (64%)
3.5, p = 0.062
Other
18 (7%)
9 (7%)
9 (8%)
0.1, p = 0.702
Preferences for information to include in infographics
Information expected to see in an infographic, n (%)*
Conclusion or ‘Take away’ message
240 (95%)
131 (95%)
109 (94%)
0.1, p = 0.738
Description of intervention(s)
234 (92%)
130 (94%)
104 (90%)
1.8, p = 0.180
Description of outcome(s)
220 (87%)
122 (88%)
98 (85%)
0.8, p = 0.360
Description of population
206 (81%)
105 (76%)
101 (87%)
5.0, p = 0.026*
Description of comparison(s)
188 (74%)
96 (70%)
92 (79%)
3.1, p = 0.078
Sample size
166 (65%)
84 (61%)
82 (71%)
2.7, p = 0.101
Statistics summarising the effect size
147 (58%)
79 (57%)
68 (59%)
0.0, p = 0.825
Some study limitations
100 (39%)
56 (41%)
44 (38%)
0.2, p = 0.667
Conflicts of interest
65 (26%)
31 (23%)
34 (29%)
1.6, p = 0.213
Other
14 (6%)
9 (7%)
5 (4%)
0.6, p = 0.442
Barriers to reading full-text articles, n (%)*
Lack of time
196 (77%)
102 (74%)
94 (81%)
1.8, p = 0.178
Lack of access
180 (71%)
115 (83%)
65 (56%)
22.7, p < 0.001*
Unsure how to determine study quality
67 (26%)
48 (35%)
19 (16%)
11.0, p = 0.001*
Unsure how to interpret results
60 (24%)
37 (27%)
23 (20%)
1.7, p = 0.192
Unsure how to interpret methods
59 (23%)
35 (25%)
24 (21%)
0.8, p = 0.380
Other
14 (6%)
9 (7%)
5 (4%)
0.6, p = 0.442
No barriers experienced
6 (2%)
3 (2%)
3 (3%)
0.0, p = 0.829
Never attempted to access full text
3 (1%)
2 (2%)
1 (1%)
0.2, p = 0.666
NB: the ranking question was not included in the Table because the results were almost identical to the question about what information people expect to see in an
infographic
IQR Interquartile range, n number of participants satisfying the item, N number of participants with data, SD standard deviation
*Percentages do not add to 100% because participants could select multiple options
**Comparison between those not involved in research or academia (N = 138) and those involved in researcher and/or academia (N = 116) to quickly scan for studies that align with their research
interests or for studies that have features indicating low
risk of bias (e.g. Barriers to reading full text articlesh randomisation, low loss to follow-up). Barriers to reading full text articlesh BMC Medical Education (2022) 22:677 Page 5 of 9 Table 2 Use of and opinions about infographics, preferences for information in infographics, and barriers to reading full text articles
in the total sample (N = 254 participants) and compared between those not involved in research or academia (N = 138) and those
involved in researcher and/or academia (N = 116) Table 2 Use of and opinions about infographics, preferences for information in infographics, and barriers to reading full text articles
in the total sample (N = 254 participants) and compared between those not involved in research or academia (N = 138) and those
involved in researcher and/or academia (N = 116) Total sample
Not involved in
research/ academia
Involved in
research/ academia
Chi2, p-value**
Use of infographics (primary outcomes)
Likely to read full text after viewing an infographic, n (%)
Extremely unlikely
6 (2%)
3 (2%)
3 (3%)
4.5, p = 0.474
Somewhat unlikely
25 (10%)
17 (12%)
8 (7%)
Neither likely nor unlikely
30 (12%)
14 (10%)
16 (14%)
Somewhat likely
139 (55%)
72 (52%)
67 (58%)
Extremely likely
54 (21%)
32 (23%)
22 (19%)
Use infographics as a substitute for reading full text, n (%)
Never
30 (12%)
9 (7%)
21 (18%)
20.8, p < 0.001*
Sometimes
119 (47%)
57 (41%)
62 (54%)
About half the time
49 (19%)
31 (23%)
18 (16%)
Most of the time
53 (21%)
40 (29%)
13 (11%)
Always
3 (1%)
1 (1%)
2 (2%)
Accessing infographics, n (%)*
Twitter
170 (67%)
80 (58%)
90 (78%)
11.0, p = 0.001*
Instagram
111 (44%)
78 (57%)
33 (29%)
20.2, p < 0.001*
Journal website
87 (34%)
44 (32%)
43 (37%)
0.8, p = 0.386
Facebook
77 (30%)
47 (34%)
30 (26%)
2.0, p = 0.157
Non-journal website
42 (17%)
21 (15%)
21 (18%)
0.4, p = 0.537
Other
28 (11%)
9 (7%)
19 (16%)
6.2, p = 0.012*
Device used to view infographics, n (%)*
Smart phone
225 (89%)
124 (90%)
101 (87%)
0.5, p = 0.487
Laptop
137 (54%)
65 (47%)
72 (62%)
5.7, p = 0.017*
Desktop
63 (25%)
31 (23%)
32 (28%)
0.9, p = 0.346
iPad
38 (15%)
18 (13%)
20 (17%)
0.9, p = 0.350
Other
3 (1%)
1 (1%)
2 (2%)
0.5, p = 0.463
Opinions about infographics
Infographics should be detailed enough so readers don’t have to read the full-text, n (%)
Definitely not
20 (8%)
7 (5%)
13 (11%)
14.0, p = 0.007*
Probably not
39 (15%)
17 (12%)
22 (19%)
Might or might not
55 (22%)
24 (17%)
31 (27%)
Probably yes
87 (34%)
53 (38%)
34 (29%)
Definitely yes
53 (21%)
37 (27%)
16 (14%)
Infographics are useful tools to communicate research, n (%)
Definitely not
1 (< 1%)
0 (0%)
1 (1%)
11.4, p = 0.022*
Probably not
3 (1%)
1 (1%)
2 (2%)
Might or might not
16 (6%)
4 (3%)
12 (10%)
Probably yes
49 (19%)
22 (16%)
27 (23%)
Definitely yes
185 (73%)
111 (80%)
74 (64%)
Infographics increase the attention research receives, n (%)
Definitely not
0 (0%)
0 (0%)
0 (0%)
1.1, p = 0.776
Probably not
4 (2%)
2 (2%)
2 (2%)
Might or might not
8 (3%)
3 (2%)
5 (4%)
Probably yes
53 (21%)
28 (20%)
25 (22%)
Definitely yes
189 (74%)
105 (76%)
84 (72%)
Functions of an infographic, n (%)*
Communicate research in a more user-friendly way
226 (89%)
127 (92%)
99 (85%)
2.9, p = 0.090 Zadro et al. Meaning of the study Participants who were not involved in research or aca-
demia were more likely to use infographics as a substitute
for reading the full-text article, think infographics should
be detailed enough so they don’t have to read the full-text
article, and view infographics as tools to reduce the time
burden of reading the full-text article. Given that most
infographics don’t allow readers to adequately interpret
and apply study findings [1], these findings confirm our
initial concern that some health professionals may be
making poor preventative or treatment decisions because
they use infographics as a substitute for reading the full-
text article [11]. Nearly one-third of researchers and aca-
demics use infographics as a substitute for reading the
full-text article at least half of the time, which might be
due to a lack of time (reported by 4 in 5 researchers and
academics). However, there could be other explanations. Some researchers and academics may use infographics Participants who were not involved in research or aca-
demia were more likely to report lack of access as a bar-
rier to reading the full-text article, which may explain
why they appear more reliant on the content of info-
graphics. However, they were not less likely to read the
full-text article after viewing an infographic compared
to researchers and academics. One explanation is that
participants may have interpreted our question about
their likelihood of reading a full-text article after view-
ing an infographic in the context of having access to the
full-text article. In addition, the question about barriers
was framed in a way that anyone who has been unable to
access a full-text article on at least one occasion would
have reported ‘lack of access’ as a barrier. This may have
inflated the prevalence of this barrier in our sample, Zadro et al. BMC Medical Education (2022) 22:677 Page 7 of 9 particularly among those not involved in research or
academia. lack of effect may be explained by low article views over-
all. For example, a prospective, case-control crossover
study took 40 articles published over a 11-month period
in American Journal of Nephrology and tweeted each arti-
cle in three formats (citation only vs. citation plus key fig-
ure from the article vs. citation plus visual abstract) [8]. Tweets including visual abstracts received similar arti-
cle visits compared to the other formats (9.0 for visual
abstracts vs. 8.1 for citation only vs. Comparison to existing literature Our participants’ view that infographics can be useful
tools to communicate research is consistent with previ-
ous research highlighting that visual abstracts (a type
of infographic) require less cognitive load and are more
preferred over traditional abstract summarises [13] and
are rated as more user-friendly [14] (without negatively
impacting knowledge [14] or information retention [13]). However, it needs to be acknowledged that considerable
care is required when examining research on the benefits
and limitations of infographics since there is substantial
variation in the design of infographics and the quality
and impact of the research they summarise. In The BMJ between June and December 2018, tweets
including visual abstracts received fewer URL clicks (29
vs. 60) compared to the average of all tweets from The
BMJ. However, this finding was only specific to visual
abstracts. Other types of infographics performed better
(e.g. more detailed full page visual summaries, interactive
graphics) [15]. Over the past 3 years, The BMJ has found
that 64.9% of their tweets including visual abstracts
(87.5% in 2021) have been at least 15% above the average
tweet in most metrics (unpublished data). This suggests
improving the quality of visual abstracts may improve
their impact. Other studies have also found a positive
effect of infographics on article visits [6]. Nevertheless,
the total available evidence may challenge the assump-
tion that infographics increase the attention research
receives and may suggest many people are solely relying
on the infographic summary of some studies to inform
their research, clinical practice or healthcare choices. Participants’ view that infographics increase the atten-
tion research receives is also consistent with several
studies that have found some infographics increased
the number of impressions and re-tweets Twitter posts
receive [6, 8] and increased abstract views [3, 10]. How-
ever, there are some infographics that have reduced
impressions, re-tweets, abstract views and citations [9,
15]. There are also examples of some infographics that
may not be superior to other research communication
tools. For example, a survey of 58 physicians and nurses
who were shown a one-page text summary and an info-
graphic of a systematic review on pain medication for
acute migraine found the infographic had substantially
greater visual appeal, but lower clarity and comprehen-
sibility [2]. Meaning of the study 6.7 for citation plus
key figure). Another study took 12 articles published in
the Canadian Journal of Emergency Medicine that were
promoted on Facebook and Twitter without infographics
over a 3-month period in 2015/16 and compared these to
11 articles published in this journal and additionally pro-
moted using infographics over the same time [3]. Articles
promoted using infographics received slightly less full-
text views compared to articles promoted without info-
graphics (65 vs. 73). Another interesting finding is that participants not
involved in research or academia were more likely to
access infographics via Instagram (nearly twice as much)
and less likely to access them via Twitter. This should be
considered by journal editors and researchers trying to
increase the reach of their research, particularly among
non-academic audiences. In addition, a similar propor-
tion of people involved vs. not involved in research or
academia access infographics on journal websites (37%
vs. 32%) which might be explained by some publishers
making their infographics open access even when the
full-text article is behind a paywall [12]. Ethics approval and consent to participate All recruitment and data collection procedures were approved by the
University of Sydney Human Research Ethics Committee (Reference number:
2021/542) and all methods were carried out in accordance with relevant
guidelines and regulations. Before starting the survey, all participants provided
consent by checking a box that confirmed they had read the Participant Infor‑
mation Statement and Consent Form and agreed to participate. Participants’
rights were protected. Informed consent was obtained from each participant. Availability of data and materials y
All data relevant to the study are available upon reasonable request to the
corresponding author. Acknowledgements
N/A Acknowledgements
N/A Competing interests All authors declare: no support from any organisation for the submitted work;
no financial relationships with any organisations that might have an interest
in the submitted work; no other relationships or activities that could appear to
have influenced the submitted work. Authors’ contributions All authors critically revised the manuscript for important intellectual content
and approved the final manuscript. Please find below a detailed description
of the role of each author: Joshua R Zadro: conception and design, analysis
and interpretation of data, drafting and revision of the manuscript, and final
approval of the version to be published. Giovanni Ferreira: conception and
design, interpretation of data, drafting and revision of the manuscript and
final approval of the version to be published. Mary O’Keeffe: conception and
design, interpretation of data, drafting and revision of the manuscript and
final approval of the version to be published. Will Stahl-Timmins: conception
and design, interpretation of data, drafting and revision of the manuscript and
final approval of the version to be published. Mark R Elkins: conception and
design, interpretation of data, drafting and revision of the manuscript and final
approval of the version to be published. Christopher G Maher: conception and
design, interpretation of data, drafting and revision of the manuscript and final
approval of the version to be published. The Corresponding Author (JZ) attests
that all listed authors meet authorship criteria and that no others meeting the
criteria have been omitted. Funding
h
d This study did not receive any funding. Unanswered questions and future researchh There are limited data on people’s preference for what
information to include in infographics and why people
have certain preferences. When The BMJ were develop-
ing their template for visual abstracts, they sought feed-
back on a visual abstract of tai chi for fibromyalgia from
77 health professionals who treat patients with fibro-
myalgia (e.g. primary care physicians) [15]. Participants
wanted more detailed statistics so they could quickly
judge how effective tai chi was and for the conclusion
to be at the top of the visual abstract. Possible explana-
tions for this feedback include that these health profes-
sionals wanted infographics to reduce the time burden
of reading the full-text article or that more detailed sta-
tistics may have helped them decide whether they need
to read the full-text article. Our study adds further data Many participants in our study reported regularly
using infographics as a substitute for reading the full-text
article, thought infographics should be detailed enough
so readers can translate the findings to their context
without having to read the full-text article, and viewed
infographics as tools to reduce the time burden of read-
ing the full-text article. This appears to be consistent with
previous research showing that some infographics either
have no effect or a negative effect on article visits and
full-text downloads [3, 8–10]. However, in some cases a Zadro et al. BMC Medical Education (2022) 22:677 Zadro et al. BMC Medical Education (2022) 22:677 Page 8 of 9 Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12909-022-03744-6. Additional file 1. Survey. Additional file 2. Other comments from participants related to how they
use or view infographics. Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12909-022-03744-6. Additional file 1. Survey. Additional file 2. Other comments from participants related to how they
use or view infographics. to what people want to see in infographics, but it does
not explain why people have certain preferences. Future
qualitative research could be useful to explore this
issue. Supplementary Information Supplementary Information Given that most people sometimes use infographics
as a substitute for reading the full-text article and that
most broadly agree that infographics should be detailed
enough so they do not have to read the full-text article,
a checklist or ‘minimum standard’ for certain types of
infographics may facilitate clear, transparent, and suf-
ficiently detailed infographics summarising health and
medical research. We are currently conducting a Delphi
study to develop a checklist to improve the reporting
of infographics summarising the main findings of com-
parative studies of health and medical research (e.g. randomised controlled trials, systematic reviews) [16]. Such a checklist will likely outline the minimum items
necessary to include in these types of infographics
(e.g. PICO items, effect size, 95% CIs) but give design-
ers flexibility on how to present these items visually or
through text. A checklist could improve the accuracy
with which research findings are communicated and
avoid research findings being mis-interpreted if con-
sumers (e.g. health professionals, researchers) do not
refer to the full-text article. Once developed, we will
evaluate the acceptability of the checklist and whether
infographics developed according to the checklist are
more acceptable and lead to wiser healthcare choices. These findings will determine whether checklists for
other types of infographics could be useful, but we
acknowledge that checklists may be inappropriate for
some types of infographics (e.g. an infographic focusing
on the interaction of different participant characteris-
tics on a secondary outcome). Conclusion
l h
h Although most participants were somewhat/extremely
likely (76%) to read the full-text article after viewing an
infographic, some used infographics as a substitute for
reading the full-text article at least half of the time (41%),
thought infographics should be detailed enough so they
don’t have to read the full-text article (55%), and viewed
infographics as tools to reduce the time burden of read-
ing the full-text article (64%). These behaviours and
beliefs were more common among those who were not
involved in research or academia. A checklist to facilitate
clear, transparent, and sufficiently detailed infographics
summarising some types of health and medical research
could improve the accuracy with which research find-
ings are communicated and avoid research findings being
mis-interpreted if readers do not refer to the full-text
article. References Buljan I, Malički M, Wager E, Puljak L, Hren D, Kellie F, et al. No difference
in knowledge obtained from infographic or plain language summary of
a Cochrane systematic review: three randomized controlled trials. J Clin
Epidemiol. 2018;97:86–94. 15. Stahl-Timmins W, Black J, Simpson P. Pragmatic evaluation of The BMJ’s
visual abstracts. Inf Des J. 2019;25(1):101–9. 16. Equator Network. Reporting guidelines under development for other
study designs. https://www.equator-network.org/library/reporting-guide
lines-under-development/reporting-guidelines-under-development-for-
other-study-designs/#TERRI. Accessed 7 Dec 2021. Author details 1 Institute for Musculoskeletal Health, The University of Sydney and Sydney
Local Health District, PO Box M179, Level 10 North, King George V Building,
Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050,
Australia. 2 Data Graphics Designer, The BMJ, London, UK. 3 Faculty of Medicine
& Health, The University of Sydney, Sydney, Australia. Received: 18 May 2022 Accepted: 6 September 2022 Received: 18 May 2022 Accepted: 6 September 2022 Page 9 of 9 Zadro et al. BMC Medical Education (2022) 22:677 Zadro et al. BMC Medical Education (2022) 22:677 References 1. Ferreira GE, Elkins MR, Jones C, O’Keeffe M, Cashin AG, Becerra RE, et al. Reporting characteristics of journal infographics: a cross-sectional study. BMC Med Educ. 2022;22(1):326. 2. Crick K, Hartling L. Preferences of knowledge users for two formats of
summarizing results from systematic reviews: infographics and critical
appraisals. PLoS One. 2015;10(10):e0140029. pp
3. Thoma B, Murray H, Huang SYM, Milne WK, Martin LJ, Bond CM, et al. The
impact of social media promotion with infographics and podcasts on
research dissemination and readership. CJEM. 2018;20(2):300–6. 4. Murray IR, Murray AD, Wordie SJ, Oliver CW, Murray AW, Simpson AHRW. Maximising the impact of your work using infographics. Bone Joint Res. 2017;6(11):619–20. 5. Scott H, Fawkner S, Oliver C, Murray A. Why healthcare profes‑
sionals should know a little about infographics. Br J Sports Med. 2016;50(18):1104. 6. Ibrahim AM, Lillemoe KD, Klingensmith ME, Dimick JB. Visual abstracts
to disseminate research on social media: a prospective, case-control
crossover study. Ann Surg. 2017;266(6):e46–e8. 6. Ibrahim AM, Lillemoe KD, Klingensmith ME, Dimick JB. Visual abstracts
to disseminate research on social media: a prospective, case-control
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ics are more effective at increasing social media attention in comparison
with original research articles: an altmetrics-based analysis. Arthroscopy. 2021;37(8):2591–7. 7. Kunze KN, Vadhera A, Purbey R, Singh H, Kazarian GS, Chahla J. Infograph‑
ics are more effective at increasing social media attention in comparison
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crossover trial of visual abstracts to examine their impact on research
dissemination. J Med Internet Res. 2020;22(12):e22327. 9. Pferschy-Wenzig E-M, Pferschy U, Wang D, Mocan A, Atanasov AG. Does
a graphical abstract bring more visibility to your paper? Molecules. 2016;21(9):1247. 10. Huang S, Martin LJ, Yeh CH, Chin A, Murray H, Sanderson WB, et al. The
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ership: a randomized controlled trial. CJEM. 2018;20(6):826–33. 11. The PME. The impact of open access upon public health. PLoS Med. 2006;3(5):e252. 12. NEJM. Visual abstracts. https://www.nejm.org/multimedia/visual-abstr
acts. Accessed 9 Aug 2022. 13. Martin LJ, Turnquist A, Groot B, Huang SYM, Kok E, Thoma B, et al. Explor‑
ing the role of Infographics for summarizing medical literature. Health
Prof Educ. 2019;5(1):48–57. 14. Publisher’s Note
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Protective effects of coconut water against the intraperitoneal infused carbon tetrachloride-induced toxicity—evaluations of biochemical, haematological and histopathological profiles in rats
|
Bulletin of the National Research Centre/Bulletin of the National Research Center
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© 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/. Background Coconut water is a relished beverage gotten from coco-
nuts (Cocos nucifera L) (Gordon and Jackson 2017). Previously, coconut water was mostly acquired as a
fresh beverage in regions where it was accessible, by
cutting open a green nut fresh off the tree; today, it is a
$300 + million business (Gordon and Jackson 2017). It is Abstract Background: Coconut water is a relished beverage traditionally used as a remedy for childhood diarrhoea and gas‑
troenteritis. It can be given to an individual who has hangover or suspected to have ingested a toxic substance. In this
study, we evaluated the protective effect of coconut water against carbon tetrachloride (CCl4)-induced toxicity in rats. Results: Administration of coconut water decreased significantly albumin, aspartate transaminase (AST), alanine
transaminase (ALT), urea, creatinine, bicarbonate (HCO3
−), total cholesterol, triglycerides, low-density lipoprotein, very
low-density lipoprotein and malondialdehyde (MDA) levels compared to the non-pretreated group. Furthermore,
high-density lipoprotein, glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) increased significantly
(p < 0.05) in coconut water-pretreated groups compared to the negative control. There were no observed pathologi‑
cal changes in the coconut water-pretreated groups, but slight enlargement of the central veins and tubules was
evident when compared to the negative control. Conclusions: Therefore, intake of coconut water may be protective against toxicity induced by CCl4 as its pretreat‑
ment elevated antioxidant parameters (GSH, SOD and CAT) and did not alter biochemical parameters in rats. Keywords: Antioxidant, Carbon tetrachloride, Coconut water, Haematology, Toxicity Keywords: Antioxidant, Carbon tetrachloride, Coconut water, Haematology, Toxicity a perennial plant that bears fruit 12–13 times a year for
up to 60–70 years, providing between 30 and 75 fruits
per year. The fruit, in the shape of a nut, grows on trees
of different heights depending on the type of coconut
and is an important source of money for many farm-
ers and countries in the far East in particular, where it
is utilized in a number of ways and exported to other
countries across the world (Gordon and Jackson 2017). The fruit is a significant source of oilseeds, particularly
in many developing nations such as the Philippines, Sri
Lanka, India, Malaysia and portions of West Africa, Latin Emmanuel et al.
Bulletin of the National Research Centre (2022) 46:206
https://doi.org/10.1186/s42269-022-00893-y Emmanuel et al. Bulletin of the National Research Centre (2022) 46:206
https://doi.org/10.1186/s42269-022-00893-y Emmanuel et al. Bulletin of the National Research Centre (2022) 46:206
https://doi.org/10.1186/s42269-022-00893-y Bulletin of the National
Research Centre Bulletin of the National
Research Centre Protective effects of coconut water
against the intraperitoneal infused
carbon tetrachloride‑induced toxicity—
evaluations of biochemical, haematological
and histopathological profiles in rats Okezie Emmanuel1* , Ifeanyichukwu Elekwa1, Chidiebere Paul‑Joseph1, Victor C. Ude2, Ozioma G. Egedeuzu1,
Solomon N. Ijioma3, Victor Obioma Amachaghi1, Chikezie Uche‑Ikonne4 and Eziuche A. Ugbogu1 *Correspondence: emmanuelokezie7@gmail.com
1 Department of Biochemistry, Abia State University, PMB 2000, Uturu, Abia
State, Nigeria
Full list of author information is available at the end of the article Emmanuel et al. Bulletin of the National Research Centre (2022) 46:206 Page 2 of 11 America and the Caribbean (Gordon and Jackson 2017;
Ghosh et al. 2014). America and the Caribbean (Gordon and Jackson 2017;
Ghosh et al. 2014). America and the Caribbean (Gordon and Jackson 2017;
Ghosh et al. 2014). system’s reductive dehalogenation to create trichlorome-
thyl free radical, which rapidly interacts with molecular
oxygen to produce trichloromethylperoxyl radicals (Li
et al. 2004). Excessive formation of CCl4 free radicals
can result in membrane lipid oxidation, which can pro-
gress to hepatorenal injury (Li et al. 2004). In this study,
we investigated the protective effects of coconut water
against CCl4-induced toxicity in rats. The chemical composition of coconut water has been
widely studied (Yong et al. 2009; Awua et al. 2011; Prades
et al. 2012). Compositionally, it is made of about 96%
water and 6% solids. The major components of the sol-
ids in coconut water are soluble sugars, proteins and
minerals with relatively high potassium content and low
sodium content. Phytochemical compositions of coconut
water have shown to contain phenolic compounds, flavo-
noids, tannins and phytates (Akpro et al. 2019). However,
the content of coconut water is highly influenced by the
type of cultivar of coconut and the stage of maturation
of the nut (Gordon and Jackson 2017). The reports of
Yong et al. (2009) revealed the presence of some water-
soluble vitamins in coconut water such as thiamine, ribo-
flavin, nicotinic acid, pantothenic acid, pyridoxine, biotin,
folic acid and ascorbic acid. Salicylic acid, syringic acid,
m-coumaric acid, p-hydroxybenzoic acid, p-coumaric
acid, caffeic acid and catechin are among the phenolic
constituents found in coconut water (Mahoyothee et al. 2016). Aside from amino acids and L-arginine, coconut
water contains plant hormones such as auxin, 1, 3-diphe-
nylurea and cytokinin. It also contains acid phosphatase,
catalase, dehydrogenase, diastase, peroxidase, RNA poly-
merases and growth factors. More so, it contains some
minerals including sodium, potassium, magnesium, iron,
calcium, copper and phosphorous (Kaliyamoorthy et al. 2015). Collection and preparation of coconut water p
p
Healthy tender coconuts were gathered from Ndi Ekpere
Compound, Amaiyi Igbere in Bende Local Government
Area of Abia State, Nigeria. The coconut was de-husked,
and the epicarp was gently opened to avoid losing or
contaminating the coconut water. Every day, new coco-
nut water was collected and filtered in a sterile condition
using a ready-to-use Whatman filter No 1. Animal use and care
( Healthy rats (male and female) (8 weeks old; weight
151.78 ± 19.45 g, n = 60) bred locally at the Department
of Biochemistry, Faculty of Biological Sciences, Abia
State University, Uturu, were used for the study. The rats
were housed in a metal cage for at least a week before the
commencement of the study to allow for acclimatization
under natural atmospheric conditions. They were fed
with standard rat chow (Vital Feed) and water ad libitum. The laboratory procedures and tests were carried out
with the permission of the Abia State University Ethical
Committee (ABSU/REC/BMR/0026) and in accordance
with World Health Organization recommendations for
‘good laboratory practice’. Coconut water is unstable to air, and it is best con-
sumed fresh in tropical climates. Fresh coconut water
has traditionally been used for oral rehydration, treat-
ment of childhood diarrhoea and gastroenteritis (Mandal
et al. 2009). It provides several nutritional, physiological
and medicinal benefits including antifungal, antioxidant,
antibacterial, hypoglycaemic, anti-cancer, antitumor,
anti-dermatophyte, antiviral, anti-parasitic and hepato-
protective properties (Loki and Rajamohan 2003;
Mohamad et al. 2019; Elekwa et al. 2021). These biologi-
cal activities have been attributed to their high concen-
tration of bioactive chemicals. Researchers have also
uncovered that virgin coconut oil has anti-inflammatory,
analgesic, antipyretic, immune-stimulatory and anti-can-
cer (Intahpuak et al. 2010; Varma et al. 2019). Group 1: normal control- (distilled water; 1 mL/kg;
p.o.)
Groups 2: negative control- CCl4 only (1 mL/kg; i.p.)
Group 3: Coconut water (2 mL/kg; p.o.) + CCl4
(1 mL/kg; i.p.)
Group 4: Coconut water (4 mL/kg; p.o.) + CCl4
(1 mL/kg; i.p.)
Group 5: Coconut water (6 mL/kg; p.o.) + CCl4
(1 mL/kg; i.p.) Haematological and biochemical assays Red blood cell (RBC), white blood cell (WBC), platelets,
RBC indices and WBC indices were measured in accord-
ance with the procedure previously described by Bain
et al. (2017). Randox diagnostic kits (Randox Laboratory
Ltd., Co., Antrim, UK) were used to evaluate spectropho-
tometrically the biomarkers of kidney, liver and lipids. Urea, creatinine and electrolytes (Na+, K+, HCO3
− and
Cl−) were the renal biomarkers studied. The hepatic bio-
markers measured were alanine transaminase (ALT),
aspartate transaminase (AST), alkaline phosphatase
(ALP), bilirubin, total protein and albumin. Furthermore,
lipids calculated included triglycerides (TAG), high-den-
sity lipoprotein (HDL), total cholesterol (TC) and low-
density lipoprotein (LDL). Histopathological assessmentsh The liver and kidneys were fixed in 10% v/v forma-
lin before being encased in paraffin wax and sectioned. Each specimen was cut at 5 µm and stained with hae-
matoxylin and eosin dyes. Using X40 magnification light
microscope, the stained portions were examined for his-
tological changes and photomicrographs were taken. Statistical analysis
Th
d d The generated data were represented as mean ± stand-
ard deviation. To determine the difference between the
groups, statistical analysis was performed with R™ statis-
tical program (https://cran.r-project.org/src/base/R3/),
version 3.0.3. A significant relationship was determined
using the Tukey test post hoc at a 95% level of confidence. Experimental procedure Healthy Wistar rats (male, n = 30 and female, n = 30)
were randomly divided into five groups. To prevent mat-
ing, the male and female rats were placed in separate
cages. Groups 3–5 were treated with different doses of
coconut water for 14 days. After the 14th day pretreat-
ment with coconut water, groups 2–5 were given 1 mL/
kg of CCl4 (Merck, UK) diluted in olive oil at a 1:1 ratio
by intraperitoneal (i.p.) route. The procedure is shown as: Group 1: normal control- (distilled water; 1 mL/kg;
p.o.) Pollutants and chemical xenobiotics emitted by indus-
trial waste, such as CCl4, can harm the liver and kidney
by producing reactive oxygen species (ROS) (Dutta et al. 2018). ROS promotes oxidative stress, which has been
linked to hepatorenal damages (Liu et al. 2011). ROS
has also shown to induce cellular damage by lipid per-
oxidation and covalent binding, and an increase in ROS
decreases oxidative stress defence enzymes. Its mode of
action is evident in the liver cytochrome P450 enzyme Group 4: Coconut water (4 mL/kg; p.o.) + CCl4
(1 mL/kg; i.p.) Group 5: Coconut water (6 mL/kg; p.o.) + CCl4
(1 mL/kg; i.p.) Emmanuel et al. Bulletin of the National Research Centre (2022) 46:206 Page 3 of 11 Emmanuel et al. Bulletin of the National Research Centre (2022) 46:206 glutathione were evaluated using the methods described
by Buege and Aust (1978) and Sedlak and Lindsay (1968),
respectively. The rats were sacrificed after 24 h of CCl4 treatment. The rats were weighed and humanely sacrificed by cer-
vical dislocation. The blood samples were collected
through cardiac puncture and distributed into dry plain
bottles for biochemical tests and ethylenediaminetet-
raacetic acid (EDTA) bottles for haematological investi-
gations. The liver and kidney were harvested and used to
assess their histopathology. Protective effect of coconut water on histology section
of liver and kidneyh In the CCl4 group, the values of RBC, PCV, HB, platelet,
MCV and neutrophils were decreased compared to the
coconut water-treated groups. Furthermore, the values
of WBC and lymphocytes were increased in the negative The liver and kidney histology sections of the male and
female rats are presented in Figs. 2, 3, 4 and 5. The photo-
micrograph of the normal control showed a normal liver
architectural design, but the treated rats showed a slight Table 1 Protective effect of coconut water on liver biomarkers of carbon tetrachloride-induced toxicity in male (A) and female (B) rats
AST, aspartate aminotransferase; ALT, alanine transaminase; and ALP, alkaline phosphatase. Values are presented as mean ± SD, n = 6. Values in the row bearing the
same letters of the alphabet are not statistically significant (p > 0.05) from one another. The data were analysed with one-way ANOVA and expressed as mean ± SD,
then compared to controls, n = 6. Protective effect of coconut water on lipid profiles
of CCl4‑induced toxicity Table 4 shows the result obtained from the protective
effect of coconut water on lipid profiles of carbon tetra-
chloride-induced toxicity in both male and female rats. Groups administered with coconut water had a signifi-
cant decrease (p < 0.05) in the values of TC, triglycerides,
LDL and VLDL. The values of the HDL decreased signifi-
cantly in the CCl4 group compared to the coconut water-
treated groups. Protective effect of coconut water on liver biomarkers
of CCl4‑induced toxicity control compared to coconut water-treated groups
(Table 3). Intake of coconut water decreased the values of albu-
min, AST and ALT compared to the negative control. However, total protein and bilirubin levels had a dose-
dependent significant difference (p < 0.05) between the
coconut water-pretreated groups (4 and 6 mL/kg) and
the negative (CCl4) control. ALP values did not show any
significant difference in the male rats. In the female rats,
there were significant differences (p < 0.05) in the val-
ues of albumin, AST, ALT, ALP and bilirubin among the
coconut water-pretreated groups compared to the CCl4
group (Table 1). Protective effect of coconut water on kidney biomarkers
of CCl4‑induced toxicity The result presented in Table 5 is the protective effect of
coconut water on antioxidant enzymes of CCl4-induced
toxicity in both male and female rats. Administration of
coconut water caused a significant elevation in the values
of GSH, SOD and CAT. The values of MDA decreased in
the treatment group compared to its untreated counter-
parts in both male and female rats. Table 2 shows the data set obtained from the protec-
tive effect of coconut water on kidney function of
CCl4-induced toxicity in both sexes. There were sig-
nificant differences observed in the values of urea,
creatinine, Na+, K+ and HCO3
− in the coconut water-
pretreated groups compared to the CCl4-treated group. Antioxidant markers Other researchers’ methodologies were used to deter-
mine oxidative stress indicators. Techniques for meas-
uring superoxide dismutase (SOD) were used as
reported by Sun and Zigma (1978). Catalase was esti-
mated as revealed by Aebi (1984). Malondialdehyde and Figure 1 represents the protective effect of coconut water
on body weight of CCl4-induced toxicity in both male
and female rats. No significant changes (p > 0.05) in body
weight were observed in both sexes in all the treatment
groups. Fig. 1 Body weight of 14 days treatment of coconut water on CCl4-induced toxicity in male (A) and female (B) rats. Values represent the mean ± SD
for n = 6 Page 4 of 11 Emmanuel et al. Bulletin of the National Research Centre (2022) 46:206 AST, aspartate aminotransferase; ALT, alanine transaminase; and ALP, alkaline phosphatase. Values are presented as mean ± SD, n = 6. Values in the row bearing the
same letters of the alphabet are not statistically significant (p > 0.05) from one another. The data were analysed with one-way ANOVA and expressed as mean ± SD,
then compared to controls, n = 6. Different letters (a, b or c) p < 0.05 both male and female rats show significant difference between the tested groups (2, 4 and 6 mL/
kg) compared to the negative control e; ALT, alanine transaminase; and ALP, alkaline phosphatase. Values are presented as mean ± SD, n = 6. Values in the row bearing the
e not statistically significant (p > 0.05) from one another. The data were analysed with one-way ANOVA and expressed as mean ± SD, e; ALT, alanine transaminase; and ALP, alkaline phosphatase. Values are presented as mean ± SD, n = 6. Values in the row bearing the
t t ti ti
ll
i
ifi
t (
0 05) f
th
Th d t
l
d
ith
ANOVA
d
d
±SD et are not statistically significant (p > 0.05) from one another. The data were analysed with one way ANOVA and expressed as mean ± SD,
, n = 6. Different letters (a, b or c) p < 0.05 both male and female rats show significant difference between the tested groups (2, 4 and 6 mL
ive control Protective effect of coconut water on histology section
of liver and kidneyh Different letters (a, b or c) p < 0.05 both male and female rats show significant difference between the tested groups (2, 4 and 6 mL/
kg) compared to the negative control
Parameters
Control
CCl4 only
Coconut water (mL/kg)
2 mL/kg
4 mL/kg
6 mL/kg
A
Total protein (g/dl)
7.20 ± 0.07a
5.65 ± 0.11b
5.79 ± 0.09b
6.03 ± 0.25c
6.12 ± 0.15a
Albumin (g/dl)
4.22 ± 0.04a
3.37 ± 0.20b
3.36 ± 0.43b
3.40 ± 0.37b
3.34 ± 0.56b
AST (U/L)
18.67 ± 3.06a
80.33 ± 2.52b
74.67 ± 4.16c
71.67 ± 0.58c
70.67 ± 3.06c
ALT (U/L)
10.33 ± 1.53a
74.67 ± 4.16b
64.67 ± 0.58c
63.00 ± 1.00c
63.00 ± 2.65c
ALP (U/L)
58.70 ± 3.87a
71.67 ± 0.58b
72.83 ± 1.19b
72.97 ± 0.50b
71.97 ± 1.39b
Bilirubin (g/dl)
0.50 ± 0.08a
1.65 ± 0.10b
1.41 ± 0.09c
1.31 ± 0.09c
1.27 ± 0.04c
B
Total protein (g/dl)
6.56 ± 0.40a
5.16 ± 0.22b
5.83 ± 0.04c
5.46 ± 0.66d
5.57 ± 0.33c
Albumin (g/dl)
4.13 ± 0.29b
2.91 ± 0.25a
3.09 ± 0.06c
2.97 ± 0.13a
3.05 ± 0.07c
AST (U/L)
22.33 ± 2.52b
87.67 ± 1.53a
80.00 ± 2.00c
79.67 ± 4.73c
70.00 ± 2.00d
ALT (U/L)
12.00 ± 2.00a
78.67 ± 3.51b
65.67 ± 6.03c
61.33 ± 1.15c
60.00 ± 2.00c
ALP (U/L)
58.57 ± 4.30a
81.37 ± 3.81b
71.33 ± 1.12c
72.23 ± 1.72c
72.10 ± 2.33c
Bilirubin (g/dl)
0.45 ± 0.06a
1.60 ± 0.25b
1.06 ± 0.23c
1.07 ± 0.29c
0.98 ± 0.10c Table 1 Protective effect of coconut water on liver biomarkers of carbon tetrachloride-induced toxicity in ma Emmanuel et al. Bulletin of the National Research Centre (2022) 46:206 Page 5 of 11 Table 2 Protective effect of coconut water on kidney biomarkers of carbon tetrachloride-induced toxicity in male (A) and female (B)
rats
Na + , sodium ion; Cl−, chloride ion; K + , potassium ion; and HCO3
−, bicarbonate. Values in the row bearing the same letters of the alphabet are not statistically
significant (p > 0.05) from one another. The data were analysed with one-way ANOVA and expressed as mean ± SD, then compared to controls, n = 6. Protective effect of coconut water on histology section
of liver and kidneyh Different letters
(a, b or c) p < 0.05 both male and female rats show significant difference between the tested groups (2, 4 and 6 mL/kg) compared to the negative control levels (Emmanuel et al. 2021). In the present study, the
negative control had elevated levels of liver enzymes and
administration of coconut water decreased the values
of albumin, AST and ALT. Our findings correlate with
the results of other researchers (Anurag and Rajamo-
han 2003). Anurag and Rajamohan (2003) showed that
coconut water counteracted an increased level of liver
enzymes caused by isoproterenol exposure. The reported
hepatoprotective activities of coconut water might be
linked to its antioxidant activities (Tsai et al. 2009). levels (Emmanuel et al. 2021). In the present study, the
negative control had elevated levels of liver enzymes and
administration of coconut water decreased the values
of albumin, AST and ALT. Our findings correlate with
the results of other researchers (Anurag and Rajamo-
han 2003). Anurag and Rajamohan (2003) showed that
coconut water counteracted an increased level of liver
enzymes caused by isoproterenol exposure. The reported
hepatoprotective activities of coconut water might be
linked to its antioxidant activities (Tsai et al. 2009). enlarged central vein compared to the negative control. More so, the hepatocytes appeared a bit distorted and
many microcysts within the stroma were found (A-J). In
the kidney histology (K-T), no pathological change was
observed in the normal control unlike the CCl4-treated
groups. While the negative control had a slight dilated
and shrunken glomerulus, a slight enlargement of the
tubules was observed in the coconut water-treated group,
although there were no pathological changes observed
amongst the groups (Figs. 4, 5). A functional renal system may be related to the kid-
ney’s filtration capacity, commonly known as the glo-
merular filtration rate (GFR). A reduction in GFR may
produce an increase in urea and creatinine in the serum,
indicating renal disease (Jose 2014). Additionally, in kid-
ney test, a high serum urea level indicates renal failure,
which usually progresses to severe kidney damage in
long-term instances (Gowda et al. 2010). In our study,
urea, creatinine and HCO3
− significantly decreased in
the coconut water-treated groups when compared to the
negative control. Abnormal elevations of creatinine, urea
and electrolytes levels are implicated in kidney insults
(Ugbogu et al. 2019; Emmanuel et al. 2021). Protective effect of coconut water on histology section
of liver and kidneyh Different letters
(a, b or c) p < 0.05 both male and female rats show significant difference between the tested groups (2, 4 and 6 mL/kg) compared to the negative control
Parameters
Control
CCl4 only
Coconut water (mL/kg)
2 mL/kg
4 mL/kg
6 mL/kg
A
Urea (mg/dl)
25.47 ± 1.36a
27.13 ± 0.85b
23.10 ± 0.62a
24.77 ± 0.91a
22.50 ± 1.48c
Creatinine (mg/dl)
0.58 ± 0.05a
0.68 ± 0.02b
0.54 ± 0.03a
0.51 ± 0.02a
0.53 ± 0.06a
Na + (mEq/L)
130.00 ± 1.48a
120.57 ± 0.87b
133.43 ± 1.56a
134.67 ± 5.31a
147.83 ± 0.99c
Cl- (mEq/L)
89.45 ± 1.28a
85.67 ± 0.75a
87.66 ± 0.39a
88.80 ± 0.70a
96.54 ± 4.71b
K+ (mEq/L)
4.82 ± 0.08b
4.12 ± 0.18a
4.50 ± 0.22c
4.61 ± 0.13c
5.27 ± 0.40a
HCO3
− (mEq/L)
23.53 ± 0.67b
29.50 ± 1.01a
25.83 ± 0.85b
26.13 ± 0.64c
25.23 ± 0.40b
B
Urea (mg/dl)
25.24 ± 0.83a
23.77 ± 1.26b
23.70 ± 0.28b
24.78 ± 0.35a
25.42 ± 0.30a
Creatinine(mg/dl)
0.47 ± 0.03a
0.66 ± 0.04b
0.58 ± 0.03c
0.61 ± 0.02c
0.60 ± 0.05c
Na+ (mEq/L)
126.87 ± 1.68a
118.10 ± 2.41b
139.37 ± 0.75c
144.27 ± 1.97c
146.30 ± 1.05c
Cl− (mEq/L)
94.90 ± 0.46a
92.20 ± 1.71a
97.30 ± 0.90b
97.77 ± 1.21b
101.43 ± 1.86c
K+ (mEq/L)
4.43 ± 0.32a
4.10 ± 0.10b
4.80 ± 0.10c
5.27 ± 0.08d
5.15 ± 0.15d
HCO3
− (mEq/L)
24.50 ± 0.26a
28.80 ± 0.70b
25.63 ± 1.40a
24.40 ± 1.31a
23.70 ± 1.10a Table 2 Protective effect of coconut water on kidney biomarkers of carbon tetrachloride-induced toxicity in male (A) and female (B)
rats Table 2 Protective effect of coconut water on kidney biomarkers of carbon tetrachloride-induced toxicity in male (A) and female (B)
rats t of coconut water on kidney biomarkers of carbon tetrachloride-induced toxicity in male (A) and female (B) t of coconut water on kidney biomarkers of carbon tetrachloride-induced toxicity in male (A) and female (B) Na + , sodium ion; Cl−, chloride ion; K + , potassium ion; and HCO3
−, bicarbonate. Values in the row bearing the same letters of the alphabet are not statistically
significant (p > 0.05) from one another. The data were analysed with one-way ANOVA and expressed as mean ± SD, then compared to controls, n = 6. Discussion In this study, we examined the protective effect of coco-
nut water against CCl4-induced toxicity in rats. The find-
ings of the study revealed no significant changes in body
weight in the experimental rats. This might be attribut-
able to the brief period of CCl4 delivery following coco-
nut water pretreatment. In other reports, body weight
measurement has been described as plausible evidence in
toxicological research (Emmanuel et al. 2021). g
Elevations of liver biomarkers; enzymes (AST, ALT and
ALP), bilirubin, albumin and total proteins are strong
indicators for liver insults (Orieke et al. 2019; Ugbogu
et al. 2019; Emmanuel et al. 2021). When the liver is
under severe attack, these enzymes are released into the
blood and thus give insight on the hepatic status of the
liver (Ugbogu et al. 2019). Dutta et al. (2018) reported
that CCl4 is hepatotoxic by producing ROS. Increased
ROS has been associated with decreased antioxidant Haematological parameters are those that relate to
blood and blood-forming organs. The quantity and
shape of RBC, WBC and platelets are important in dis-
ease diagnosis and monitoring (Owoeye et al. 2011). In
this work, groups pretreated with coconut water prior to
CCl4 induction had a significant haematoprotective effect Emmanuel et al. Bulletin of the National Research Centre (2022) 46:206 Page 6 of 11 Table 3 Protective effect of coconut water on haematological parameters of carbon tetrachloride-induced toxicity in male (A) and
female (B) rats
RBC, Red blood cells; PCV, packed cell volume; HB, haemoglobin; MCV, mean corpuscular volume; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular
haemoglobin concentration; and WBC, white blood cell. Values in the row bearing the same letters of the alphabet are not statistically significant (p > 0.05) from one
another. The data were analysed with one-way ANOVA and expressed as mean ± SD, then compared to controls, n = 6. Discussion Different letters (a, b or c) p < 0.05 both male
and female rats show significant difference between the tested groups (2, 4 and 6 mL/kg) compared to the negative control
Parameters
Control
CCl4 only
Coconut water (mL/kg)
2 mL/kg
4 mL/kg
6 mL/kg
A
RBC (× 1012/L)
5.13 ± 0.15a
4.47 ± 0.15a
5.00 ± 0.10a
5.30 ± 0.10a
6.07 ± 0.15a
PCV (%)
46.67 ± 1.53a
40.00 ± 1.00b
47.67 ± 3.21a
50.00 ± 1.00c
52.67 ± 1.53c
HB (g/dL)
14.43 ± 0.38a
13.27 ± 0.25b
14.60 ± 0.53a
14.97 ± 0.25a
15.07 ± 0.15a
WBC (× 109/L)
7.23 ± 0.35a
10.13 ± 0.35b
6.93 ± 0.31a
8.43 ± 0.38a
7.50 ± 0.30a
Platelet (× 109/L)
29.00 ± 2.65a
17.67 ± 2.52b
31.67 ± 1.53a
29.33 ± 4.16a
26.33 ± 4.04a
MCV (fl)
90.93 ± 1.14a
89.57 ± 0.99a
95.30 ± 5.15a
94.30 ± 0.10a
87.37 ± 3.16a
MCH (pg)
28.10 ± 0.46a
29.70 ± 0.46a
29.20 ± 0.72a
28.23 ± 0.12a
24.87 ± 0.90b
MCHC (g/L)
309.33 ± 2.64a
331.70 ± 2.12b
306.73 ± 9.98a
299.33 ± 1.15a
286.57 ± 6.85a
Neutrophil (%)
60.67 ± 2.31a
41.33 ± 3.06b
48.33 ± 1.53c
50.00 ± 1.00c
52.00 ± 1.00c
Lymphocyte (%)
32.00 ± 2.00a
51.33 ± 3.21b
45.33 ± 2.52c
42.67 ± 1.53c
41.00 ± 1.00c
Monocyte (%)
4.67 ± 0.58a
3.67 ± 0.58a
3.33 ± 0.58a
4.00 ± 0.00a
3.33 ± 0.58a
Eosinophil (%)
2.67 ± 0.58a
3.33 ± 0.58a
2.67 ± 0.58a
2.67 ± 1.15a
2.67 ± 0.58a
Basophil (%)
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
1.00 ± 0.00
B
RBC (× 1012/L)
6.80 ± 0.50a
5.90 ± 0.40a
6.10 ± 0.60a
6.90 ± 0.06a
7.00 ± 0.90a
PCV (%)
48.30 ± 2.50a
43.60 ± 2.00b
48.30 ± 3.50a
52.00 ± 3.60c
57.70 ± 3.00c
HB (g/dL)
15.80 ± 0.81a
14.80 ± 0.20b
15.70 ± 0.50a
16.00 ± 0.60a
16.10 ± 0.90a
WBC (× 109/L)
9.90 ± 0.82a
11.67 ± 0.78b
8.90 ± 0.42a
10.00 ± 0.67a
9.10 ± 0.20a
Platelet (× 109/L)
51.30 ± 2.50a
44.60 ± 3.70b
37.80 ± 3.60c
45.00 ± 2.40b
37.30 ± 1.58c
MCV (fl)
90.30 ± 3.70a
88.70 ± 3.10a
93.20 ± 2.80a
86.80 ± 2.40a
93.40 ± 4.70a
MCH (pg)
29.10 ± 1.60a
29.30 ± 0.81a
29.37 ± 0.80a
26.20 ± 1.30b
26.70 ± 1.25b
MCHC (g/L)
313.40 ± 3.50a
325.50 ± 9.90b
307.70 ± 12.40a
295.50 ± 8.80c
271.20 ± 7.10d
Neutrophil (%)
65.60 ± 3.00a
36.60 ± 2.00b
44.30 ± 3.20c
45.60 ± 1.50c
47.00 ± 3.60c
Lymphocyte (%)
27.60 ± 1.50a
56.60 ± 2.15b
49.30 ± 2.80c
48.30 ± 2.80c
45.60 ± 2.90d
Monocyte (%)
4.60 ± 0.58a
4.30 ± 0.58a
4.30 ± 0.58a
4.30 ± 0.58a
4.00 ± 0.60a
Eosinophil (%)
4.33 ± 0.58a
3.33 ± 0.58b
3.67 ± 0.58b
3.67 ± 0.58b
3.33 ± 1.15b
Basophil (%)
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00 Table 3 Protective effect of coconut water on haematological parameters of carbon tetrachloride-induced toxicity in male (A) and
female (B) rats
Parameters
Control
CCl4 only
Coconut water (mL/kg) Table 3 Protective effect of coconut water on haematological parameters of carbon tetrachloride-induced toxicity in male (A) and
female (B) rats RBC, Red blood cells; PCV, packed cell volume; HB, haemoglobin; MCV, mean corpuscular volume; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular
haemoglobin concentration; and WBC, white blood cell. Discussion Different letters (a, b or c) p < 0.05 both male and female rats show significant difference between the
tested groups (2, 4 and 6 mL/kg) compared to the negative control
Parameters
Control
CCl4 only
Coconut water (mL/kg)
2 mL/kg
4 mL/kg
6 mL/kg
A
TC (mg/dL)
133.57 ± 4.04a
152.57 ± 2.65b
144.20 ± 2.13c
142.83 ± 1.96c
135.47 ± 2.12a
HDL-C (mg/dL)
76.83 ± 1.21a
65.43 ± 1.70b
68.10 ± 3.35b
72.47 ± 2.60c
73.80 ± 3.35c
Triglycerides (mg/dL)
155.00 ± 6.85a
179.47 ± 3.05b
170.50 ± 1.08c
172.43 ± 2.90c
167.97 ± 2.62c
LDL-C (mg/dL)
25.73 ± 3.41a
45.24 ± 35.89b
42.19 ± 5.13b
36.55 ± 3.58c
28.07 ± 5.36a
VLDL-C (mg/dL)
31.00 ± 1.37a
35.89 ± 0.61a
34.10 ± 0.22a
33.82 ± 0.70a
33.59 ± 0.52a
B
TC (mg/dL)
142.20 ± 4.40a
149.80 ± 5.70b
144.20 ± 2.80a
144.20 ± 2.20a
142.30 ± 3.40a
HDL-C (mg/dL)
77.20 ± 2.10a
67.80 ± 1.80b
70.90 ± 2.20c
72.90 ± 2.00c
73.70 ± 2.90c
Triglycerides (mg/dL)
171.70 ± 4.50a
186.80 ± 4.10b
178.00 ± 2.50c
178.20 ± 2.20c
178.70 ± 2.30c
LDL-C (mg/dL)
30.70 ± 3.20a
45.30 ± 3.80b
37.70 ± 2.80c
35.50 ± 1.60c
32.90 ± 1.90a
VLDL-C (mg/dL)
34.30 ± 1.80a
37.30 ± 1.84b
35.60 ± 1.50c
35.60 ± 1.40c
35.70 ± 1.90c Table 4 Protective effect of coconut water on lipid profiles of carbon tetrachloride-induced toxicity in male (A) and female (B) rats
Parameters
Control
CCl4 only
Coconut water (mL/kg) Table 4 Protective effect of coconut water on lipid profiles of carbon tetrachloride-induced toxicity in male (A) and female (B) rats TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; and VLDL-C, very low-density lipoprotein cholesterol. Values in the row bearing the same letters of the alphabet are not statistically significant (p > 0.05) from one another. The data were analysed with one-way ANOVA
and expressed as mean ± SD, then compared to controls, n = 6. Different letters (a, b or c) p < 0.05 both male and female rats show significant difference between the
tested groups (2, 4 and 6 mL/kg) compared to the negative control Table 5 Protective effect of coconut water on antioxidant enzymes of carbon tetrachloride-induced toxicity in male (A) and female (B)
rats
GSH, glutathione; SOD, superoxide dismutase; CAT, catalase; and MDA, malondialdehyde. Discussion Values in the row bearing the same letters of the alphabet are not statistically significant (p > 0.05) from one
another. The data were analysed with one-way ANOVA and expressed as mean ± SD, then compared to controls, n = 6. Different letters (a, b or c) p < 0.05 both male
and female rats show significant difference between the tested groups (2, 4 and 6 mL/kg) compared to the negative control this present study (Saba et al. 2010; Emmanuel et al. 2021).h in both male and female rats compared to the negative
control. In the negative control, the levels of RBC, PCV
and HB were decreased, and this may be associated with
the toxic free radicals generated from CCl4 which conse-
quently affected haematopoiesis in the experimental ani-
mals as revealed in other studies (Rahmouni et al. 2011). Therefore, haematological parameters are very sensi-
tive and are crucial for predicting the toxicological and
pathological effect of potentially toxic substances in both
human and animals (Kong et al. 2016). Previous study on
the effects of CCl4 on haematological parameters showed
that acute CCl4 toxicity led to transient decrease in the
HB concentration and reticulocyte count as well as PCV
and RBC counts by extension which is in consonant with The rats administered with coconut water prior CCl4
induction had decreased levels TC, triglycerides, LDL
and VLDL, while HDL levels increased compared to
the untreated group. This demonstrates that CCl4 could
disrupt lipid metabolism. Atherosclerosis and coronary
heart disease have been linked to higher levels of cho-
lesterol, triglycerides, LDL and VLDL (Sandhya and
Rajamohan 2008; Emmanuel et al. 2021). Our findings
confirmed the previously reported evidence that coconut
water lowered the increased levels of TC, triglycerides,
LDL and VLDL in rats induced by a high cholesterol fat
feed (Sandhya and Rajamohan 2006). Emmanuel et al. Bulletin of the National Research Centre (2022) 46:206 Page 7 of 11 Page 7 of 11 Table 4 Protective effect of coconut water on lipid profiles of carbon tetrachloride-induced toxicity in male (A) and female (B) rats
TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; and VLDL-C, very low-density lipoprotein cholesterol. Values in the row bearing the same letters of the alphabet are not statistically significant (p > 0.05) from one another. The data were analysed with one-way ANOVA
and expressed as mean ± SD, then compared to controls, n = 6. Discussion Values in the row bearing the same letters of the alphabet are not
statistically significant (p > 0.05) from one another. The data were analysed with one-way ANOVA and expressed as mean ± SD, then compared to controls, n = 6. Different letters (a, b or c) p < 0.05 both male and female rats show significant difference between the tested groups (2, 4 and 6 mL/kg) compared to the negative
control
Parameters
Normal control
CCl4 only
Coconut water (mL/kg)
2 mL/kg
4 mL/kg
6 mL/kg
A
GSH (U/L)
49.53 ± 1.81a
46.98 ± 2.67b
48.13 ± 3.06a
49.35 ± 1.16a
49.67 ± 2.35a
SOD (U/L)
21.97 ± 2.48a
17.94 ± 1.52b
18.52 ± 1.03b
21.82 ± 0.65a
22.44 ± 0.81a
CAT (U/L)
12.52 ± 2.12a
10.35 ± 0.75b
10.89 ± 0.79b
12.63 ± 0.77a
11.62 ± 0.85a
MDA (U/L)
0.51 ± 0.06a
0.99 ± 0.10b
0.98 ± 0.13b
0.81 ± 0.06b
0.81 ± 0.05b
B
GSH (U/L)
48.55 ± 4.03a
45.25 ± 2.71b
49.49 ± 3.47a
51.36 ± 0.88a
52.49 ± 1.71a
SOD (U/L)
22.85 ± 1.60a
19.81 ± 1.55b
22.37 ± 0.53a
22.57 ± 1.31a
24.26 ± 0.81c
CAT (U/L)
12.31 ± 1.11a
10.56 ± 0.60b
11.05 ± 1.31a
12.17 ± 0.19a
11.89 ± 1.00a
MDA (U/L)
0.66 ± 0.05a
1.09 ± 0.11b
0.88 ± 0.04c
0.87 ± 0.06c
0.77 ± 0.03c GSH, glutathione; SOD, superoxide dismutase; CAT, catalase; and MDA, malondialdehyde. Values in the row bearing the same letters of the alphabet are not
statistically significant (p > 0.05) from one another. The data were analysed with one-way ANOVA and expressed as mean ± SD, then compared to controls, n = 6. Different letters (a, b or c) p < 0.05 both male and female rats show significant difference between the tested groups (2, 4 and 6 mL/kg) compared to the negative
control CCl4 has been found to be toxic by causing the pro-
duction of free radicals capable of interacting with
sulphur-containing proteins, which can impair antioxi-
dant activity (Tsai et al. 2009). This study revealed that
CCl4 has a negative effect on antioxidants, as seen by
a decrease in SOD, GSH and CAT and an increase in
MDA. Our findings are consistent with the findings of
Rahmouni et al. (2011) that discovered that CCl4 may
reduce erythrocyte GSH, SOD and CAT activities while
increasing erythrocyte MDA levels. Values in the row bearing the same letters of the alphabet are not statistically significant (p > 0.05) from one another. The data were analysed with one-way ANOVA
and expressed as mean ± SD, then compared to controls, n = 6. Different letters (a, b or c) p < 0.05 both male and female rats show significant difference between the
tested groups (2, 4 and 6 mL/kg) compared to the negative control Discussion In the histology of the liver and kidney, pathological
changes were evident; however, there were slight enlarge-
ment of the central veins and tubules, respectively,
compared to the negative control. Coconut water admin-
istration revealed a dose-dependent protection compared
to the untreated group. Injuries in the liver included
deformed hepatocytes, dilated sinusoids, microvascular
steatosis and an enlarged central vein, while congested
blood vessels and dilated tubules were detected in the
kidney of the negative control. These findings support the Emmanuel et al. Bulletin of the National Research Centre (2022) 46:206 Page 8 of 11 Fig. 2 Protective effect of coconut water on histology section of liver against CCl4-induced toxicity in male rats (A–E). A Control, B CCl4only, C
2 mL/kg, D 4 mL/kg, E 6 mL/kg. V: central vein, S: sinusoids, NS: unmarkable sinusoids, M: multicystic space. The liver sections were stained with
haematoxylin and eosin staining (H&E) solution and observed under an Olympus light microscope fixed with camera, at a magnification (40×) ogy section of liver against CCl induced toxicity in male rats (A E) A Control B CCl only C Fig. 2 Protective effect of coconut water on histology section of liver against CCl4-induced toxicity in male rats (A–E). A Control, B CCl4only, C
2 mL/kg, D 4 mL/kg, E 6 mL/kg. V: central vein, S: sinusoids, NS: unmarkable sinusoids, M: multicystic space. The liver sections were stained with
haematoxylin and eosin staining (H&E) solution and observed under an Olympus light microscope fixed with camera, at a magnification (40×) Fig. 3 Protective effect of coconut water on histology section of liver against CCl4-induced toxicity in female rats (A–E). A Control, B CCl4only, C
2 mL/kg, D 4 mL/kg, E 6 mL/kg. V: central vein, S: sinusoids, NS: unmarkable sinusoids, M: multicystic space, D: dilated cystic space, H: distorted
hepatocytes. The liver sections were stained with haematoxylin and eosin staining (H&E) solution and observed under an Olympus light microscope
fixed with camera, at a magnification (40×) ogy section of liver against CCl -induced toxicity in female rats (A–E) A Control B CCl only C Fig. 3 Protective effect of coconut water on histology section of liver against CCl4-induced toxicity in female rats (A–E). A Control, B CCl4only, C
2 mL/kg, D 4 mL/kg, E 6 mL/kg. V: central vein, S: sinusoids, NS: unmarkable sinusoids, M: multicystic space, D: dilated cystic space, H: distorted
hepatocytes. Discussion The liver sections were stained with haematoxylin and eosin staining (H&E) solution and observed under an Olympus light microscope
fixed with camera, at a magnification (40×) CAT levels and decreased MDA levels. These data
show that coconut water may protect against lipid
peroxidation by scavenging the harmful radicals pro-
duced by CCl4 and thereby normalizing the quantity
and distribution of lipids in rats’ systemic circulation. The observed increase in these antioxidants may be
related to coconut water’s hepato/reno-protective
potentials and reduced cellular oxidative damage. assertion that CCl4 has haematotoxic, hepatotoxic and
nephrotoxic potentials (Rahmouni et al. 2011; Elshater
et al. 2013; Emmanuel et al. 2021). Conclusionsi Our findings show that coconut water protects rats
from CCl4-induced toxicity. Its pretreatment for
14 days following CCl4 toxicity elevated GSH, SOD, Emmanuel et al. Bulletin of the National Research Centre (2022) 46:206 Page 9 of 11 Notwithstanding the health benefits of coconut water,
it can be noted that its contents may be influenced and
varied by the type of cultivar, geographical location and
the stage of maturation of the nut. Abbreviations
ALT: Alanine transaminase; AST: Aspartate transaminase; ALP: Alanine phos‑
phatase; CAT: Catalase; CCl4: Carbon tetrachloride; Cl−: Chloride ion; CVD:
Cardiovascular disease; EDTA: Ethylenediaminetetraacetic acid; HB: Haemo‑
globin; HCO3
−: Bicarbonate; HDL: High-density lipoprotein; GSH: Glutathione;
K+: Potassium ion; LDL: Low-density lipoprotein; MCV: Mean corpuscular
volume; MCH: Mean corpuscular haemoglobin; MCHC: Mean corpuscular
Fig. 4 Protective effect of coconut water on histology section of kidney against CCl4-induced toxicity in male rats (A–E). A Control, B CCl4only, C
2 mL/kg, D 4 mL/kg, E 6 mL/kg. G: glomeruli, T: tubules, D: glomerular degeneration, GS: shrunken glomerula, BCS: Bowman’s space, B: Bowman’s
capsule, D: dilated tubules, GN: network of the glomerulus. The kidney sections were stained with haematoxylin and eosin staining (H&E) solution
and observed under an Olympus light microscope fixed with camera, at a magnification (40×)
Fig. 5 Protective effect of coconut water on histology section of kidney against CCl4-induced toxicity in female rats (A–E). A Control, B CCl4only,
C 2 mL/kg, D 4 mL/kg, E 6 mL/kg. G: glomeruli, T: tubules, D: glomerular degeneration, GS: shrunken glomerula, BCS: Bowman’s space, B: Bowman’s
capsule, D: dilated tubules, GN: network of the glomerulus. The kidney sections were stained with haematoxylin and eosin staining (H&E) solution
and observed under an Olympus light microscope fixed with camera, at a magnification (40×) Fig. 4 Protective effect of coconut water on histology section of kidney against CCl4-induced toxicity in male rats (A–E). A Control, B CCl4only, C
2 mL/kg, D 4 mL/kg, E 6 mL/kg. G: glomeruli, T: tubules, D: glomerular degeneration, GS: shrunken glomerula, BCS: Bowman’s space, B: Bowman’s
capsule, D: dilated tubules, GN: network of the glomerulus. The kidney sections were stained with haematoxylin and eosin staining (H&E) solution
and observed under an Olympus light microscope fixed with camera, at a magnification (40×) Fig. 4 Protective effect of coconut water on histology section of kidney against CCl4-induced toxicity in male rats (A–E). Conclusionsi A Control, B CCl4only, C
2 mL/kg, D 4 mL/kg, E 6 mL/kg. G: glomeruli, T: tubules, D: glomerular degeneration, GS: shrunken glomerula, BCS: Bowman’s space, B: Bowman’s
capsule, D: dilated tubules, GN: network of the glomerulus. The kidney sections were stained with haematoxylin and eosin staining (H&E) solution
and observed under an Olympus light microscope fixed with camera, at a magnification (40×) Fig. 5 Protective effect of coconut water on histology section of kidney against CCl4-induced toxicity in female rats (A–E). A Control, B CCl4only,
C 2 mL/kg, D 4 mL/kg, E 6 mL/kg. G: glomeruli, T: tubules, D: glomerular degeneration, GS: shrunken glomerula, BCS: Bowman’s space, B: Bowman’s
capsule, D: dilated tubules, GN: network of the glomerulus. The kidney sections were stained with haematoxylin and eosin staining (H&E) solution
and observed under an Olympus light microscope fixed with camera, at a magnification (40×) Fig. 5 Protective effect of coconut water on histology section of kidney against CCl4-induced toxicity in female rats (A–E). A Control, B CCl4only,
C 2 mL/kg, D 4 mL/kg, E 6 mL/kg. G: glomeruli, T: tubules, D: glomerular degeneration, GS: shrunken glomerula, BCS: Bowman’s space, B: Bowman’s
capsule, D: dilated tubules, GN: network of the glomerulus. The kidney sections were stained with haematoxylin and eosin staining (H&E) solution
and observed under an Olympus light microscope fixed with camera, at a magnification (40×) Notwithstanding the health benefits of coconut water,
it can be noted that its contents may be influenced and
varied by the type of cultivar, geographical location and
the stage of maturation of the nut. Consent for publication
Not applicable. Li JX, Pang YZ, Tang CS, Li ZQ (2004) Protective effect of taurine on hypochlor‑
ous acid toxicity to nuclear nucleoside triphosphatase in isolated nuclei
from rat liver. World J Gastroenterol 10:694–698 Received: 23 December 2021 Accepted: 29 June 2022 Mandal SM, Dey S, Mandal M, Sarkar S, Maria-Neto S, Franco OL (2009) Iden‑
tification and structural insights of three novel antimicrobial peptides
isolated from green coconut water. Peptides 30:633–637 Mohamad NE, Yeap SK, Abu N, Lim KL, Zamberi NR, Nordin N, Sharifuddin SA,
Alitheen LK, NB, (2019) In vitro and in vivo antitumour effects of coconut
water vinegar on 4T1 breast cancer cells. Food Nutr Res 10:63 Funding This study was sponsored by Tertiary Education Trust Fund Institutional Base
Research Grant from Abia State University, Uturu, Nigeria. Gordon A, Jackson J (2017) Case study: application of appropriate technolo‑
gies to improve the quality and safety of coconut water. Food Safety Qual
Syst Dev Countries. https://doi.org/10.1016/B978-0-12-801226-0.00007-4 Availability of data and materials All the data used for this manuscript are available on request from the cor‑
responding author. Gowda S, Desai PB, Kulkarni SS, Hull VV, Math AAK, Vernekar SN (2010) Markers
of renal function tests. North Am J Med Sci 2:170–173 Intahphuak S, Khonsung P, Panthong A (2010) Anti-inflammatory, analge‑
sic, and antipyretic activities of virgin coconut oil. Pharmaceut Biol
48(2):151–157 The authors declare none whatsoever. The authors declare none whatsoever. Liu Q, Kong B, Li G, Liu N, Xia X (2011) Hepatoprotective and antioxidant
effects of porcine plasma protein hydrolysates on carbon tetrachloride-
induced liver damage in rats. Food Chem Toxicol 49(6):1316–1321 Acknowledgements
Not applicable. Elshater AEA, Salman MMA, Mohamed SA (2013) The hepato-ameliorating
effect of Solanum nigrum against CCl4 induced liver toxicity in albino rats. Egypt Acad J Biol Sci 5(1):59–66 Author contributions Emmanuel O, Okezie UM, Iweala EJ, Ugbogu EA (2021) Pretreatment of red
palm oil extracted from palm fruit (Elaeis guineensis) attenuates carbon
tetrachloride induced toxicity in Wistar rats. Phytomed plus 1:100079. https://doi.org/10.1016/j.phyplu.2021.100079 OE, IE and EAU conceptualized the work, sourced the literature and wrote the
original draft. CP, VCU, OGE, SNI, VOA and CU wrote the original draft and read
the manuscript. All authors have read and approved the manuscript. Ghosh PK, Bhattacharjee P, Mitra S, Poddar-Sarkar M (2014) Physicochemical
and phytochemical analyses of copra and oil of Cocos nucifera L. (West
Coast Tall Variety). Inter J Food Sci. https://doi.org/10.1155/2014/310852 Abbreviations ALT: Alanine transaminase; AST: Aspartate transaminase; ALP: Alanine phos‑
phatase; CAT: Catalase; CCl4: Carbon tetrachloride; Cl−: Chloride ion; CVD:
Cardiovascular disease; EDTA: Ethylenediaminetetraacetic acid; HB: Haemo‑
globin; HCO3
−: Bicarbonate; HDL: High-density lipoprotein; GSH: Glutathione;
K+: Potassium ion; LDL: Low-density lipoprotein; MCV: Mean corpuscular
volume; MCH: Mean corpuscular haemoglobin; MCHC: Mean corpuscular Page 10 of 11 Emmanuel et al. Bulletin of the National Research Centre (2022) 46:206 Emmanuel et al. Bulletin of the National Research Centre (2022) 46:206 by antioxidant rich leaf extract of Croton bonplandianus Baill. PLoS ONE
13(4):e0196411 haemoglobin concentration; MDA: Malondialdehyde; Na + : Sodium ion; PCV:
Packed cell volume; RBC: Red blood cell; ROS: Reactive oxygen species; SOD:
Superoxide dismutase; TC: Total cholesterol; VLDL: Very low-density lipopro‑
tein; WBC: White blood cell. haemoglobin concentration; MDA: Malondialdehyde; Na + : Sodium ion; PCV:
Packed cell volume; RBC: Red blood cell; ROS: Reactive oxygen species; SOD:
Superoxide dismutase; TC: Total cholesterol; VLDL: Very low-density lipopro‑
tein; WBC: White blood cell. Elekwa I, Ude VC, Emmanuel O, Amachaghi VO, Ugbogu EA (2021) In vivo
studies on the ameliorative effect of coconut water against carbon
tetrachloride induced toxicity in rats. Biomarkers. https://doi.org/10.1080/
1354750X.2021.1946848 Author details
1 1 Department of Biochemistry, Abia State University, PMB 2000, Uturu, Abia
State, Nigeria. 2 Department of Medical Biochemistry, College of Medicine,
Enugu State University of Science and Technology, PMB 01660, Enugu, Nigeria. 3 Department of Zoology and Environmental Biology, Michael Okpara Univer‑
sity of Agriculture, Umudike, Abia State, Nigeria. 4 Department of Public Health,
Abia State University, PMB 2000, Uturu, Abia State, Nigeria. Loki AL, Rajamohan T (2003) Hepatoprotective and antioxidant effect of ten‑
der coconut water on CCl4 induced liver injury in rats. Indian J Biochem
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J (2016) Phenolic compounds, antioxidant activity, and medium chain
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S Springer Nature remains neutral with regard to jurisdictional claims in pub‑
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No evidence for trade‐offs between bird diversity, yield and water table depth on oil palm smallholdings: Implications for tropical peatland landscape restoration
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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 Authors. Journal of Applied Ecology published by John Wiley & Sons Ltd on behalf of British Ecological Society. No evidence for trade-offs between bird diversity, yield and
water table depth on oil palm smallholdings: Implications for
tropical peatland landscape restoration We examined oil palm fruit yields and bird diversity on 41 smallholder farms in
Jambi (Sumatra, Indonesia), which varied in drainage intensity (12-month mean
water table per plot from August 2018 to August 2019: −52 to −3 cm below-
ground). We also compared farm bird diversity with a neighbouring area of pro-
tected peat swamp forest (11,000 ha, 21 plots; mean water table per plot −3 to
+15 cm). 3. Bird species richness (3–18 species per plot), species composition and oil
palm yields (4.5–19.2 t fresh fruit bunch ha−1 year−1) varied among farms,
but were not detectably affected by water table depth, although ground-
level vegetation was more complex on wetter farms. Bird richness in oil palm
(mean = 10.3 species per plot) was <50% of that in forest (26 species per provided the original work is properly cited.
© 2022 The Authors. Journal of Applied Ecology published by John Wiley & Sons Ltd on behalf of British Ecological Society. 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 R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E No evidence for trade-offs between bird diversity, yield and
water table depth on oil palm smallholdings: Implications for
tropical peatland landscape restoration J Appl Ecol. 2022;00:1–17. | 1
wileyonlinelibrary.com/journal/jpe No evidence for trade-offs between bird diversity, yield and
water table depth on oil palm smallholdings: Implications for
tropical peatland landscape restoration Eleanor Warren-Thomas1,2,3
| Fahmuddin Agus4
| Panji Gusti Akbar5 |
Merry Crowson6 | Keith C. Hamer7
| Bambang Hariyadi8
| Jenny A. Hodgson9
|
Winda D. Kartika8 | Mailys Lopes6
| Jennifer M. Lucey10
| Dedy Mustaqim8 |
Nathalie Pettorelli6
| Asmadi Saad11
| Widia Sari8 | Gita Sukma8 |
Lindsay C. Stringer1,12,13
| Caroline Ward1,13
| Jane K. Hill1 Eleanor Warren-Thomas1,2,3
| Fahmuddin Agus4
| Panji Gusti Akbar5 |
Merry Crowson6 | Keith C. Hamer7
| Bambang Hariyadi8
| Jenny A. Hodgson9
|
Winda D. Kartika8 | Mailys Lopes6
| Jennifer M. Lucey10
| Dedy Mustaqim8 |
Nathalie Pettorelli6
| Asmadi Saad11
| Widia Sari8 | Gita Sukma8 |
Lindsay C. Stringer1,12,13
| Caroline Ward1,13
| Jane K. Hill1 1Leverhulme Centre for Anthropocene Biodiversity, Department of Biology, University of York, York, UK; 2School of Natural Sciences, Bangor University,
Bangor, UK; 3Biodiversity and Natural Resources Program, International Institute for Applied Systems Analysis (IIASA), Laxenburg, Austria; 4Indonesian Center
for Agricultural Land Resources Research and Development, Bogor, Indonesia; 5Birdpacker, Batu, Indonesia; 6Institute of Zoology, Zoological Society of
London, London, UK; 7School of Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK; 8Biology Education Program, Faculty of Education and
Teacher Training, Jambi University, Jambi, Indonesia; 9Department of Evolution, Ecology and Behaviour, University of Liverpool, Liverpool, UK; 10Department
of Zoology, University of Oxford, Oxford, UK; 11Faculty of Agriculture, Jambi University, Jambi, Indonesia; 12Department of Environment and Geography,
University of York, York, UK and 13School of Earth and Environment, University of Leeds, Leeds, UK This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
Correspondence
Eleanor Warren-Thomas
Email: em.warren.thomas@gmail.com
Funding information
Natural Environment Research Council,
Grant/Award Number: NE/M006840/1-2,
NE/P014658/1, NE/R009597/1 and NE/
T009306/1
Handling Editor: Júlio Louzada
Abstract
1. Tropical peat swamp forests retain large carbon stocks and support unique bio-
diversity, but clearance and drainage for agriculture have resulted in fires, car-
bon emissions and biodiversity losses. Initiatives to re-wet cultivated peatlands
may benefit biodiversity if this protects remaining forests from fire and agricul-
tural encroachment, but there are concerns that re-wetting could reduce yields
and damage livelihoods, as relationships between drainage, on-farm biodiver-
sity, and crop yields have not been studied. 2. Received: 28 June 2021 | Accepted: 7 January 2022
DOI: 10.1111/1365-2664.14135 Received: 28 June 2021 | Accepted: 7 January 2022 DOI: 10.1111/1365-2664.14135 1 | INTRODUCTION industrial plantations, primarily of oil palm and pulpwood (Miettinen
et al., 2012, 2016). Drainage is considered necessary to maintain
oil palm yields, because prolonged flooding reduces fruit produc-
tion as roots cannot respire under prolonged inundation (Woittiez
et al., 2017). However, peatland drainage means Sumatra is now a
hotspot for peat fires (Page & Hooijer, 2016). Peatland restoration
activities are now underway across Indonesia, mostly focused on
re-wetting cultivated areas by blocking drainage canals to reduce
the risk of fires, driven by the establishment of new legislative re-
quirements (Dohong et al., 2018). Tropical peatlands in Southeast Asia contain large below-ground car-
bon stocks, and peat swamp forests contain unique and threatened
biodiversity. However, peat forests have been cleared and peatlands
drained for cultivation, resulting in carbon emissions, biodiversity
losses and land subsidence (Posa et al., 2011; Wijedasa et al., 2018). Peat deposits are formed when waterlogged conditions prevent
microbial decomposition of vegetation, creating accumulations of
extremely carbon-rich, water-retentive and acidic soils (peatlands
cover just 3% of global land area, but contain 32%–46% of all soil
carbon) that accumulate over thousands of years, forming a net car-
bon sink (Page & Hooijer, 2016). Drainage of tropical peatlands to
allow access for agriculture or development, via digging of drainage
canals, draws the water table down below the soil surface, expos-
ing peat to the air where it decomposes and releases stored car-
bon, leading to land subsidence, peat loss and carbon emissions. Dry
peat is also very flammable, making drained tropical peatlands vul-
nerable to fires; in Southeast Asia peat fires cause trans-boundary
haze, particularly during droughts associated with El Niño Southern
Oscillation (ENSO) years, damaging human health and the regional
economy, threatening local livelihoods, and putting remaining peat
forests and their biodiversity at risk of loss (Page & Hooijer, 2016). Legislation in Indonesia now mandates average water table
depths of −40 cm (below the surface) in cultivated peatlands
within active agricultural land concessions (Wijedasa et al., 2018). Regulations stipulate that water tables in the centre of plantation
blocks should be maintained above −100 cm at all times, and −40 cm
for half of the year, in contrast to the −70 cm drainage depth used in
standard operating procedures for plantations (Evans et al., 2019). Abstract 1. Tropical peat swamp forests retain large carbon stocks and support unique bio-
diversity, but clearance and drainage for agriculture have resulted in fires, car-
bon emissions and biodiversity losses. Initiatives to re-wet cultivated peatlands
may benefit biodiversity if this protects remaining forests from fire and agricul-
tural encroachment, but there are concerns that re-wetting could reduce yields
and damage livelihoods, as relationships between drainage, on-farm biodiver-
sity, and crop yields have not been studied. 2. We examined oil palm fruit yields and bird diversity on 41 smallholder farms in
Jambi (Sumatra, Indonesia), which varied in drainage intensity (12-month mean
water table per plot from August 2018 to August 2019: −52 to −3 cm below-
ground). We also compared farm bird diversity with a neighbouring area of pro-
tected peat swamp forest (11,000 ha, 21 plots; mean water table per plot −3 to
+15 cm). 3. Bird species richness (3–18 species per plot), species composition and oil
palm yields (4.5–19.2 t fresh fruit bunch ha−1 year−1) varied among farms,
but were not detectably affected by water table depth, although ground-
level vegetation was more complex on wetter farms. Bird richness in oil palm
(mean = 10.3 species per plot) was <50% of that in forest (26 species per | 1
wileyonlinelibrary.com/journal/jpe J Appl Ecol. 2022;00:1–17. 2 Journal of Applied Ecology WARREN-THOMAS et al. plot), and only 3 of 35 conservation-priority species found in forest were
recorded in oil palm. 4. Synthesis and applications. Tropical peatlands in Indonesia have been drained to
allow farmer access and improve farm yields, but we found no trade-offs be-
tween drainage depth, yields and bird diversity on smallholder oil palm farms
in our study landscape within the studied range of drainage depths. Current
restoration initiatives to re-wet peat may benefit farmers by reducing fire risk,
without affecting yields. Wetter farms had increased understorey vegetation
complexity, but this did not affect bird diversity, so we find no evidence that re-
wetting improves on-farm biodiversity. However, on-farm fire reduction efforts
in cultivated peatlands, including re-wetting, will be vital for reducing the risk of
fires escaping into nearby forests, which contain unique and diverse bird species
assemblages. Protection of remaining peatland forests from fire and clearance
is key for biodiversity conservation, and for providing a source of seed dispers-
ers and genetic material for future forest and landscape restoration efforts. Abstract Restoration of more biodiversity-friendly land covers will improve landscape
permeability and help conserve species and the ecosystem services they deliver. K E Y W O R D S
birds, canal blocking, deforestation, Indonesia, Jambi, peat swamp forest, peatlands, Sumatra 1 | INTRODUCTION The extent and severity of recent fires on tropical peatlands neg-
atively impact health and livelihoods, making restoration activities
vital, but the consequences of re-wetting and any trade-offs with
yield or biodiversity must be understood. The ecological effects of
re-wetting are poorly understood, and in many cases canal block-
ing activities have not yet been completed. However, variation in
drainage depth across peatland landscapes provides opportunities
to study yields and biodiversity in areas that vary from shallow to
more deeply drained peat. In this study, we examine how variation
in water table depth affects vegetation structure, avian diversity
and oil palm yields on smallholder farms across a peatland landscape
in Sumatra. We ask whether bird species richness, abundance and
community composition are related to water table depth (drainage
intensity) via changes in vegetation structural complexity on farms,
and whether oil palm yields are reduced in shallowly drained (wet-
ter) oil palm farms relative to more deeply drained farms. We also
compare bird species in oil palm with a nearby protected peat for-
est fragment (~10,000 ha), to understand changes in diversity and
ecological function following conversion of peat swamp forest to oil
palm, and to assess the importance of conserving forest fragments
for future restoration initiatives. Smallholders produce around 40% of palm oil globally, but yields
vary greatly (Woittiez et al., 2017) and tend to be much lower than
those of industrial plantations (Lee et al., 2014). On smallholder
farms, drainage depth could affect management intensity of oil
palm, shifting biodiversity-yield trade-offs (e.g. as outlined by Grass
et al., 2020) towards lower intensity systems, while flooding may
damage oil palm trees (Woittiez et al., 2017). Farms that are more
shallowly drained (and periodically flood) may be challenging to ac-
cess, increasing the difficulty of harvesting and management activ-
ities such as weeding and fertiliser application that could increase
yields. Thus, shallowly drained (wetter) farms could be expected to
have lower yields but greater on-farm biodiversity. However, despite
evidence from Sumatra for biodiversity-yield trade-offs (Teuscher
et al., 2015), the wide variation in smallholder oil palm yields is diffi-
cult to explain (Lee et al., 2014), and evidence from other smallholder
production systems in the tropics indicate space for biodiversity im-
provements through management changes without yield declines
(Clough et al., 2011). 1 | INTRODUCTION The −40 cm standard is also used as a target in other restoration
activities, such as the Indonesia Peat Restoration Agency’s (Badan
Restorasi Gambut; BRG) work in smallholder oil palm, although
resource limitations strongly constrain capacity to monitor water
table depths outside of large concessions. Existing restoration proj-
ects in Central Kalimantan, Indonesia, have used -40 cm as a min-
imum threshold, rather than a mean, water table depth (Dohong
et al., 2018), given evidence that the risk of peat ground fires is
greater when water tables fall below this level (Page et al., 2009). Thus, there are compelling reasons to raise water tables, but the Indonesia is estimated to contain 47% of global tropical
peatlands, chiefly on the islands of Borneo and Sumatra (Page
et al., 2011). Forests covered 76% of Sumatra’s peatland in 1990,
but by 2015 66% was covered by smallholder agriculture or WARREN-THOMAS et al. Journal of Applied Ecology 3 consequences of re-wetting peatlands for oil palm cultivation on
peat have been little studied. favourable for peatland plant species. These drainage-induced
changes may indirectly affect bird diversity on oil palm farms. Birds are also an excellent taxon for examining the effects of
habitat change on community structure and ecosystem functioning
through changes in functional traits, such as feeding guild and body
mass (Darras et al., 2018; Edwards et al., 2013; Prabowo et al., 2016). These avian traits are particularly important in the context of peat-
land restoration at the landscape scale. Therefore, examining bird
species diversity, composition and functional traits in remaining peat
swamp forest fragments is also important for understanding impacts
of drainage in predominantly cultivated peatland landscapes. In the longer term, complete withdrawal of drainage-dependent
agriculture from peatlands in Sumatra may take place, and indeed is
necessary if a substantial decrease in carbon emissions from peat
degradation is to be achieved (Afriyanti et al., 2019), provided that
alternative systems can offer comparable livelihood and economic
benefits (Tan et al., 2021). Subsidence of cultivated peatland due to
drainage may also mean cultivation is forced to cease, due to regular
inundation (Wijedasa et al., 2018). In this case, forest restoration on
degraded peatlands may become a more viable land-management
option. The presence of remaining intact peat forest in the land-
scape will be essential for restoration success, by providing sources
of seeds and seed-dispersing agents, such as frugivorous birds
(Wijedasa et al., 2020). 1 | INTRODUCTION Recent work has also shown that maintenance
of understorey vegetation may be beneficial for oil palm by improv-
ing soil health (Darras et al., 2019). To our knowledge, there is cur-
rently no information on responses of peatland oil palm yields to
water table depth and farm management. A better understanding
of possible trade-offs between oil palm yields and biodiversity is,
however, key to appreciating the possible ecological and livelihood
impacts of re-wetting restoration schemes and to guide farm man-
agement practices. 2 | MATERIALS AND METHODS Permission to collect data was granted by Ristekdikti, The Ministry
of Research, Technology and Higher Education, The Republic of
Indonesia, under research permit numbers 198/SIP/FRP/E5/Dit.KI/
VII/2018 and 199/SIP/FRP/E5/Dit.KI/VII/2018. Birds are an important study taxon for understanding biodi-
versity responses to peatland oil palm management, as their habi-
tat requirements and conservation status are well understood. For
example, bird diversity in oil palm landscapes varies in relation to
understorey vegetation, native tree diversity, canopy cover and dis-
tance to forest (Aratrakorn et al., 2006; Azhar et al., 2014; Hamer
et al., 2021; Teuscher et al., 2015). In the context of peatland oil
palm, canopy cover may be affected by drainage depth because
oil palms can lean or fall in very wet ground (Woittiez et al., 2017). Additionally, vegetation structure of the herbaceous understorey
on smallholder farms may be more complex in wetter areas, due to
altered weeding practices, or if soil moisture conditions are more 2.1 | Study location The study was conducted in Jambi province, Sumatra, Indonesia. The study landscape lies on a single peat dome (a hydrological unit
of connected peatland) that had been mostly drained for cultiva-
tion, leaving a single remaining peat forest fragment (Figure 1A). Drainage canals 2–3 m wide ran alongside the main access roads,
and smaller hand-dug canals were found within the oil palm Elaeis
guineensis smallholdings. The forest contained a patchy distribution
of shallow hand-dug drainage canals, formerly used to extract tim-
ber, with wider drainage canals adjacent to the perimeter road and Journal of Applied Ecology WARREN-THOMAS et al. 4 |
FI G U R E 1 (A) Map of study area showing plots in forest and at three oil palm sites that vary in water table depth (Site 1, Site 2 and Site
3); land cover classification based on fusion of optical and radar sentinel data at 10 m resolution, with temporal information and ground-
truthing conducted specifically for this study landscape; please refer to Crowson et al. (2018) and Lopes et al. (2020) for specific methods,
error rates and definitions of land cover categories; inset aerial photographs of plots at each site/forest provided under licence via the
Education and Research Program of Planet Labs PBC (2017). 2.1 | Study location (B) sampling design for water table depth, bird diversity (50 m radius) leaf area
index (LAI photo) and vegetation structure (20 m radius), shown within an oil palm smallholder farm, but an identical sampling design was
used in forest; (C) location of study landscape on Sumatra
(A)
(B)
Focal farm
Neighbouring farm
100 m
20m radius vegetation plot
50m bird count radius
Ground cover
Understorey density pole
Dipwell/bird point count centre
LAI photo
Land cover
Forest
Water
Urban/cloud
Cultivated palm
Acacia plantation
Fern
Bare ground/young plantation
Plot locations
Forest
Oil palm site 1
Oil palm site 2
Oil palm site 3
(C)
Sumatra A) (B)
Focal farm
Neighbouring farm
100 m
20m radius vegetation plot
50m bird count radius
Ground cover
Understorey density pole
Dipwell/bird point count centre
LAI photo (B) (C)
Sumatra (C) FI G U R E 1 (A) Map of study area showing plots in forest and at three oil palm sites that vary in water table depth (Site 1, Site 2 and Site
3); land cover classification based on fusion of optical and radar sentinel data at 10 m resolution, with temporal information and ground-
truthing conducted specifically for this study landscape; please refer to Crowson et al. (2018) and Lopes et al. (2020) for specific methods,
error rates and definitions of land cover categories; inset aerial photographs of plots at each site/forest provided under licence via the
Education and Research Program of Planet Labs PBC (2017). (B) sampling design for water table depth, bird diversity (50 m radius) leaf area
index (LAI photo) and vegetation structure (20 m radius), shown within an oil palm smallholder farm, but an identical sampling design was
used in forest; (C) location of study landscape on Sumatra WARREN-THOMAS et al. Journal of Applied Ecology 5 an access road that ran to the centre of the forest patch (established
during surveys by a petrochemical company). Industrial Acacia spp. plantations lay south of the forest, while smallholdings of palm lay to
the north, east and west. To the east and south, where study plots
were located, the farms were all monocultural oil palm smallhold-
ings (photographs Figure S1 in Supporting Information), while to
the west some palm areas also contained betel Areca catechu and
coconut Cocos nucifera which are indistinguishable using remote
sensing (Lopes et al., 2020). We only studied monocultural oil palm
smallholdings. 2.2 We collected data on bird species at each oil palm and forest plot to
establish species richness, abundance, biomass and species compo-
sition, and to establish the representation of species of conservation
concern, habitat specificity and functional traits that are important
in the context of ecosystem function and future landscape restora-
tion. Birds were surveyed at all 62 plots using 15-min point counts on
four consecutive mornings (06:00–10:00) during August–October
2018 (Edwards et al., 2014). Visits were rotated so each plot was sur-
veyed early, middle and late in the morning. All birds seen or heard
within 50 m were recorded, but flyovers and fly-throughs were not
included in analysis, because we could not confirm that they were
using the study plot. All surveys were recorded (Olympus LS-10 digi-
tal recorder) to confirm species' identifications and check unknown
sounds against verified recordings on Xeno-Canto (xeno-canto.org). All surveys were conducted by the same experienced ornithologist
(PGA), who used a 2-week pilot phase to become familiar with birds
in the area. Bird species richness per plot was calculated as the total
of all species recorded at each plot across all four visits (including
migratory species recorded within the plots, highlighted in Table S2). Total abundance per plot was calculated as the sum of the maximum
number of individuals detected on any single visit per species, to
avoid the risk of double-counting individuals. We also established 21 plots in an adjacent area of protected for-
est (Sungai Buluh Peat Protection Forest/Hutan Lindung Gambut)
to use as reference site to indicate water table variation in the land-
scape under minimal drainage (i.e. to understand the potential for
water table recovery in this landscape), and to capture data on the
biodiversity value of unprotected forest fragments in cultivated
peatland landscapes for bird diversity, composition and functional
traits. The forest had been commercially selectively logged some-
time after 1980, but the date of cessation is unknown (see Galudra
et al., 2014 for history of neighbouring district) and is the largest
block of remaining forest (~10,000 ha in 2018) in the locality, be-
cause larger extents of neighbouring forest were cleared after 2000. Some illegal logging activity has taken place in the fragment since
commercial logging ceased, and areas of forest were lost to fires
in 1997 (the southeast corner) and 2015 (central-northwest patch;
Figure 1A). 2.1 | Study location from existing analysis (Crowson et al., 2018; Lopes et al., 2020) to
calculate the distance from plot centroids to the edge of the contig-
uous forest cover within the forest fragment, to use as a co-variate
in analyses of bird diversity in oil palm. Understorey vegetation of
oil palm farms (27 plant species recorded across all plots; Table S1)
was dominated by the fern Nephrolepis falcate and the herb Asystasia
gangetica. The forest included peat-specialist trees Dyera lowii and
Shorea uliginosa, the critically endangered Vatica teysmanniana and
Gonystylus bancanus, and the gap specialist species Macaranga pru-
inosa, reflecting its disturbance history. Canal blocking activities to re-wet parts of the drained small-
holder oil palm areas had been planned by the BRG, but no canal
blocks had yet been installed at the time of our study. We there-
fore used a space-for-time approach to assess the effect of water
table variation, which also allowed us to assess the longer term ef-
fects of water table variation that may not manifest immediately
after canal blocking activities. We established 41 sampling plots in
oil palm farms in three areas (one plot per farm; farm area 2–15 ha,
sites termed Site 1, Site 2 and Site 3) known from pilot fieldwork to
show variation in water table depth, both within and between sites. Farmers were contacted through the head of their farmer group and
were invited to voluntarily participate in the study, resulting in a
clustered sampling design (Figure 1A). Those famers who agreed to
participate permitted the field team to conduct surveys and install
dipwells on their farms (Figure 1B), and verbally completed a ques-
tionnaire about their peatland farms (details below). 2.2 Bird species habitat dependence was defined based on IUCN
status (IUCN, 2019) and the Handbook of Birds of the World (del
Hoyo et al., 2017), and was used to determine the capacity for oil
palm plots to support species usually dependent on forests. Species
were defined as ‘forest dependent’ if found in primary, secondary
or disturbed forest, as ‘high tree-cover dependent’ if also reported
from plantations, ‘generalist’ if reported from both forest and non-
forest habitats, and ‘open habitat species’ if found only in non-forest
habitats. Conservation-priority species were defined as those
with IUCN status of Near Threatened, Vulnerable, Endangered or
Critically Endangered. The centres of plots were at least 200 m apart and located in
the centre of each farm. Peat depth was measured in each plot with
a peat auger until a mineral clay layer was found, up to the maxi-
mum measurable depth of 5 m. Peat depths ranged from 1.15 m to
>5.00 m across all plots, and were similar between forest and oil
palm sites (mean = 3.3 m forest, 2.8 m Site 1, 4.1 m Site 2 and 3.8 m
Site 3). For forest-oil palm comparisons, each bird species was assigned
to one of five diet groups based on the Elton Traits database vari-
able ‘Diet-5Cat’, according to their predominant diets (Wilman
et al., 2014). Total bird abundance, as calculated above, was assigned
to the feeding guilds, to give plot-level abundance per feeding guild. Community-weighted avian body mass was calculated per plot by Bird species presence on farms can be affected by the distance
to areas of forest remaining in the wider cultivated landscape (Azhar
et al., 2011), which act as population sources (Hamer et al., 2021). We therefore used land cover data and aerial imagery (Figure 1A) WARREN-THOMAS et al. Journal of Applied Ecology 6 6 plot, Figure S5), so we analysed them using principal component
analysis (PCA) to produce two principal components that together
explained 70% of variance (43% by PC1 and 27% by PC2; Table S2),
and enabled a reduced number of variables to be included in our sta-
tistical models, reducing the risk of over-fitting. We interpret PC1 as
measuring complexity of the herbaceous ground vegetation (higher
values mean greater complexity), and PC2 as measuring vertical veg-
etation structure and canopy openness (higher values mean greater
vertical complexity, fewer fallen/leaning oil palms and a more open
canopy). 2.6 | Statistical analysis Statistical analyses were conducted using plots as independent sam-
ples (excluding one of the 41 plots for which complete water table
data were unavailable, n = 40 plots), as each farm is a unit managed
independently by farmers (affecting vegetation and water tables),
and bird survey points at the centre of plots were sufficiently far
apart to be considered independent samples. We also used site as
an explanatory variable in all models, to ask whether changes in bird
occurrence, vegetation complexity or water table depths occurred
at a wider spatial scale, that is, between sites. All analyses were
conducted in R (R Core Team, 2018). We compared models using
Akaike’s information criterion corrected for small sample sizes (AICc;
Burnham & Anderson, 2002). The best model was defined as having
the lowest AICc of the model set (whether multiple alternative mod-
els, or a null model compared to a single alternative model). 2.2 Soil pH (method in Table S3) was acidic, ranging from 2.83
to 3.81 in the forest, and 3.06 to 4.30 on oil palm farms. multiplying the abundance of each species by a mass value for an
individual, and summing across all species (Darras et al., 2018). Body
mass for each species was taken from the Handbook of Birds of the
World (del Hoyo et al., 2017). 2.5 | Oil palm yields All farms contained mature fruiting oil palms (age 6–26 years since
planting). All farmers were independent smallholders, who sold
their fresh fruit bunches to oil palm mills through a local broker. Questionnaires, conducted with each farmer, collected informa-
tion about annual oil palm yield estimates per plot, and other infor-
mation on farm management, including frequency and amount of
chemical applications, oil palm age, harvesting frequency, weeding
practices and removal of dead fronds from palms (questionnaire,
Supplementary Text S2 and Ward et al., 2021). Questionnaires were
conducted by research assistants from the University of Jambi. The questionnaire was approved by the University of Leeds Ethics
Committee before data collection started. 2.3 | Water table monitoring Water table depths were manually recorded at each plot every fort-
night from August 2018 to August 2019, using a 2 m dipwell installed
at the centre of each plot (further methodology and schematic, Figure
S2). Southern Sumatra has a marked dry season from July to September
(Aldrian & Dwi Susanto, 2003), meaning our sampling period captured
the end of the 2018 dry season, the 2018–2019 wet season and the
start of the 2019 dry season. Rainfall in this region is reduced during
ENSO events (Qian, 2019). A strong ENSO event occurred in 2015–
2016, and a weaker event was underway for the duration of this study
(NOAA/National Weather Service, 2019). A range of water table depth
indices were calculated for each plot for the whole monitoring period
(including maximum depth, number of records below 40 cm, and oth-
ers) to test whether indices other than mean average water table
could be informative, but these correlated strongly with each other
(correlation plot, Figure S3) so we used mean water table depth in our
study because this is used in legislation (Wijedasa et al., 2018), and is
often reported in other peat studies. Manual measurements were sup-
plemented by four water table depth loggers recording every 15 min
(Seametrics/Van Walt LevelSCOUT 10 m), installed in one dipwell at
each of the three oil palm sites and the forest site. Together with rain-
fall data (manually recorded daily at Sites 1 and 2) these acted as a
sense-check, and showed the response of water table to precipitation,
including the rapid decline in water tables with the onset of the dry
season in June/July 2019 (logger vs. manual water table depths and
rainfall, Figure S4). 2.4 | Vegetation structure and soil pH We assessed the ecological impacts of farm management by quan-
tifying the structural complexity of vegetation at each oil palm plot,
and measuring soil pH. We measured six variables within a 20 m ra-
dius of the bird sampling point (Figure 1B): identity and diameter at
breast height (DBH) of all non-oil palm trees ≥10 cm DBH (all trees
were cultivated species); number of fallen or leaning oil palms; num-
ber of sections of a 2 m pole visible at a distance of 14 m from the plot
centre (understorey density index, a measure of vertical complexity,
recorded four times to the NE, NW, SE and SW (Barlow et al., 2007);
herbaceous ground vegetation cover (estimated in a 1 m2 quadrat)
and height (to the nearest 10 cm, recorded four times N, S, E and W
of the plot centre), and leaf area index (LAI) and canopy cover (Global
LAI Project protocol, Supplementary Text S1). Maximum density of
non-oil palm trees was only 0.5 stems ha−1 and most farms had none,
so this variable was excluded from analysis (photograph of farms,
Figure S1). Correlations existed among these variables (correlations To examine whether bird species richness, abundance and
composition were related to water table depth (drainage intensity)
on oil palm farms via changes in vegetation structural complex-
ity, we first fitted a general linear model of the two vegetation
complexity principal components (PC1, PC2), with mean water
table depth and site as explanatory variables. These models tested
whether drainage affected on-farm vegetation at the plot or site
scales. WARREN-THOMAS et al. Journal of Applied Ecology 7 effect (R2, proportion of variance explained) we would be able to
detect with our dataset, given specified Type 2 error thresholds. Di
Stefano (2003) shows that the risk of making Type 2 errors (i.e. a
false negative result) can be as harmful as making a Type 1 error (i.e. a false positive). In our case, erroneously concluding that there is no
effect of drainage on oil palm yields, or avian biodiversity, is equally
as concerning as erroneously concluding that there are trade-offs. 2.4 | Vegetation structure and soil pH In
the case of oil palm yields, the risk of making a Type 2 error (conclud-
ing there is no effect, when there is one) is that further management
to raise water tables within the ranges measured in this study could
have unforeseen negative impacts on farmer yields, damaging liveli-
hoods and reducing income. The risk of a Type 2 error in conclusions
around bird diversity is less serious, but a Type 1 error (concluding
there is an effect, when there is not one) could result in expected
conservation gains from re-wetting not being realised. Next, we examined the response of bird species richness and total
bird abundance to vegetation complexity on oil palm farms, as well
as distance to forest edge, and site. We fitted models with a full set
of explanatory variables, sub-models of all combinations of the vari-
ables, and a null model to identify the most informative model struc-
ture (based on comparison of AICc of standardised models). All models
were Poisson log-link generalised linear models fitted using the bbmle
package (Bolker & R Development Core Team, 2017). Predictions were
made from the most informative model. Bird species composition response to the same variables was
analysed using the Bray–Curtis dissimilarity index and non-metric
multi-dimensional scaling (NMDS; based on abundance data with
a Wisconsin double standardisation; Edwards et al., 2014). NMDS
scores were correlated with environmental variables (vectors), or
averages compared between factor levels, with ‘significance’ test-
ing using 999 permutations of the environmental factors. Analysis
was conducted using the metaMDS and envfit functions in the vegan
package (Oksanen et al., 2017). We therefore assessed the size of effect (R2, proportion of ex-
plained variance) that would be detectable with a sample size of 40,
39 and 33 farms (reflecting the sample sizes outlined above), assum-
ing significance levels of 0.05 (alpha, Type 1 error rates, of 5%), and
power of 80% (beta, Type 2 error rates, of 20%) are acceptable, but
also considering power thresholds of 90% and 95%, (Type 2 error
rates of 10% and 5% respectively). We examined variation in oil palm yields (fresh fruit bunch
tonnes ha−1 year−1) in relation to water table depth on farms, by fit-
ting a general linear model and assessing AICc compared to a null
model (n = 39 farms, omitting one farm for which yield data were not
available). 3 | RESULTS Finally, to consider the role played by the forest fragment on
bird diversity in the study landscape, total bird species richness
was compared between the forest plots and oil palm plots using
individual-based rarefaction to account for differing sample sizes,
including calculation of asymptotic species richness, using the
iNEXT package in r (Chao et al., 2014). Bird abundance, species rich-
ness, abundance per feeding guild and (natural-log-transformed)
community-weighted body mass (used as a measure of ecosystem
function, with body size an important indicator of species function)
per plot were also compared between forest and oil palm using linear
models, as above, and an NMDS ordination was applied to test for
differences in species composition. 2.4 | Vegetation structure and soil pH Nine a-priori defined alternative models of yield response
to management practices were also fitted (variables Table S3, model
structures Table S4) designed to investigate multiple aspects of oil
palm management previously found to affect yields (Lee et al., 2014;
Woittiez et al., 2017), including tree age, competition from weed
growth, fertiliser application, plot management (frond removal and
harvest frequency), soil pH and tree health (using leaf area index and
the number of fallen or leaning trees). We were unable to use ques-
tionnaire responses on weed management in any models, as most
respondents did not quantify their weeding practices. These models
excluded plots for which yield data (n = 1), complete water table data
(n = 1) and oil palm tree age (n = 5) were unknown, leaving n = 33
farms. We used the pwr package in r (Champely, 2021) to estimate the
sample size that would be needed to detect 10%, 20%, 30%, 40%
or 50% of explained variance in a response variable to either a sin-
gle predictor, or two predictor variables in a linear model (such as
the response of oil palm yields to water table depth, or bird species
richness response to vegetation complexity and site), using a signif-
icance level of 0.05 (5% Type 1 error rate) and a power of 80%, 90%
or 95% (20%, 10% or 5% Type 2 error rates). These results are shown
in Figure S6 and Table S5, and are used to assist interpretation of our
model results. 3.1 | Drainage and water table depth on farms The entire study landscape had been drained with canals, but fort-
nightly water table depth measures across the 40 smallholder farms
were highly variable, ranging from −169 cm to +23 cm during the
study period. Hence, our farm study sites spanned a range of drain-
age intensities, from wet to moderately drained, representing the
variation found within smallholder oil palm farms in the study land-
scape. Twelve-month mean (± SD) water table depths per oil palm
plot ranged from −51.5 ± 29.8 cm to −6.0 ± 18.9 cm, meaning some
farms were drained below the recommended -40 cm depth (as
calculated on an annual average basis), and many farms had water
tables below the 40 cm threshold for part of the year (Figure 2B). Site 1 farms were relatively less severely drained than those at
Sites 2 or 3 (Figure 2A), but the overall mean water table depth of
−27.8 ± 25.9 cm across all plots means that the surveyed landscape
as a whole complied with legislative requirements for agricultural 2.7 | Power analysis Water tables were above the soil surface (black line at 0 cm) during the rainy season in forest, but in oil palm, generally remained below the
soil surface. Reduced rainfall in 2019 relative to 2018 explains the rapid drop in water tables at all three sites (Figure S4). Light grey line at
−40 cm indicates legislated mean water table depth for active agricultural concessions in Indonesia (Wijedasa et al., 2018) 2.7 | Power analysis We conducted a power analysis to support the interpretation of the
modelled relationships in our study, in order to quantify the size of 8 WARREN-THOMAS et al. Journal of Applied Ecology FI G U R E 2 (A) Data points show mean water table depth per plot (across the 12 month monitoring period), point and range shows model
predictions with 95% CI; mean water tables differed between forest and all three farm sites, and between Site 1 and Sites 2 and 3 (model of
water table response to site and forest ∆AICc −104.2 relative to null model; letters are used to show differences; coefficients and CI: forest
5.6 ± 3.3 cm; relative to forest, Site 1 –24.1 ± 5.34 cm, Site 2 –35.5 ± 5.23 cm, Site 3 –41.3 ± 5.34 cm); (B) water table depths in forest and
oil palm sites are shown from 7 September 2018 to 24 August 2019, with a loess smoothed curve (span = 0.2) and standard error per site. Water tables were above the soil surface (black line at 0 cm) during the rainy season in forest, but in oil palm, generally remained below the
soil surface. Reduced rainfall in 2019 relative to 2018 explains the rapid drop in water tables at all three sites (Figure S4). Light grey line at
−40 cm indicates legislated mean water table depth for active agricultural concessions in Indonesia (Wijedasa et al., 2018)
(A)
(B) (B) (A) (B) (A) FI G U R E 2 (A) Data points show mean water table depth per plot (across the 12 month monitoring period), point and range shows model
predictions with 95% CI; mean water tables differed between forest and all three farm sites, and between Site 1 and Sites 2 and 3 (model of
water table response to site and forest ∆AICc −104.2 relative to null model; letters are used to show differences; coefficients and CI: forest
5.6 ± 3.3 cm; relative to forest, Site 1 –24.1 ± 5.34 cm, Site 2 –35.5 ± 5.23 cm, Site 3 –41.3 ± 5.34 cm); (B) water table depths in forest and
oil palm sites are shown from 7 September 2018 to 24 August 2019, with a loess smoothed curve (span = 0.2) and standard error per site. 3.3 | Variation in avian diversity in response to
farm vegetation, distance to forest and among sites concessions to maintain mean water table depths −40 cm over the
study period. However, due to reduced rainfall (Figure S4) water
tables on farms were much lower in the dry season of September
2019, when levels mostly dropped below −40 cm from July onwards,
compared with September 2018 when most farms stayed above the
−40 cm threshold (Figure 2B). As expected, smallholdings were drier
than forest plots, which had a mean depth of −5.6 ± 16.7 cm over
the same period. In total, we observed 1093 individual birds in oil palm (maximum ob-
served abundance of each species per plot on any single sampling
day, summed across all plots), comprising 48 species (Table S2). Bird
species richness varied among farms (3–18 species per farm), and the
majority of species in oil palm were defined as generalist or open-
habitat species (NMDS plot with habitat dependence, Figure S7). We found no relationships between species richness of birds
per plot on oil palm farms and any of the measured variables, with
no models performing better than the null model (Table 1). There
was also no difference in cumulative species richness among sites
(rarefaction shown in Figure S8). Power analysis suggests that with
our sample size (n = 40) we should have been able to detect ef-
fects where the R2 of a model containing one or two predictors
was approximately 0.20 or below. Effect sizes smaller than this (i.e. resulting in a lower R2, or explaining less than 20% of variance in
bird species richness) would likely be undetectable with our study
design, so there is a possibility that smaller changes in bird spe-
cies richness were present but not detected in this analysis. We
conclude that vegetation complexity, moderated by water table
depth, is not a strong driver of local bird species richness on oil
palm farms. 3.2 | Vegetation structure and drainage of farms The model of PC1 score (complexity of herbaceous ground vegeta-
tion) response to water table depth and site (∆AICc −7.4 relative to
null model, adjusted R2 0.25) showed that there was no overall de-
tectable relationship with water table depth across the measured
gradient (coefficient and 95% CI: 0.02 ± 0.04 change in PC1 score
per 1 cm of water table depth reduction). However, plots at Site
1 tended to have higher PC1 scores, and thus greater herbaceous
ground vegetation complexity than Sites 2 or 3 (compared to Site 1,
Site 2 PC1 score −1.44 ± 1.08, and Site 3 PC1 score −2.59 ± 1.23;
Figure 3A,B). The PC2 score, representing vertical vegetation complexity and
canopy openness, also varied among sites, but not with water table
depth (∆AICc −4.9 relative to null model, adjusted R2 0.20). PC2
score differed by 1.62 ± 0.88 at Site 2 and 1.15 ± 0.99 at Site 3,
relative to Site 1 (Figure 3C,D). Plots in Site 1, which was the wetter
site, therefore tended to have reduced vertical complexity, a greater
number of fallen/leaning palms and a less open canopy. However, the best model explaining variation in species abun-
dance on farms revealed that abundance was lower on farms with
lower vegetation complexity (PC1 score), greater distance to forest
and at the driest Site 3 (∆AICc −15.49 relative to null, r2 = 0.30;
Table 1, Figure 4), although all effect sizes were small. Fitting environmental variables to an NMDS, based on Bray–
Curtis dissimilarity, of bird species composition on farms showed
that site had a small but significant influence on species composition,
but there was considerable overlap in species composition of plots
across all three sites (Table S6; Figure S9). Taking these results together with the relationship between site
and water tables (see above) shows that water tables and vegetation
complexity co-varied at the site scale, but not farm scale. Therefore,
we conclude that vegetation complexity could be influenced by
other site-level effects that covary with water table effects. Journal of Applied Ecology WARREN-THOMAS et al. 3.2 | Vegetation structure and drainage of farms 9 FI G U R E 3 (A) Predicted values of vegetation principal component 1 (ground vegetation complexity) response to mean water table depth
and site; dotted lines show predicted relationship between PC1 and water table depth per site, points are original data; coefficient for overall
relationship: 0.02 ± 0.02 95% CI, that is, no relationship; (B) predicted values of PC1 per site relative to Site 1, Site 2 PC1 score −1.44 ± 0.55,
and Site 3 PC1 score −2.59 ± 0.01, that is, greater understorey vegetation complexity at wetter Site 1 as indicated by letters; (C) predicted
values of vegetation principal component 2 (vertical vegetation structure and canopy openness) response to water table depth and site;
dotted lines show predicted relationship between PC2 and water table depth per site, points are original data; coefficient for overall
relationship: 0.03 ± 0.04, that is, no relationship; (D) predicted values of PC2 per site relative to Site 1, Site 2 PC2 score 1.62 ± 0.88, and Site
3 PC2 score 1.15 ± 0.99, that is, reduced representing vertical vegetation complexity and canopy openness at wetter Site 1 as indicated by
letters
(A)
(B)
(C)
(D) (A) (B) (B) (A) (D) (C) (C) FI G U R E 3 (A) Predicted values of vegetation principal component 1 (ground vegetation complexity) response to mean water table depth
and site; dotted lines show predicted relationship between PC1 and water table depth per site, points are original data; coefficient for overall
relationship: 0.02 ± 0.02 95% CI, that is, no relationship; (B) predicted values of PC1 per site relative to Site 1, Site 2 PC1 score −1.44 ± 0.55,
and Site 3 PC1 score −2.59 ± 0.01, that is, greater understorey vegetation complexity at wetter Site 1 as indicated by letters; (C) predicted
values of vegetation principal component 2 (vertical vegetation structure and canopy openness) response to water table depth and site;
dotted lines show predicted relationship between PC2 and water table depth per site, points are original data; coefficient for overall
relationship: 0.03 ± 0.04, that is, no relationship; (D) predicted values of PC2 per site relative to Site 1, Site 2 PC2 score 1.62 ± 0.88, and Site
3 PC2 score 1.15 ± 0.99, that is, reduced representing vertical vegetation complexity and canopy openness at wetter Site 1 as indicated by
letters 3.4 | Oil palm yields responses to water table
depth and management asymptote = 113 ± 12 SE; Figure 6A). Richness and abundance of
birds per plot in oil palm (mean 10.3 ± 2.9 SD species; 26.9 ± 8.1 SD
individuals) were also about half those of forest (mean 26 ± 3.9 SD
species; 48.2 ± 15.3 SD individuals (Figure 6B,C, ∆AICc −195.6 rela-
tive to null richness model, ∆AICc −164.3 relative to null abundance
model)). Oil palm yields varied considerably among farms from 4.5 to
19.2 t ha−1 year−1 (mean 11.7 ± 4.2 SD t ha−1 year−1). However, yields
were not detectably related to drainage within the measured range of
drainage depths (Figure 5), nor to any other measured variables rep-
resenting management strategies, oil palm age, tree health chemical
applications or by site (Table S4; Figure S10). Our power analysis indi-
cated that smaller changes in yields would not have been detectable
(i.e. we would not be able to detect effects that explained less than
20% of variance in oil palm yields; Figure S6). Thus, we conclude that
variation in yields was driven by factors not examined in our study. The conservation value of the forest fragment for birds was
confirmed by the occurrence of 35 conservation-priority bird
species in forest, and only three in oil palm. Forest supported a
very different avian community to farms (Figure 6D); none of the
18 forest-dependent bird species were found on farms, and only
26/123 bird species (21%) were recorded in both oil palm and for-
est (Table S2). Birds on farms were typically generalist and open-
habitat species (Figure 6D), with smaller body mass (Figure S11). Oil palm supported fewer invertebrate-feeding birds, but a higher
abundance of frugivores, nectarivores and granivores (Figure
S12). These body mass patterns were primarily due to the absence
of large-bodied frugivorous families (Bucerotidae and Trogonidae)
on farms, which are important seed dispersers, as well as the
absence or rarity of predominantly insectivorous bird families 3.5 | Forest bird diversity and composition Bird species richness on oil palm farms (total observed richness = 48
species, predicted asymptote 56 ± 6 SE) was about half of that in
forest (observed = 90 species from 1,013 individuals, predicted WARREN-THOMAS et al. Journal of Applied Ecology 10 |
of bird species richness response to vegetation complexity, distance to forest and site on oil palm farms. No model of bird species richness was more informative than the
est model of bird abundance included site, forest distance and vegetation PC1 score
Model
Intercept
Variables included
df
logLik
AICc
Delta AICc
Weight
Site
Distance to
forest
Veg PC1
Veg PC2
Best
2.34
0.15
2
−97.34
199.01
0.00
0.23
Null
2.34
1
−98.46
199.02
0.01
0.23
2.34
0.06
2
−98.30
200.92
1.91
0.09
2.34
0.05
2
−98.36
201.04
2.02
0.08
2.34
0.05
0.14
3
−97.23
201.13
2.12
0.08
2.34
0.05
0.15
3
−97.24
201.14
2.13
0.08
2.26
+
3
−97.83
202.32
3.31
0.04
2.34
0.09
0.08
3
−98.03
202.73
3.72
0.04
2.34
0.07
0.07
0.14
4
−96.99
203.13
4.12
0.03
2.20
+
0.15
4
−97.05
203.23
4.22
0.03
2.30
+
0.13
4
−97.18
203.49
4.48
0.02
2.27
+
0.01
4
−97.83
204.80
5.78
0.01
2.24
+
0.12
0.10
5
−96.71
205.18
6.17
0.01
2.28
+
0.18
0.16
5
−96.98
205.71
6.70
0.01
2.32
+
0.06
0.13
5
−97.17
206.10
7.09
0.01
2.32
+
0.18
0.13
0.10
6
−96.63
207.81
8.80
0.00
Best
2.98
+
0.41
0.07
5.00
−132.70
277.17
0.00
0.54
2.95
+
0.40
0.08
−0.04
6.00
−132.08
278.71
1.54
0.25
3.07
+
0.06
4.00
−135.88
280.90
3.74
0.08
3.04
+
0.06
−0.04
5.00
−135.20
282.17
5.00
0.04
3.09
+
0.29
4.00
−136.71
282.57
5.40
0.04
3.14
+
3.00
−138.37
283.41
6.24
0.02
3.08
+
0.28
−0.01
5.00
−136.67
285.10
7.93
0.01
3.13
+
−0.01
4.00
−138.26
285.66
8.49
0.01
3.24
0.07
2.00
−143.23
290.78
13.61
0.00
3.24
0.07
0.03
3.00
−142.66
291.98
14.81
0.00
3.23
0.08
0.02
3.00
−142.93
292.52
15.35
0.00
Null
3.31
1.00
−145.27
292.65
15.49
0.00
3.31
0.03
2.00
−144.57
293.47
16.30
0.00
3.23
0.08
0.02
0.03
4.00
−142.39
293.92
16.75
0.00
3.31
0.00
2.00
−145.27
294.87
17.70
0.00
3.31
0.00
0.03
3.00
−144.57
295.81
18.64
0.00 g
0.23
0.23
0.09
0.08
0.08
0.08
0.04
0.04
0.03
0.03
0.02
0.01
0.01
0.01
0.01
0.00
0.54
0.25
0.08
0.04
0.04
0.02
0.01
0.01
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00 FI G U R E 4 Results of model of total bird abundance per plot in response to site, distance to forest and vegetation structure; predictions
made from whole model for each variable, holding other variables at mean/mid values: (A) predicted bird abundance per plot in Site 1 was
19.7 ± 2.7 individuals (95% CI), increased relative to Site 1 by 1.2 ± 0.2 in Site 2, and decreased by 0.6 ± 0.2 in Site 3; (B) predicted change in
bird abundance with increasing distance to forest was 1.5 ± 0.4 individuals per km, with no detectable relationship within each site (dotted
lines); (C) predicted change in bird abundance with increasing values of vegetation structure measure PC1 was 1.1 ± 0.1 individuals per unit
of PC1, with no detectable relationship within each site (dotted lines); (D) all effect sizes were therefore small, and in many cases close to
zero (effect sizes relative to intercept = Site 1)
(A)
(B)
(C)
(D) (A) (B) (B) (A) (D) (C) (D) (C) FI G U R E 4 Results of model of total bird abundance per plot in response to site, distance to forest and vegetation structure; predictions
made from whole model for each variable, holding other variables at mean/mid values: (A) predicted bird abundance per plot in Site 1 was
19.7 ± 2.7 individuals (95% CI), increased relative to Site 1 by 1.2 ± 0.2 in Site 2, and decreased by 0.6 ± 0.2 in Site 3; (B) predicted change in
bird abundance with increasing distance to forest was 1.5 ± 0.4 individuals per km, with no detectable relationship within each site (dotted
lines); (C) predicted change in bird abundance with increasing values of vegetation structure measure PC1 was 1.1 ± 0.1 individuals per unit
of PC1, with no detectable relationship within each site (dotted lines); (D) all effect sizes were therefore small, and in many cases close to
zero (effect sizes relative to intercept = Site 1) FI G U R E 5 Oil palm yield response to water table depth, with
prediction and SE from general linear model (n = 39) showing
no relationship between reported yields and water table depth
(model performed no better than null model, ∆AICc = 2.00,
coefficient = 0.03 ± 0.12 t ha−1 year−1)
4
8
12
16
−50
−40
−30
−20
−10
Mean water table depth (cm)
Fresh fruit bunch yield
(t per ha per year)
Site 1
Site 2
Site 3
Oil palm site 4
8
12
16
−50
−40
−30
−20
−10
Mean water table depth (cm)
Fresh fruit bunch yield
(t per ha per year)
Site 1
Site 2
Site 3
Oil palm site between them within the range of drainage intensities we studied. g
0.23
0.23
0.09
0.08
0.08
0.08
0.04
0.04
0.03
0.03
0.02
0.01
0.01
0.01
0.01
0.00
0.54
0.25
0.08
0.04
0.04
0.02
0.01
0.01
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00 11 Journal of Applied Ecology WARREN-THOMAS et al. g
0.23
0.23
0.09
0.08
0.08
0.08
0.04
0.04
0.03
0.03
0.02
0.01
0.01
0.01
0.01
0.00
0.54
0.25
0.08
0.04
0.04
0.02
0.01
0.01
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00 Less heavily drained (i.e. wetter) farms had more complex ground
vegetation, but there was no detectable relationship between the
increased vegetation complexity on wetter farms and on-farm
avian diversity, and no relationship between water table depth
and oil palm yields. Power analysis indicated that our sample size
was sufficient to detect effects resulting in a model R2 (proportion
of explained variance) of 0.20 or above, but that we would have
been unable to detect small effects that explained less than 20% of
overall variation. Hence, we conclude that even with larger sample
sizes, while we may have had better statistical power to explain
small amounts of variation in response, the effect size would still
be very small, and our overall conclusion would qualitatively be the
same: that water tables had minimal influence on oil palm yields or
bird diversity on oil palm farms in our study system. Avian richness
and abundance were ~50% lower on farms than in neighbouring
forest, and farm bird community composition was characterised
by more generalist species, fewer invertebrate-feeding birds,
higher abundance of frugivores, nectarivores and granivores, and
species with smaller body masses. The forest fragment retained
large-bodied frugivorous bird species that are important for future
peatland restoration potential, and for retaining genetic diversity
and gene flow among isolated forest fragments. However, the for-
est fragment is vulnerable to drainage and fires in surrounding cul-
tivated land. FI G U R E 5 Oil palm yield response to water table depth, with
prediction and SE from general linear model (n = 39) showing
no relationship between reported yields and water table depth
(model performed no better than null model, ∆AICc = 2.00,
coefficient = 0.03 ± 0.12 t ha−1 year−1) (Aegithinidae, Campephagidae, Chloropseidae, Dicruridae and
Timaliidae) (Figure S13). Timaliidae) (Figure S13). (Aegithinidae, Campephagidae, Chloropseidae, Dicruridae and
Timaliidae) (Figure S13). 4.1 | Oil palm water tables, vegetation, yields and
bird diversity heterogeneity of oil palm management across farms in different
studies. For example, in Peninsular Malaysia, a positive effect of
understorey vegetation on bird richness was seen only in poly-
culture oil palm farms, not in monocultures (Azhar et al., 2014). In
our study landscape, the predominance of monocultural oil palm
may result in fewer bird species capable of responding to local
variation in understorey vegetation. While we sampled across
the full range of understorey vegetation complexity in the study
landscape (Figure S1) it is also possible that this range is limited
relative to other landscapes. An alternative explanation is that the
heterogeneity of farm management practices in a fine-grained
mosaic (most plots were 100 m × 200 m in size) meant that birds
were easily able to find relatively complex understorey vegetation
within the wider landscape, and so local plot-level effects were
not detectable. Our finding that understorey vegetation complexity was greater on
farms with shallower water tables could be driven by at least two
processes: increased soil moisture could favour peat-dependent her-
baceous plants that might otherwise be suppressed by unfavourable
dry soil conditions, or farmers could be reducing weed management
due to access difficulties on wetter peatland. Given the similarity
of plant species found across the 10 plots that had botanical sur-
veys (Table S1), an effect of farm management seems more likely. However, farms with both higher water tables and greater vegetation
complexity occurred at the same site (Site 1), that is, were spatially
autocorrelated. Therefore, there may be differences in farm man-
agement practices among sites that are not linked to water tables. For example, farmer access to inputs, capital or differences in land
tenure may affect management, and so our conclusions about the
response of on-farm vegetation to variation in water table depths
remain tentative. While we have focussed on birds, variation in local vegetation
complexity may influence other taxa, such as ants, termites, moths,
beetles, butterflies, mammals or reptiles that respond to environmen-
tal change at different spatial or temporal scales (Neoh et al., 2017;
Yong et al., 2016). For example, understorey vegetation affects
mammal occurrence in oil palm in Colombia (Pardo et al., 2019). Peat
swamp forests are also home to numerous fish species, including
local endemics (Posa et al., 2011), and we know little about their
responses to peat management. 4 | DISCUSSION points further apart are less similar in species composition); species
points are positioned in relation to plot scores, and represent the optimum position of each species in the NMDS space, and show that few
species used both habitats. Species habitat dependence categories are defined in the methods Bird species: (A) sample-based rarefaction in forest versus oil palm extrapolated to the largest sample size (oil FI G U R E 6 Bird species: (A) sample-based rarefaction in forest versus oil palm extrapolated to the largest sample size (oil palm, n = 40
farms/plots; FO = forest, OP = oil palm), (B) points show observed bird species richness per plot, error bar shows model prediction of
25.9 ± 2.0 species per plot in forest, reduced by a factor of 0.4 ± 2.1 in oil palm; (C) points show observed bird abundance per plot, error
bar shows model prediction of 48.2 ± 1.1 individuals per plot in forest, reduced by a factor of 0.6 ± 2.0 in oil palm; (D) NMDS ordination of
bird species composition across all plots, based on Bray–Curtis dissimilarity index and Wisconsin double standardisation (stress <0.01); plot
points (grey triangles or circles) are positioned in space such that the distances between points match the dissimilarity ranking of the plots
in multi-dimensional space according to Bray–Curtis dissimilarity (i.e. points further apart are less similar in species composition); species
points are positioned in relation to plot scores, and represent the optimum position of each species in the NMDS space, and show that few
species used both habitats. Species habitat dependence categories are defined in the methods 4 | DISCUSSION We found variation among farms in water table depths, oil palm
yields and avian biodiversity, but no evidence for any trade-offs WARREN-THOMAS et al. Journal of Applied Ecology 12 FI G U R E 6 Bird species: (A) sample-based rarefaction in forest versus oil palm extrapolated to the largest sample size (oil palm, n = 40
farms/plots; FO = forest, OP = oil palm), (B) points show observed bird species richness per plot, error bar shows model prediction of
25.9 ± 2.0 species per plot in forest, reduced by a factor of 0.4 ± 2.1 in oil palm; (C) points show observed bird abundance per plot, error
bar shows model prediction of 48.2 ± 1.1 individuals per plot in forest, reduced by a factor of 0.6 ± 2.0 in oil palm; (D) NMDS ordination of
bird species composition across all plots, based on Bray–Curtis dissimilarity index and Wisconsin double standardisation (stress <0.01); plot
points (grey triangles or circles) are positioned in space such that the distances between points match the dissimilarity ranking of the plots
in multi-dimensional space according to Bray–Curtis dissimilarity (i.e. points further apart are less similar in species composition); species
points are positioned in relation to plot scores, and represent the optimum position of each species in the NMDS space, and show that few
species used both habitats. Species habitat dependence categories are defined in the methods
(A)
(B)
(C)
(D) (A) (B) (B) (D) (C) (D) (C) FI G U R E 6 Bird species: (A) sample-based rarefaction in forest versus oil palm extrapolated to the largest sample size (oil palm, n = 40
farms/plots; FO = forest, OP = oil palm), (B) points show observed bird species richness per plot, error bar shows model prediction of
25.9 ± 2.0 species per plot in forest, reduced by a factor of 0.4 ± 2.1 in oil palm; (C) points show observed bird abundance per plot, error
bar shows model prediction of 48.2 ± 1.1 individuals per plot in forest, reduced by a factor of 0.6 ± 2.0 in oil palm; (D) NMDS ordination of
bird species composition across all plots, based on Bray–Curtis dissimilarity index and Wisconsin double standardisation (stress <0.01); plot
points (grey triangles or circles) are positioned in space such that the distances between points match the dissimilarity ranking of the plots
in multi-dimensional space according to Bray–Curtis dissimilarity (i.e. 4.2 | Peat swamp forest supports a distinct and
more diverse bird community than oil palm We found that bird species richness in the peat swamp forest frag-
ment was double that of surrounding smallholder oil palm farms,
with an almost completely distinct species composition, and at least
35 conservation-priority species. This finding highlights the conser-
vation value of large patches of forest in agricultural landscapes. Drainage of cultivated land affects neighbouring forests (e.g. up to
300 m away in the nearby Kampar Peninsula; Evans et al., 2019), and
in dry years (such as during ENSO events) fires in cultivated lands
have escaped into unusually dry, drainage-affected neighbouring
forest, as occurred in the western portion of our study forest frag-
ment in 2015. Raising water tables in surrounding oil palm farms may
therefore provide protective co-benefits for biodiversity by reduc-
ing the risk of future forest loss or degradation from fires. Our finding that peat swamp forests contained about twice the
avian biodiversity of oil palm smallholdings adds support to other
studies emphasising their importance for bird conservation (e.g. Azhar et al., 2011; Posa et al., 2011). Total bird species richness of
the Sungai Buluh forest fragment in our study (90 species) is simi-
lar to the total species richness recorded in nearby Berbak National
Park (88 species across primary, secondary and swamp forest hab-
itats; Darras et al., 2018). Another study of logged peat swamp for-
est in Peninsular Malaysia recorded 194 bird species over a 9-month
sampling period (Azhar et al., 2011), suggesting Sungai Buluh may
harbour greater bird diversity than we were able to record in our
relatively short survey. Our findings of greater avian body mass, and
reduced abundance of invertebrate-feeding birds, in forest plots
compared to oil palm reflects findings of other studies (Edwards
et al., 2013), although responses to land use can vary among land-
scapes (Prabowo et al., 2016). The increased abundance of frugiv-
orous and/or nectarivorous birds in oil palm may be explained by
the presence of relatively complex understorey vegetation on some
farms and small patches of uncultivated land between oil palm farms,
that could support flowering and fruiting understorey plants. This
information could be used to boost bird diversity on farms without
reducing yields. All sites surveyed were relatively shallowly drained in the year
of study, although there was an indication of a relatively dry pe-
riod leading to deeper drainage towards the end of the study. 4.1 | Oil palm water tables, vegetation, yields and
bird diversity Therefore, the impact of peatland The lack of detectable bird species richness and composition
responses to vegetation complexity contrasts with other research
emphasising the importance of understorey vegetation for en-
hanced bird richness in oil palm (e.g. Aratrakorn et al., 2006; Azhar
et al., 2011). This lack of consensus may be due to differences in WARREN-THOMAS et al. Journal of Applied Ecology 13 re-wetting on other taxa will differ from the responses of birds, and
our results cannot be generalised to all biodiversity. Comparisons of yield-drainage relationships between smallholders
and industrial scale oil palm would be very informative, especially
in the context of sustainability initiatives such as guidance from the
Roundtable on Sustainable Oil Palm. Comparisons of yield-drainage relationships between smallholders
and industrial scale oil palm would be very informative, especially
in the context of sustainability initiatives such as guidance from the
Roundtable on Sustainable Oil Palm. We found highly variable oil palm yields among farms, but
there was no evidence that drainage depth or vegetation complex-
ity accounted for this, and variation was not explained by oil palm
age, farm management or chemical and fertiliser applications. At
11 t ha−1 year−1, average reported yields in this landscape were below
regional averages (Indonesian smallholders 13 t ha−1 year−1, national
average 17 t ha−1 year−1, while best performing Malaysian industrial
estates yield up to 40 t ha−1 year−1; Hoffmann et al., 2014; Woittiez
et al., 2017). A wider study of smallholder oil palm farmers in Sumatra
also found that agronomy practices explained little variation in self-
reported yields, although lack of fertiliser reduced yields (although
effects of fertiliser can take 2 years to appear; Darras et al., 2019),
and that independent smallholders (as in this study) tend to have the
lowest yields (Lee et al., 2014). Retaining some ‘weed’ vegetation
cover on non-peat soils may reduce the risk of soil compaction and
erosion, enhance porosity and water infiltration, providing benefits
for oil palm production relative to weeding practices that clear all
vegetation (Darras et al., 2019). Similar benefits might be expected
for peat soils. However, these previous studies support our finding
that the presence of understorey vegetation has little relationship
with oil palm yields. 5 | CONCLUSIONS The loss of ~50% of bird species richness following conversion
of logged peat swamp forest to smallholder oil palm in this study is
similar to other studies reporting conversion of logged peat swamp
forest to smallholder or industrial oil palm in Peninsula Malaysia
(48%–60%; Azhar et al., 2011), non-peat rainforest to smallholder oil
palm in Jambi, Sumatra (43%–45%, Prabowo et al., 2016) and small-
holder oil palm in Thailand (60%; Aratrakorn et al., 2006). This sug-
gests that smallholder oil palm landscapes are not necessarily any
better than industrial oil palm estates for conserving bird diversity. Our finding that distance to the nearest continuous forest block
(0.13–3 km) had no effect on bird diversity in oil palm farms could
be explained by the presence of a paved road and wide canal sur-
rounding the forest, which could act as an impermeable barrier to
the movement of many forest birds into surrounding plantations
(Johnson et al., 2017). This fits with our finding that very few species
were shared between forest and oil palm. This concurs with existing
evidence that distance to large blocks of continuous forest had no
effect on bird diversity on oil palm smallholdings in Malaysia at dis-
tances of 8–50 km (Azhar et al., 2011), but contrasts with findings
from Ghana, where bird diversity on oil palm smallholdings increased
at proximities of 0.6 km compared to 9.6 km from contiguous for-
est (Hamer et al., 2021), and evidence that smaller forest patches
near oil palm smallholdings increased bird diversity at distances of
0.01–5 km (Azhar et al., 2011). This suggests that some bird dis-
persal from forest into oil palm may occur for several km, if land-
scapes are relatively permeable. Landscape permeability for bird
movement was not addressed directly in our study, and we did not
survey other land covers including Acacia, homegardens or patches
of scrub vegetation that could facilitate bird movement. However,
our findings that dispersal of forest birds into oil palm smallholdings
appears very limited in this landscape has important implications for
forest recovery or restoration at the landscape scale in the longer
term. We conclude that without interventions to improve landscape
permeability for birds, processes such as seed dispersal and gene
flow across the landscape may remain limited. 5 | CONCLUSIONS of agriculture (Wijedasa et al., 2018), while targets to reduce car-
bon emissions from Sumatra’s peatlands may also incentivise with-
drawal of oil palm in the longer term (Afriyanti et al., 2019). This
withdrawal of agriculture could result in opportunities for low-cost
forest restoration. However, successful restoration is on the as-
sumption that peat subsidence ceases, because regular inundation
of sites would make forest recovery impossible (Giesen, 2018). If
preserved, remaining forest fragments, such as Sungai Buluh in this
study, could act as reservoirs of peat swamp forest animals, plants
and their seed dispersers, including large-bodied frugivores with
long dispersal abilities such as hornbills, thus facilitating forest re-
covery (Wijedasa et al., 2020), and potentially providing gene flow
between peat-specialist plant and animal populations in isolated
forest fragments. 5 Declining oil palm yields could drive livelihood losses and further
deforestation, but we find that re-wetting peatlands to the levels
assessed in this study is unlikely to negatively impact yields. Hence,
we conclude from our study that re-wetting will conserve both for-
est biodiversity and livelihoods if fires are reduced. We found no
evidence for trade-offs between yields, water table depth and on-
farm bird diversity, and our power analysis indicates that missed ef-
fects are likely to be small. There are challenges in understanding
drivers of yield variation on heterogeneous smallholder farms, and
more studies are needed, but we conclude that re-wetting should
have net positive effects for smallholders by reducing the risk of
fires that can damage property, plantations and human health. It is
important that any prolonged flooding following re-wetting does not
negatively impact farm access or yield, given that oil palms can only
tolerate temporary flooding (palms can develop root pneumatodes,
but submerged roots cannot respire normally; Woittiez et al., 2017). The long-term success of canal blocking varies, and requires long-
term local support (Dohong et al., 2018), to mitigate any impacts
on farmer livelihoods (Ward et al., 2021). However, even the wet-
test farms still had water tables below the ground most of the time,
meaning carbon emissions and land subsidence continue. Full hydro-
logical restoration would involve sustained flooding (as observed in
the nearby forest) which is incompatible with oil palm production. 4.2 | Peat swamp forest supports a distinct and
more diverse bird community than oil palm So,
we cannot draw conclusions about the effect of deeper drainage
on yields or biodiversity. We analysed self-reported yields and data
from questionnaires rather than taking measurements over time,
due to logistical constraints, and were unable to account for the po-
tential time-lag between management or weather effects on palms
and changes in yield. A time-lag of 20–30 months has been reported
between stress factors impacting palms, and resulting variation in
yields (Woittiez et al., 2017), while fertiliser applications increase oil
palm root biomass but can take 2 years to impact fruit yields (Darras
et al., 2019). However, discussions with farmers indicated that water
table and farm management had not been substantially altered in the
months prior to data collection. The water table data collected in this study, and made publicly
available by this project, offers a good opportunity to monitor yield
outcomes in coming months, as part of wider restoration planning. This information will be vital for helping to close yield gaps, produce
oil palm to meet growing demands and improve smallholder liveli-
hoods, without increasing plantation area (Afriyanti et al., 2016). However, increased yields on farms may not result in avoided de-
forestation unless there is accompanying forest governance and
enforcement of protection, but higher yields are required if produc-
tion is to match increasing demand without further deforestation
(Woittiez et al., 2017). Our results highlight the huge variability in
smallholder yields, and the low productivity in the area. There is
also an urgent need for studies of industrial plantations, where man-
agement, oil palm age, water tables and yields are likely to be more
closely controlled, allowing potential trade-offs to be examined. Together, our findings emphasise the biodiversity value of re-
maining large peat swamp forest fragments for bird conservation
in Sumatra, and the importance of efforts to reconnect them with
other fragments, especially those recently isolated that are early
in the ‘relaxation’ stage of the extinction debt process (Wearn
et al., 2012). Conservation of bird species diversity and compo-
sition is also essential from the perspective of future peatland
landscape restoration efforts. Cultivated peatlands are subsiding,
and in coastal areas will become inundated, forcing a withdrawal WARREN-THOMAS et al. Journal of Applied Ecology 14 5 | CONCLUSIONS For example, findings
from the same province as our study indicate potential for the use
of ‘framework’ species, such as abandoned or unmanaged Acacia
plantations, to encourage forest recovery processes including seed
dispersal (Wijedasa et al., 2020). Nearly all farms in our study met the legislative requirement for
maintaining a mean water table depth of −40 cm. However, −40 cm
is considered a threshold below which surface peat can become dry
enough to combust (Page et al., 2009), meaning this threshold should
be considered a minimum, rather than mean, value above which the
risk of fires is minimised. We note that in the 2019 ENSO event most
farms dropped below −40 cm from July onwards, a pattern not found
in 2018. This inter-annual variation highlights the need for monitor-
ing and adaptive management of water tables during any re-wetting
scheme, to enable responses to long-term rainfall variation, espe-
cially that associated with ENSO events when severe droughts are
common. Further data on oil palm yield–water table relationships
are urgently needed, including from industrial plantations, to en-
able quantification of trade-offs between production, water tables,
carbon emissions and land subsidence, in order to develop more
sustainable agricultural practices. In the long term, withdrawal of
drainage-dependent agriculture from peatland is necessary to avoid
carbon emissions and land loss to subsidence (Wijedasa et al., 2017),
with forest restoration or flood-tolerant agriculture the sustainable
alternatives (Tan et al., 2021). Our findings highlight that peat forest protection in these land-
scapes is vital for conserving biodiversity, because we find no ev-
idence that re-wetting of peatland oil palm improves on-farm bird
diversity, and only 3/35 conservation-priority bird species occurred
on farms. Recent research indicates that drainage impacts extend
300 m into forest from a plantation boundary (Evans et al., 2019),
highlighting the fundamental connections between management
of drained cultivated peatlands and forest conservation. Thus, WARREN-THOMAS et al. Journal of Applied Ecology 15 Bambang Hariyadi
https://orcid.org/0000-0001-8492-2974
Jenny A. Hodgson
https://orcid.org/0000-0003-2297-3631
Mailys Lopes
https://orcid.org/0000-0002-3804-7950
Jennifer M. Lucey
https://orcid.org/0000-0001-5224-091X
Nathalie Pettorelli
https://orcid.org/0000-0002-1594-6208
Asmadi Saad
https://orcid.org/0000-0002-2761-3072
Lindsay C. Stringer
https://orcid.org/0000-0003-0017-1654
Caroline Ward
https://orcid.org/0000-0001-8362-4713
Jane K. Hill
https://orcid.org/0000-0003-1871-7715 management practices to reduce on-farm fires in peatland oil palm,
including re-wetting, are crucial to protect forests from escaped
fires. ACKNOWLEDGEMENTS Research funding was provided by NERC/The Newton Fund, grant
number NE/P014658/1. E.W.-T. was also supported by NERC-
IIASA Fellowship NE/T009306/1. J.L. was supported by NERC
Knowledge Exchange Fellowship NE/M006840/1-2. J.A.H. was
supported by NERC Innovation grant NE/R009597/1. Thanks to
Phillip Platts and the Global LAI Project for guiding LAI/canopy
cover methodology, and for valuable help image processing. We
also thank Lasmito for providing plant identification expertise,
who did not wish to be a co-author of this work. This work would
not have been possible without the work of Aan, Atok, Agus and
Udin who arranged introductions to local farmer groups, guided
the Indonesian-UK research team around the study landscape,
and accurately and diligently conducted water table monitor-
ing. All authors extend heartfelt thanks to the communities who
hosted the research team in Jambi, agreed to participate in the
study, and gave time to join us and share their ideas at a summary
workshop at the end of the research. 5 | CONCLUSIONS Given the halving of bird diversity in smallholder oil palms rel-
ative to forest, restoration of more biodiversity-friendly land covers
that could improve landscape permeability, and improve connec-
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2-Hydroxyoleic Acid Induces ER Stress and Autophagy in Various Human Glioma Cell Lines
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PloS one
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Abstract Background: 2-Hydroxyoleic acid is a synthetic fatty acid with potent anti-cancer activity which does not induce undesired
side effects. However, the molecular and cellular mechanisms by which this compound selectively kills human glioma
cancer cells without killing normal cells is not fully understood. The present study was designed to determine the molecular
bases underlying the potency against 1321N1, SF-767 and U118 human glioma cell lines growth without affecting non
cancer MRC-5 cells. Methodology/Principal Findings: The cellular levels of endoplasmic reticulum (ER) stress, unfolded protein response (UPR)
and autophagy markers were determined by quantitative RT-PCR and immunoblotting on 1321N1, SF-767 and U118 human
glioma cells and non-tumor MRC-5 cells incubated in the presence or absence of 2OHOA or the ER stress/autophagy
inducer, palmitate. The cellular response to these agents was evaluated by fluorescence microscopy, electron microscopy
and flow cytometry. We have observed that 2OHOA treatments induced augments in the expression of important ER stress/
UPR markers, such as phosphorylated eIF2a, IRE1a, CHOP, ATF4 and the spliced form of XBP1 in human glioma cells. Concomitantly, 2OHOA led to the arrest of 1321N1 cells in the G2/M phase of the cell cycle, with down-regulation of cyclin
B1 and Cdk1/Cdc2 proteins in the three glioma cell lines studied. Finally, 2OHOA induced autophagy in 1321N1, SF-767 and
U118 cells, with the appearance of autophagic vesicles and the up-regulation of LC3BI, LC3BII and ATG7 in 1321N1 cells,
increases of LC3BI, LC3BII and ATG5 in SF-767 cells and up-regulation of LC3BI and LC3BII in U118 cells. Importantly, 2OHOA
failed to induce such changes in non-tumor MRC-5 cells. Conclusion/Significance: The present results demonstrate that 2OHOA induces ER stress/UPR and autophagy in human
glioma (1321N1, SF-767 and U118 cell lines) but not normal (MRC-5) cells, unraveling the molecular bases underlying the
efficacy and lack of toxicity of this compound. Citation: Marcilla-Etxenike A, Martı´n ML, Noguera-Salva` MA, Garcı´a-Verdugo JM, Soriano-Navarro M, et al. (2012) 2-Hydroxyoleic Acid Induces ER Stress and
Autophagy in Various Human Glioma Cell Lines. PLoS ONE 7(10): e48235. doi:10.1371/journal.pone.0048235 Editor: Nai Sum Wong, University of Hong Kong, Hong Kong Received January 30, 2012; Accepted September 28, 2012; Published October 25, 2012 Received January 30, 2012; Accepted September 28, 2012; Pub Copyright: 2012 Marcilla-Etxenike et al. Abstract 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. Copyright: 2012 Marcilla-Etxenike et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by fellowships from The University of the Balearic Islands and the Programa de Formacio´n de Profesorado Universitario
(FPU), Ministerio de Educacio´n, Gobierno de Espan˜a. Grants [BFU2007-61071, BIO2010-21132] from the Ministerio de Educacio´n y Ciencia, Spain, and by the
Marathon Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. he author ID is employed by the company LMBRI LLC. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing Competing Interests: The author ID is employed by the company LMBRI LLC. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing
data and materials. * E-mail: xavier.busquets@uib.es 2-Hydroxyoleic Acid Induces ER Stress and Autophagy in
Various Human Glioma Cell Lines Amaia Marcilla-Etxenike1, Maria Laura Martı´n1, Maria Anto` nia Noguera-Salva` 1, Jose´ Manuel Garcı´a-
Verdugo2,3, Mario Soriano-Navarro3, Indranil Dey4, Pablo V. Escriba´ 1, Xavier Busquets1* 1 Departmento de Biologı´a - Instituto Universitario de Investigacio´n en Ciencias de la Salud, Universidad de las Islas Baleares, Palma de Mallorca, Spain, 2 Instituto
Cavanilles, Universidad de Valencia, Valencia, Spain, 3 Unidad Mixta Centro de Investigacio´n Prı´ncipe Felipe - Universidad de Valencia, Centro de Investigacio´n Biome´dica
en Red sobre Enfermedades Neurodegenerativas, Valencia, Spain, 4 Lipopharma Inc, Acton, Massachusetts, United States of America October 2012 | Volume 7 | Issue 10 | e48235 ation: Marcilla-Etxenike A, Martı´n ML, Noguera-Salva` MA, Garcı´a-Verdugo JM, Soriano-Navarro M, et al. (2012) 2-Hydroxyoleic Acid
tophagy in Various Human Glioma Cell Lines. PLoS ONE 7(10): e48235. doi:10.1371/journal.pone.0048235 2OHOA activates ER stress/UPR signaling pathways in
1321N1, SF-767 and U118 but not MRC-5 cells To determine whether inhibition of 1321N1, SF-767 and U118
cell growth by 2OHOA was mediated by ER stress/UPR
signaling, we examined the expression of key molecules in the
three main signal transduction cascades activated by ER stress/
UPR. Treatment of 1321N1, SF-767 and U118 cells with either
2OHOA or palmitate (150 mM; 12 h) significantly increased the
P-eIF2a protein levels, while a similar increase in P-eIF2a protein
was only produced by palmitate in MRC-5 cells (Fig. 3 A, B, C
and D). Thus, the effects of 2OHOA on P-eIF2a accumulation
appeared to be specific to glioma cells. Phosphorylated eIF2a
attenuates general protein translation and selectively activated
transcription and translation of the ATF4 transcription factor [13]. Both 2OHOA and palmitate (150 mM; 24 h) induced a significant
increase in ATF4 gene expression in 1321N1 cells, further
demonstrating the specificity of 2OHOA against glioma cells
(Fig. 4 A). The ER stress is the starting point from which autophagy or
apoptosis can be induced. Activation of ER stress and autophagy
represents thought a promising therapeutic strategy to treat cancer
[22]. As such, we investigated the roles of ER stress and autophagy
in the anticancer effects of 2OHOA against human glioma, the
most common type of primary tumor in the CNS with one of the
highest mortality rates of all cancers [23]. Activation of IRE1a resulted in an increase in the expression of
the XBP1 transcription factor [24,25], and 2OHOA and palmitate
(150 mM; 24 h and 48 h) markedly up-regulated IRE1a protein
levels in 1321N1, SF-767 and U118 cells (Fig. 3 F, G and H) and
modestly up-regulated mRNA levels in 1321N1 astrocytoma cells
(Fig 4 B). By contrast, the same treatments produced only a mild
increase in IRE1a protein expression in MRC-5 cells (Fig. 3 E). The mRNA transcripts of the spliced activated form of the X-box
binding protein 1 gene (sXBP1), a downstream target of ATF6 and
IRE1a augmented in 1321N1 cell line after 2OHOA treatment
(150 mM; 24 h) (Fig. 4 C). These observations indicate that
2OHOA activates the UPR signaling in all cell lines, although
more weakly in the non-cancerous MRC-5 cells. We found that treatment of 1321N1, SF-767 and U118 cells
with 2OHOA provoked effects that included: the induction of ER
stress-related genes; cell cycle arrest through the accumulation of
cells in the G2/M phase and autophagic cell death. 2OHOA impairs cell proliferation and viability in 1321N1,
SF-767 and U118 human glioma cells There are three main pathways that mediate UPR signaling: the
inositol-requiring enzyme 1 (IRE1) pathway; the eukaryotic
translation initiation factor 2a kinase 3 (PERK) pathway; and
the activating transcription factor 6 (ATF6) pathway [7]. Key
proteins in these pathways include IRE1a (involved in the
regulation
of
apoptosis
and
the
differentiation/proliferation
MAPK-dependent pathways) and its ribonuclease product XBP1
(a transcription factor that induces the expression of genes
involved in restoring protein folding or degrading unfolded
proteins) [11]. Together with XBP1, ATF4 and ATF6 regulate
the expression of the C/EBP homologous protein (CHOP), one of
the main effectors of ER stress/UPR-induced apoptosis [12]. Another important element is PERK, whose intrinsic kinase
activity is induced by oligomerization, resulting in the phosphor-
ylation of the eukaryotic translation initiation factor 2a (eIF2a)
and the suppression of global mRNA translation. Under these
conditions, only selected mRNAs are translated, including ATF4
[13], which induces the expression of genes involved in the
restoration of ER homeostasis and in autophagy [13,14,15]. Accordingly, compounds that promote the sustained phosphory-
lation of eIF2a, such as salubrinal [16], may exert cytoprotective
effects. However, prolonged suppression of protein synthesis is
incompatible with cell survival, resulting in autophagy [11], and
thus eIF2a phosphorylation and ATF4 both stimulate the
expression of genes associated with autophagy [13,14]. In order to evaluate cell proliferation in the different cell lines
after the treatment with 2OHOA or palmitate, we performed the
MTT assay based on the mitochondrial function (succinate
dehydrogenase
activity). We
observed
that
2OHOA
(50–
1000 mM, 24–72 h) had modest effects on the cell proliferation
of non-cancer human fibroblast MRC-5 cells (Fig. 1 A), while
palmitate, a potent inducer of ER stress that was used as a positive
control [10] significantly impaired MRC-5 cell proliferation (Fig.1
B). By contrast, 2OHOA and palmitate, inhibited the proliferation
of 1321N1 human astrocytoma cells (Fig. 1 C and D), SF-767
(Fig. 1 E and F) and U118 (Fig. 1 G and H) human glioma cells,
demonstrating that only 2OHOA but not palmitate was specific
against these glioma cell lines. To further analyze cell viability we also used the Trypan Blue
Exclusion method, and it was observed that 2OHOA (50–
1000 mM, 24–72 h) had modest effects on the cell viability and
proliferation of non-cancer human fibroblast MRC-5 cells, with
the exception of the highest dose of 1000 mM (Fig. 2 A–C). 2OHOA impairs cell proliferation and viability in 1321N1,
SF-767 and U118 human glioma cells It
cannot be ruled out however, that 2OHOA can also kill MRC-5
non cancer cells in a dose-dependent manner albeit at higher
killing concentration. By contrast, 2OHOA (50–1000 mM, 24–
72 h) inhibited the proliferation and increased cell death in a time
and dose-dependent manner in 1321N1 (Fig. 2 D–F), SF-767
(Fig. 2 G–I) and U118 (Fig. 2 J–L) human glioma cells. Autophagy is a cellular process that mediates the recycling of
cytoplasmic macromolecules and structures through the formation
of membrane double bounded vacuoles, called autophagosomes,
that engulf and degrade large portions of cells [17,18]. Autophagy
has also been associated with the induction of non-apoptotic cell
death [11]. The accumulation of misfolded protein aggregates in
the ER that cannot be degraded by the proteosome results in the
upregulation of the UPR and the expression of autophagy-related
genes [14,19]. Although both the UPR and autophagy can
function independently, recent studies have shown that these
processes may be linked and share a common function, exerting
either cytoprotective (under basal or metabolic stress conditions) or
cytocidal effects (after acute cellular damage) [20,21]. Introduction last cellular and molecular events that cause the cancer cell death
still remain unclear. In the present study, we provide evidence of
the molecular mechanisms underlying the death of various human
glioma cell lines, which explains not only the efficacy of this
compound against cancer cells but also its safety based on a lack of
action against normal cells. 2-Hydroxyoleic
acid
(2OHOA,
Minerval),
the
a-hydroxy
derivative of oleic acid, binds to the plasma membrane and alters
the organization of its lipids [1], increasing the propensity to form
non-lamellar (hexagonal HII) lipid phases [1,2,3]. Interestingly,
this modification inhibits the growth of lung cancer (A549) cells
and it induces apoptosis in human leukemia (Jurkat) cells [2,4,5]. The changes 2OHOA produces to the structure of the membrane
influences the location and activity of amphitropic membrane
proteins that are involved in proliferation/differentiation signaling
[1,2,3], eventually leading to the down-regulation of E2F-1 and
dihydrofolate reductase (DHFR), both pivotal proteins in cancer
cell proliferation [4,6]. In this context, although the first steps in
the anticancer mechanism of action of 2OHOA are known, the In a cell, the endoplasmic reticulum (ER) fulfills three main
functions: 1) protein folding, glycosylation and sorting; 2) synthesis
of cholesterol and other lipids; and 3) maintenance of Ca2+
homeostasis [7]. Disrupting any of these processes causes ER stress
and activates the unfolded protein response (UPR) [7], which can
be achieved with a number of cytotoxic agents, such as brefeldin A
[8], tunicamycin [9] or the fatty acid palmitate [10]. The
molecular elements associated with UPR up-regulate genes that October 2012 | Volume 7 | Issue 10 | e48235 1 October 2012 | Volume 7 | Issue 10 | e48235 PLOS ONE | www.plosone.org 2OHOA Induces UPR and Autophagy in Glioma Cells support recovery from ER stress or that initiate apoptosis in cases
of severe cell damage [7]. 2OHOA activates ER stress/UPR signaling pathways in
1321N1, SF-767 and U118 but not MRC-5 cells By contrast,
2OHOA treatment of non-cancer MRC-5 human fibroblast cells
failed to induce these key mediators of ER stress, cell growth arrest
and autophagy. These findings partly explain the specificity of
2OHOA against glioma cells and the lack of undesired toxic effects
when animals are treated with this compound [4]. In addition, this
novel therapeutic approach may constitute an innovative treat-
ment for gliomas with very high mortality rates, based on the
specific induction of ER stress and autophagy. We then studied the so-called ATF6 branch of the UPR
signaling pathway, which was activated by palmitate (150 mM;
24 h) in 1321N1 cells, provoking a significant up-regulation of
ATF6 mRNA expression (Fig 4 D). In addition, 2OHOA PLOS ONE | www.plosone.org October 2012 | Volume 7 | Issue 10 | e48235 2 2OHOA Induces UPR and Autophagy in Glioma Cells Figure 1. Effects of 2OHOA and palmitate on the proliferation of MRC-5 (A, B), 1321N1 (C, D), SF-767 (E, F) and U118 (
Human glioma (1321N1, SF-767 and U118) cells and fibroblasts (MRC-5) were exposed to increasing doses (50–1000 mM) of 2OHOA or p
different periods of time (24 h, 48 h or 72 h), and cell viability was determined using the MTT method. A. Treatments with 2OHOA did
MRC-5 cell growth below 50% at the highest incubations concentrations and times, so that IC50 value could not be determined. B. By c
IC50 values for palmitate in MRC-5 cells were: 24 h, 200 mM; 48 h, 150 mM and 72 h, 120 mM (n = 6). C. The IC50 values for 2OHOA in 1
were: 24 h, 250 mM; 48 h, 150 mM and 72 h, 100 mM (n = 6). D. The IC50 values for palmitate in 1321N1 cells were: 24 h, 160 mM; 48 h,
2OHOA Induces UPR and Autophagy in G Figure 1. Effects of 2OHOA and palmitate on the proliferation of MRC-5 (A, B), 1321N1 (C, D), SF-767 (E, F) and U118 (G, H) cells. Human glioma (1321N1, SF-767 and U118) cells and fibroblasts (MRC-5) were exposed to increasing doses (50–1000 mM) of 2OHOA or palmitate for
different periods of time (24 h, 48 h or 72 h), and cell viability was determined using the MTT method. A. Treatments with 2OHOA did not inhibit
MRC-5 cell growth below 50% at the highest incubations concentrations and times, so that IC50 value could not be determined. B. 2OHOA activates ER stress/UPR signaling pathways in
1321N1, SF-767 and U118 but not MRC-5 cells 3 treatment (150 mM; 24 h) also increased significantly ATF6
mRNA expression in human glioma (1321N1) cells (Fig 4 D). J–L) and it also increased at mRNA levels in 1321N1 astrocytoma
cells (Fig 4 E). While palmitate administration also increased
CHOP protein expression in MRC-5 cells, 2OHOA did not have
such effect (Fig. 3 I). Together these findings demonstrate the
differential effect of 2OHOA in these glioma cells versus MRC-5
normal human fibroblasts, selectivity not evident with palmitate,
which induced ER stress in both normal and glioma cells. In situations of chronic ER stress, the P-eIF2a, IRE1a and
ATF6 signaling pathways induce the transcription and translation
of the proapoptotic factor CHOP. In response to treatment with
2OHOA or palmitate (150 mM) CHOP expression increased in
1321N1, SF-767 and U118 cells, at the protein level (48 h, Fig. 3 Figure 2. 2OHOA effects on cell viability in 1321N1, SF-767 and U118 human glioma cells and MRC-5 human fibroblasts (Trypan
blue exclusion method). Glioma and MRC-5 non-tumor cell viability. 1321N1, SF-767 and U118 human glioma cells and MRC-5 human fibroblasts
were exposed to increasing doses (50–1000 mM) of 2OHOA for different periods of time (24 h, 48 h or 72 h). Total number of live and dead MRC-5
cells treated with 2OHOA 24 h (A), 48 h (B), and 72 h (C), Total number of live and dead 1321N1 cells treated with 2OHOA 24 h (D), 48 h (E) and 72 h
(F). Total number of live and dead SF-767 cells treated with 2OHOA 24 h (G), 48 h (H) and 72 h (I). Total number of live and dead U118 cells treated
with 2OHOA 24 h (J), 48 h (K) and 72 h (L). The number of cells presented in the graphs is the total number of cells per well (9.6 cm2). Cells were
plated at 50% confluence at the following densities: 26104 cells/cm2 (1.866105 cells/well) for MRC-5 cells; 66104 cells/cm2 (66105 cells/well) for
1321N1cells and 36104 cells/cm2 (36105 cells/well) for SF-767 and U118 cells. After 72 h confluence was reached. (*p,0.05, **p,0.01, ***p,0.001;
n = 3). doi:10.1371/journal.pone.0048235.g002 Figure 2. 2OHOA effects on cell viability in 1321N1, SF-767 and U118 human glioma cells and MRC-5 human fibroblasts (Trypan
blue exclusion method). Glioma and MRC-5 non-tumor cell viability. 2OHOA activates ER stress/UPR signaling pathways in
1321N1, SF-767 and U118 but not MRC-5 cells By contrast, the
IC50 values for palmitate in MRC-5 cells were: 24 h, 200 mM; 48 h, 150 mM and 72 h, 120 mM (n = 6). C. The IC50 values for 2OHOA in 1321N1 cells
were: 24 h, 250 mM; 48 h, 150 mM and 72 h, 100 mM (n = 6). D. The IC50 values for palmitate in 1321N1 cells were: 24 h, 160 mM; 48 h, 200 mM and October 2012 | Volume 7 | Issue 10 | e48235 PLOS ONE | www.plosone.org 3 2OHOA Induces UPR and Autophagy in Glioma Cells 2OHOA Induces UPR and Autophagy in Glioma Cells 72 h, 160 mM (n = 6). E. The IC50 values for 2OHOA in SF-767 cells were: 24 h, 600 mM; 48 h, 350 mM and 72 h, 200 mM (n = 6). F. The IC50 values for
palmitate in SF-767 cells were: 24 h, 160 mM; 48 h, 120 mM and 72 h, 110 mM (n = 6). G. The IC50 values for 2OHOA in U118 cellswere: 24 h, 150 mM;
48 h, 265 mM and 72 h, 260 mM (n = 6). H. The IC50 values for palmitate in U118 cells were: 24 h, 250 mM; 48 h, 175 mM and 72 h, 150 mM (n = 6). doi:10.1371/journal.pone.0048235.g001 72 h, 160 mM (n = 6). E. The IC50 values for 2OHOA in SF-767 cells were: 24 h, 600 mM; 48 h, 350 mM and 72 h, 200 mM (n = 6). F. The IC50 values for
palmitate in SF-767 cells were: 24 h, 160 mM; 48 h, 120 mM and 72 h, 110 mM (n = 6). G. The IC50 values for 2OHOA in U118 cellswere: 24 h, 150 mM;
48 h, 265 mM and 72 h, 260 mM (n = 6). H. The IC50 values for palmitate in U118 cells were: 24 h, 250 mM; 48 h, 175 mM and 72 h, 150 mM (n = 6). doi:10.1371/journal.pone.0048235.g001 treatment (150 mM; 24 h) also increased significantly ATF6
mRNA expression in human glioma (1321N1) cells (Fig 4 D). In situations of chronic ER stress, the P-eIF2a, IRE1a and
ATF6 signaling pathways induce the transcription and translation
of the proapoptotic factor CHOP. In response to treatment with
2OHOA or palmitate (150 mM) CHOP expression increased in
1321N1, SF-767 and U118 cells, at the protein level (48 h, Fig. 2OHOA activates ER stress/UPR signaling pathways in
1321N1, SF-767 and U118 but not MRC-5 cells 1321N1, SF-767 and U118 human glioma cells and MRC-5 human fibroblasts
were exposed to increasing doses (50–1000 mM) of 2OHOA for different periods of time (24 h, 48 h or 72 h). Total number of live and dead MRC-5
cells treated with 2OHOA 24 h (A), 48 h (B), and 72 h (C), Total number of live and dead 1321N1 cells treated with 2OHOA 24 h (D), 48 h (E) and 72 h
(F). Total number of live and dead SF-767 cells treated with 2OHOA 24 h (G), 48 h (H) and 72 h (I). Total number of live and dead U118 cells treated
with 2OHOA 24 h (J), 48 h (K) and 72 h (L). The number of cells presented in the graphs is the total number of cells per well (9.6 cm2). Cells were
plated at 50% confluence at the following densities: 26104 cells/cm2 (1.866105 cells/well) for MRC-5 cells; 66104 cells/cm2 (66105 cells/well) for
1321N1cells and 36104 cells/cm2 (36105 cells/well) for SF-767 and U118 cells. After 72 h confluence was reached. (*p,0.05, **p,0.01, ***p,0.001;
n = 3). doi:10.1371/journal.pone.0048235.g002 an glioma cells and MRC-5 human fibroblasts (Trypa October 2012 | Volume 7 | Issue 10 | e48235 PLOS ONE | www.plosone.org 4 2OHOA Induces UPR and Autophagy in Glioma Cells Figure 3. 2OHOA activation of ER stress/UPR signaling pathways in 1321N1, SF-767 and U118 but not in MRC-5 cells. P-eIF2a, IRE1a
and CHOP protein levels in 1321N1, SF-767 and U118 human glioma cells and in non-cancer MRC-5 human fibroblast cells determined by
immunoblotting. Upper panels: a representative immunoblot showing P-eIF2a, IRE1a or CHOP and Tubulin levels in each cell line after exposure to
2OHOA (H) or palmitate (P: 150 mM). Lower panels: Bar diagram showing the mean6SEM P-eIF2a, IRE1a or CHOP expression in each cell line after
exposure to 2OHOA (H) or palmitate (P) (150 mM) compared to untreated controls (C). A. P-eIF2a expression in MRC-5 cell line B. P-eIF2a expression
in 1321N1 cell line C. P-eIF2a expression in SF-767 cell line D. P-eIF2a expression in U118 cell line after exposure to 2OHOA (H) or palmitate (P)
(150 mM; 12 h). E. IRE1a expression in MRC-5 cell line F. IRE1a expression in 1321N1 cell line G. IRE1a expression in SF-767 cell line H. IRE1a
expression in U118 cell line after exposure to 2OHOA (H) or palmitate (P) (150 mM; 48 h). I. CHOP expression in MRC-5 cell line J. 2OHOA induces Cell Cycle arrest in 1321N1, SF-767 and
U118 but not of MRC-5 cells 2OHOA induces Cell Cycle arrest in 1321N1, SF-767 and
U118 but not of MRC-5 cells induced significant decreases in the expression of both cyclin B1
(Fig. 6 B–D) and Cdk1/Cdc2 (Fig. 6 F–H), indicative of cell cycle
arrest in the G2/M phase. In contrast, this did not occur in MRC-
5 cells (Fig. 6 A and E). The proportion of cells in the different phases of the cell cycle
was evaluated by measuring the intracellular DNA content after
exposure to 2OHOA and palmitate (150 mM; 72 h). Cell cycle
progression and growth of human MRC-5 fibroblast cells was not
affected by exposure to 2OHOA (percentage of cells in the G2/M
phase: Control, 29.8263.67%; 2OHOA, 27.0960.20%; palmi-
tate, 28.2760.71%; *p,0.05. Fig. 5 A–C). By contrast, 2OHOA
treatment inhibited 1321N1 cell proliferation, and increased the
proportion of cells in the G2/M phase when compared to
untreated
controls
(Control,
19.1362.84%;
2OHOA,
32.71*61.97%; palmitate, 28.50*68.23%; *p,0.05. Fig. 5 D–
F). Indeed, 2OHOA treatment in 1321N1, SF-767 and U118 2OHOA induces autophagy in 1321N1, SF-767 and U118
but not in MRC-5 cells 2OHOA induces autophagy in 1321N1, SF-767 and U118
but not in MRC-5 cells 2OHOA activates ER stress/UPR signaling pathways in
1321N1, SF-767 and U118 but not MRC-5 cells CHOP expression in
1321N1 cell line K. CHOP expression in SF-767 cell line L. CHOP expression in U118 cell line after exposure to 2OHOA (H) or palmitate (P) (150 mM;
48 h) (*p,0.05, **p,0.01, ***p,0.001; n = 6). doi:10.1371/journal.pone.0048235.g003 Figure 3. 2OHOA activation of ER stress/UPR signaling pathways in 1321N1, SF-767 and U118 but not in MRC-5 cells. P-eIF2a, IRE1a
and CHOP protein levels in 1321N1, SF-767 and U118 human glioma cells and in non-cancer MRC-5 human fibroblast cells determined by
immunoblotting. Upper panels: a representative immunoblot showing P-eIF2a, IRE1a or CHOP and Tubulin levels in each cell line after exposure to
2OHOA (H) or palmitate (P: 150 mM). Lower panels: Bar diagram showing the mean6SEM P-eIF2a, IRE1a or CHOP expression in each cell line after
exposure to 2OHOA (H) or palmitate (P) (150 mM) compared to untreated controls (C). A. P-eIF2a expression in MRC-5 cell line B. P-eIF2a expression
in 1321N1 cell line C. P-eIF2a expression in SF-767 cell line D. P-eIF2a expression in U118 cell line after exposure to 2OHOA (H) or palmitate (P)
(150 mM; 12 h). E. IRE1a expression in MRC-5 cell line F. IRE1a expression in 1321N1 cell line G. IRE1a expression in SF-767 cell line H. IRE1a
expression in U118 cell line after exposure to 2OHOA (H) or palmitate (P) (150 mM; 48 h). I. CHOP expression in MRC-5 cell line J. CHOP expression in
1321N1 cell line K. CHOP expression in SF-767 cell line L. CHOP expression in U118 cell line after exposure to 2OHOA (H) or palmitate (P) (150 mM;
48 h) (*p,0.05, **p,0.01, ***p,0.001; n = 6). doi:10.1371/journal.pone.0048235.g003 2OHOA induces autophagy in 1321N1, SF-767 and U118
but not in MRC-5 cells Some features of apoptosis, not observed in MRC-5 cells (Fig.7
A–B), appear to be induced in human astrocytoma (1321N1) cells
upon exposure to 2OHOA, such as the flow cytometry sub-G0
peak, poly ADP ribose polymerase (PARP) (Fig. 7 C) or caspase 8
partial proteolysis (Fig. 7 D), the latter also observed in U118 cells
after treatment with 2OHOA (Fig. 7 H). However, this induction
of apoptotic features did not fully explain the cell death induced by PLOS ONE | www.plosone.org October 2012 | Volume 7 | Issue 10 | e48235 5 Figure 4. Relative mRNA levels of ER stress/UPR transcripts. q RT-PCR analysis of the mRNA expression of ATF4 (A), ; IRE1a (B); spliced form of
XBP1 (C) ; ATF6 (D) and CHOP (E) genes in 1321N1 human astrocytoma cells after treatment with 2OHOA (H) or palmitate (P) (150 mM; 24 h). Results
are expressed as ddCt values using the following formula: ddCt = E X(Ctc-Ctx)/E Bact(Ctc-Ctx). (*P,0.05; n = 6) in a bar diagram showing the
mean6SEM (standard error of the mean). doi:10.1371/journal.pone.0048235.g004
2OHOA Induces UPR and Autophagy in Glioma Cells 2OHOA Induces UPR and Autophagy in Glioma Cells Figure 4. Relative mRNA levels of ER stress/UPR transcripts. q RT-PCR analysis of the mRNA expression of ATF4 (A), ; IRE1a (B); spliced form
XBP1 (C) ; ATF6 (D) and CHOP (E) genes in 1321N1 human astrocytoma cells after treatment with 2OHOA (H) or palmitate (P) (150 mM; 24 h). Res
are expressed as ddCt values using the following formula: ddCt = E X(Ctc-Ctx)/E Bact(Ctc-Ctx). (*P,0.05; n = 6) in a bar diagram showing
mean6SEM (standard error of the mean) Figure 4. Relative mRNA levels of ER stress/UPR transcripts. q RT-PCR analysis of the mRNA expression of ATF4 (A), ; IRE1a (B); spliced form of
XBP1 (C) ; ATF6 (D) and CHOP (E) genes in 1321N1 human astrocytoma cells after treatment with 2OHOA (H) or palmitate (P) (150 mM; 24 h). Results
are expressed as ddCt values using the following formula: ddCt = E X(Ctc-Ctx)/E Bact(Ctc-Ctx). (*P,0.05; n = 6) in a bar diagram showing the
mean6SEM (standard error of the mean). doi:10.1371/journal.pone.0048235.g004 integrated fluorescence density of the lysosomes in MRC-5 cells
(56104 cells per experiment) was as follows: Untreated control
11.5463.36%;
2OHOA
(150 mM)
16.8862.45%;
palmitate
(150 mM) 100*63.65%; *p,0.05 (Fig. 8 D). 2OHOA induces autophagy in 1321N1, SF-767 and U118
but not in MRC-5 cells 2OHOA in 1321N1, SF-767 and U118 glioma cells as we did not
observed PARP degradation induction in SF-767 and U118 cells
treated with 2OHOA (Fig. 7 E and G), as well as Caspase 8
proteolysis in SF-767 cells (Fig. 7 E). As 2OHOA induces tumor
regression and cancer cell death [6], we also assessed the role of
autophagy in the induction of cell death by 2OHOA. Acidic
vesicles (lysosomes and autophagosomes characteristic of autoph-
agy) were not observed in non-tumor MRC-5 cells treated with
vehicle or 2OHOA (150 mM; 48 h, Figs. 8 A and B), whereas
exposure to palmitate (150 mM; 48 h) induced the formation of
acidic autophagic vesicles in these cells (Fig. 8 C) The relative Both 2OHOA and palmitate (150 mM; 48 h) induced a marked
increase in the generation of lysosome/autophagosome vesicles in
human astrocytoma (1321N1) cells (Fig. 8 F, G) compared to
untreated cells (Fig. 8 E), in which the relative integrated
fluorescence density of the lysosomes was: Untreated control
3.160.37%; 2OHOA (150 mM) 81**66.18%; palmitate (150 mM)
100**66.12%; **p,0.01 (Fig. 8H). Thus, 2OHOA specifically October 2012 | Volume 7 | Issue 10 | e48235 PLOS ONE | www.plosone.org 6 2OHOA Induces UPR and Autophagy in Glioma Cells Figure 5. 2OHOA induction of G2/M cell cycle arrest of 1321N1 cells but not of MRC-5 cells. Cell cycle assessment and G2/M phase arrest. Analysis of the DNA content (flow cytometry) of MRC-5 and 1321N1 cells exposed to 2OHOA or palmitate (150 mM for 72 hours). A. Analysis of the
DNA content in untreated MRC-5 cells. B. Analysis of the DNA content in MRC-5 cells exposed to 2OHOA (150 mM for 72 h) or (C) palmitate (150 mM
for 72 hours), showing the proportion of cells in Sub G0 and G2/M phases. D. Analysis of the DNA content of untreated 1321N1 cells. E. Analysis of the
DNA content of 1321N1 cells exposed to 2OHOA (150 mM for 72 h) or (F) palmitate (150 mM for 72 hours), showing the proportion of cells in Sub G0
and G2/M phases. Statistical analysis of the DNA content of 1321N1 cells exposed to 2OHOA or palmitate (150 mM) revealed a significant increase
(*p,0.05; n = 6) in the G2/M phase peak when compared with untreated cells (C-). No significant differences in Sub G0 values were detected in MRC-5
cells exposed to 2OHOA (150 mM)) when compared with untreated cells (C-). 2OHOA induces autophagy in 1321N1, SF-767 and U118
but not in MRC-5 cells doi:10.1371/journal.pone.0048235.g005 Figure 5. 2OHOA induction of G2/M cell cycle arrest of 1321N1 cells but not of MRC-5 cells. Cell cycle assessment and G2/M phase arrest. Analysis of the DNA content (flow cytometry) of MRC-5 and 1321N1 cells exposed to 2OHOA or palmitate (150 mM for 72 hours). A. Analysis of the
DNA content in untreated MRC-5 cells. B. Analysis of the DNA content in MRC-5 cells exposed to 2OHOA (150 mM for 72 h) or (C) palmitate (150 mM
for 72 hours), showing the proportion of cells in Sub G0 and G2/M phases. D. Analysis of the DNA content of untreated 1321N1 cells. E. Analysis of the
DNA content of 1321N1 cells exposed to 2OHOA (150 mM for 72 h) or (F) palmitate (150 mM for 72 hours), showing the proportion of cells in Sub G0
and G2/M phases. Statistical analysis of the DNA content of 1321N1 cells exposed to 2OHOA or palmitate (150 mM) revealed a significant increase
(*p,0.05; n = 6) in the G2/M phase peak when compared with untreated cells (C-). No significant differences in Sub G0 values were detected in MRC-5
cells exposed to 2OHOA (150 mM)) when compared with untreated cells (C-). doi:10.1371/journal.pone.0048235.g005 2OHOA. At both low and high 2OHOA concentrations,
distended ER membranes and a loss of ER were observed in the
cytoplasm consistent with the ER stress and the autophagic
process (Fig.10 F to 10 H). Figure 10I shows in detail early
extensions of double endoplasmic reticulum (ER) membranes
beginning to surround a mitochondrion, which is characteristic of
the
autophagic
process. These
results
further
support
the
specificity
of the
effects of
2OHOA against
glioma
cells,
implicating autophagy as the final cellular effect induced by this
compound in these cancer cells. promoted the generation of autophagosomes in cancer cells,
whereas palmitate induced unspecific production of acidic vesicles
in both normal and cancer cells. To further confirm that autophagy was induced, we assessed the
expression of the autophagy markers ATG7, ATG5, LC3B I and
LC3B II. Treatment with 2OHOA or palmitate (150 mM; 72 h)
significantly augmented both LC3B-I and LC3B-II in 1321N1,
SF-767 and U118 cells compared to MRC-5 cells (Fig. 9 A–D). ATG7 was also up-regulated in 1321N1 cells compared to MRC-5
cells (Fig.9 E and F) and ATG5 was up-regulated in SF-767 cells
(Fig.9 G). 2OHOA induces autophagy in 1321N1, SF-767 and U118
but not in MRC-5 cells However, U118 cell line did not show up-regulation of
ATG7 (Fig 9 H) nor ATG5 (data not shown) as early as 12 h after
treatment, suggesting an earlier induction of this molecules. Discussion and Conclusions Cyclin B and Cdk1/Cdc2 proteins in 1321N1, SF-767 and U118 human glioma cells and MRC-5 non cancer cells. A, B, C and D
present in the upper panels: a representative immunoblot showing cyclin B expression after exposure to 2OHOA (H) or palmitate (P: 150 mM;
24 hours). Lower panels: Bar diagram showing the mean6SEM values of cyclin B expression in (A) MRC-5, (B) 1321N1, (C) SF-767 and (D) U118 cells,
upon exposure to 2OHOA (H) or palmitate (P: 150 mM; 24 h) when compared with untreated controls (C). E, F, G and H show a representative
immunoblot of Cdk1/Cdc2 expression after exposure to 2OHOA (H) or palmitate (P: 150 mM; 48 hours, upper panels). The lower panels show the bar
diagram showing the mean6SEM values of Cdk1/Cdc2 expression in (E) MRC-5, (F) 1321N1, (G) SF-767 and (H) U118 cells after exposure to 2OHOA
(H) or palmitate (P: 150 mM; 48 h) when compared with untreated controls (C). (*p,0.05, **p,0.01, ***p,0.001; n = 6). doi:10.1371/journal.pone.0048235.g006 this further demonstrates the specificity of 2OHOA to this glioma
cells, explaining the observed lack of side-effects in animal models
of cancer. concentrations. Finally, while apoptosis has been implicated in the
general mechanism of action of 2OHOA against various types of
cancer cells [5], SF-767 glioma cells do not initiate the apoptosis
program although other lines of glioma cells seem to undergo ER
stress and apoptosis [26,27] and thus, how cell death occurs in
such cases remains unknown. Since compounds that induce sustained eIF2a phosphorylation
provide cytoprotection in situations of ER stress [16], the
maintenance of eIF2a in an inactive state is somehow beneficial. However, prolonged suppression of protein synthesis is incompat-
ible with cell survival and leads to autophagy [11,14,15]. Exposure
of MRC-5 fibroblasts to 2OHOA does not induce eIF2a and
ATF4 expression, or inhibit cell growth, further evidence of its
specificity in these glioma cells and demonstrating the role of
eIF2a and ATF4 in 2OHOA-induced cell death of 1321N1, SF-
767 and U118 cells. 2OHOA selectively inhibits glioma cells growth with an IC50 of
,100 mM in 1321N1, SF-767 and U118 cells as opposed to that of
.1000 mM in MRC-5 non cancer cells, which justifies the lack of
toxic effects at therapeutic doses. In addition, 2OHOA induces cell
cycle arrest in 1321N1, SF-767 and U118 cells, resulting in a
significant accumulation of 1321N1 cells in the G2/M phase. Discussion and Conclusions Indeed, cyclin B and cdk1/cdc2 are downregulated when glioma
cells are exposed to 2OHOA. Previous studies have shown glioma
cells to undergo autophagy when exposed to compounds that
induce cell cycle arrest in the G2/M phase [27,28]. While
autophagy provides a means of recycling cytosolic molecules/
structures involved in cell survival, it can also represent a non-
apoptotic cell death program. Autophagy involves the fragmen-
tation of cells after the engulfment of proteins, organelles and
cytosol in vesicles called autophagosomes, which eventually fuse
with lysosomes to form autolysosomes [29]. In a variety of cells
and tumors, including human glioma, autophagy signaling, the
UPR and abnormal cell growth are intimately related [11,14,15]. The high rate of cancer cell proliferation is associated with
increased protein and lipid synthesis, and active metabolism,
which in turn induces a certain level of ER stress [22,30]. Furthermore, as tumors progresses, cancer cells experience
increasing nutrient starvation and hypoxic conditions, resulting
in the accumulation of unfolded or misfolded proteins, in turn
leading to activation of UPR signaling [11,22,30]. The second ER stress pathway studied, the IRE1a signaling
pathway, was also activated by 2OHOA in 1321N1, SF-767 and
U118 cells. 2OHOA induces a significant increase in IRE1a in
1321N1, SF-767 and U118 cells when compared to the modest
increase in MRC-5 cells. Interestingly, the spliced activated form
of XBP1s, a downstream target of ATF6 and IRE1a, was up-
regulated by 2OHOA in both 1321N1 and MRC-5 cells. Strong
expression of the spliced form of XBP1 is associated with cell
survival, whereas expression of the unspliced variant of XBP1 is
associated with apoptosis [33]. Our results suggest that the up-
regulation of XBP1 is not essential for cell death, given that it was
also observed in MRC-5 cells, suggesting that the activation of
other factors besides XBP1 is necessary to induce autophagy. Under persistent ER stress, the PERK, IRE1a and ATF6
signaling pathways induce the expression of the pro-apoptotic
factor CHOP. In line with its activation of ER stress/UPR,
2OHOA induces CHOP expression in 1321N1, SF-767 and U118
human glioma cells but not in MRC-5 cells, whereas palmitate up-
regulated CHOP in glioma and non-cancer cells. As CHOP is one
of the most important downstream effector proteins of ER stress,
its specific activation by 2OHOA in 1321N1, SF-767 and U118
cells is consistent with the severe induction of ER stress. Discussion and Conclusions CHOP
activation often leads to the induction of cell death and although
CHOP is one of the main effectors of apoptosis [12], 2OHOA did
not trigger apoptosis in SF-767 cells despite inducing marked
CHOP expression. Nevertheless, activation of autophagy in
various glioma cell lines that are usually resistant to apoptosis
has recently been associated with CHOP overexpression [34]. Autophagy is triggered in certain situations of stress, with the
aim of promoting cell survival by inducing cellular adaptations to
the associated conditions [29,31,32]. However, increasing evi-
dence suggests that autophagy also serves as a trigger for cell death
[29,31,32]. As was shown above, some features of apoptosis were induced in
1321N1 and U118 cells but not in SF-767 by exposure to 2OHOA
(sub-G0 peak, poly ADP ribose polymerase [PARP] or caspase 8
partial proteolysis) (Fig.7). However, this induction of apoptotic
features did not fully explain the cell death induced by 2OHOA. Therefore we examined the role of the autophagy induced by the
ER stress/UPR signaling pathway in relation to the growth
inhibition effects of 2OHOA in 1321N1, SF-767 and U118
human glioma cells and non-cancer MRC-5 cells. Treatment with
2OHOA or palmitate activated ER stress in 1321N1, SF-767 and
U118 cells within 12 h, as evidenced by the increase in
phosphorylated eIF2a protein, a marker of ER stress. Phosphor-
ylation of eIF2a induces cellular adaptation to various stress
conditions by inhibiting protein synthesis and subsequently, by
activating expression of the ATF4 transcription factor [13]. We
found that both 2OHOA and palmitate significantly increase
ATF4 expression in 1321N1 cells, while neither eIF2a phosphor-
ylation nor ATF4 gene expression were evident in non cancer
MRC-5 cells exposed to 2OHOA. Along with previous findings, We have worked with three human glioma cell lines (1321N1,
SF-767, and U118) and as a non-cancer control we have used a
human fetal lung fibroblast-like cell line (MRC-5). Despite the
differences among the cell lines employed in this study (develop-
mental age and tissue type), a number of mounting evidence
suggest that the changes observed here are not due to the
developmental age or tissue type. Thus, the efficacy of 2OHOA
against different cancer cell types apart from glioma has been
reported elsewhere [4,5,6,35]. Discussion and Conclusions 2OHOA is a potent anticancer drug that inhibits cancer cell
growth and induces tumor regression in animal models of cancer,
with no undesired side effects. In this context, 2OHOA has been
recently granted the status of orphan drug for the treatment of
glioma by the European Medicines Agency (EMA). While previous
studies have demonstrated 2OHOA provoked cell cycle arrest
[2,4] in cancer cells, the precise molecular and cellular mecha-
nisms underlying the selective induction of glioma cell death is not
fully understood. Finally, astrocytoma cell degradation upon 2OHOA treatment
was further investigated by electron microscopy, which revealed
fragments of 1321N1 cells and dense vesicles associated with
double layered autophagosomes (Fig. 10). The cytoplasm of
control (untreated) 1321N1 cells was densely packed with
abundant polyribosomes, mitochondria, dictyosomes and inter-
mediate filament bundles (Fig. 10 A and 10 E). After 48 hours in
the presence of the lowest concentration of 2OHOA used in this
study (150 mM), the nucleus of 1321N1 cells was no different to
that of control cells. Notably, 2OHOA induced the appearance of
lipid droplets and dense bodies, the latter scattered throughout the
cytoplasm with morphological characteristics of autophagosomes
(Fig 10 B to D and 10 F to H). The abundance of these dense
bodies was concentration-dependent (Fig 10 B to D), and their
heterogeneity increased in function of the concentration of We investigated the mechanism of 2OHOA-induced cell death
in 1321N1 glioma cells for a number of reasons. Firstly, previous
studies in our laboratory have demonstrated 2OHOA-induced
glioma regression in both animal xenograft models of human
glioma and in nude mice (see below). Secondly, unlike most
chemotherapeutic agents, this drug is highly selective and it does
not induce the death of healthy cells, even at very high doses/ October 2012 | Volume 7 | Issue 10 | e48235 PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e48235 2OHOA Induces UPR and Autophagy in Glioma Cells October 2012 | Volume 7 | Issue 10 | e48235 October 2012 | Volume 7 | Issue 10 | e48235 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 8 8 2OHOA Induces UPR and Autophagy in Glioma Cells 2OHOA Induces UPR and Autophagy in Glioma Cells Figure 6. 2OHOA inhibition of Cyclin B and Cdk1/Cdc2 proteins in 1321N1, SF-767 and U118 human glioma cells but not in non
cancer MRC-5 cells. October 2012 | Volume 7 | Issue 10 | e48235 Discussion and Conclusions Lower panels: Bar diagram showing the mean6SEM values of PARP expression in MRC-5 (A), 1321N1 (C), SF-767 (E)
and U118 (G) cells or Caspase 8 in MRC-5 (B), 1321N1 (D), SF-767 (F) and U118 (H) cells after exposure to 2OHOA (H) or palmitate (P: 150 mM; 72 h)
when compared with untreated controls (C, *p,0.05, **p,0.01, ***p,0.001; n = 6). doi:10.1371/journal.pone.0048235.g007 treatment with 2OHOA. PARP and Caspase 8 proteins in 1321N1, SF 767, U118 human glioma cells and MRC 5 non cancer cells. Upper panels: a
representative immunoblot showing PARP (A, C, E and G) or Caspase 8 (B, D, F and H) expression in every cell line after exposure to 2OHOA (H) or
palmitate (P: 150 mM; 72 hours). Lower panels: Bar diagram showing the mean6SEM values of PARP expression in MRC-5 (A), 1321N1 (C), SF-767 (E)
and U118 (G) cells or Caspase 8 in MRC-5 (B), 1321N1 (D), SF-767 (F) and U118 (H) cells after exposure to 2OHOA (H) or palmitate (P: 150 mM; 72 h)
when compared with untreated controls (C, *p,0.05, **p,0.01, ***p,0.001; n = 6). doi:10.1371/journal.pone.0048235.g007
Figure 8. 2OHOA induction of acidic vesicles in 1321N1 but not in MRC-5 cells. Analysis of acidic vesicles in cells stained with Hoechst and
LysoSensor Green to visualize nuclei and lysosomes, respectively. The images were captured by live cell imaging and they all represent merged
images of Hoechst (blue) and LysoSensor Green (green). The acidic vesicles in photomicrographs were analyzed with Image J 1.38x software. Neither
the vehicle (FBS, A) nor 2OHOA (150 mM; 48 h, B) resulted in the formation of acidic vesicular organelles (lysosomes and autophagosomes) in non-
cancer MRC-5 human fibroblast cells, as detected by the LysoSensor fluorescence probe, whereas palmitate (150 mM, 48 h: C) induced acidic vesicle
formation. Graphs show the integrated fluorescence density of the lysosomes (56104 cells per experiment) in MRC-5 cells (D): Control, 11.5463.36%;
2OHOA (150 mM), 16.8862.45%; palmitate (150 mM), 100*63.65%; *p,0.05. No vesicular organelles accumulated in 1321N1 human astrocytoma cells
treated with the vehicle alone (control, E), while exposure to 2OHOA (F) or palmitate (G) (150 mM; 48 h) resulted in the appearance of acidic vesicular
organelles. Integrated fluorescence density of lysosomes in 1321N1 cells (56104 cells per experiment) (H): Control, 3.160.37%; 2OHOA (150 mM)
81**66.18%; P (150 mM) 100**66.12%; **p,0.01 (n = 6 experiments). Discussion and Conclusions Moreover, the lack of effects against
nomal (non-tumor, IMR-90) cells in vitro (cell culture) [5] or in vivo
(animal models, unpublished GLP toxicology data in mice, rats,
and dogs) and the efficacy observed in animal models of glioma
[36], supports the specificity of 2OHOA against glioma cancer
cells. October 2012 | Volume 7 | Issue 10 | e48235 October 2012 | Volume 7 | Issue 10 | e48235 PLOS ONE | www.plosone.org 9 2OHOA Induces UPR and Autophagy in Glioma Cells O O
duces U
a d
uto October 2012 | Volume 7 | Issue 10 | e48235 October 2012 | Volume 7 | Issue 10 | e48235 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 10 Figure 7. Expression of PARP and Caspase 8 in 1321N1, SF-767, U118 human glioma cells and non cancer MRC-5 cells after
treatment with 2OHOA. PARP and Caspase 8 proteins in 1321N1, SF-767, U118 human glioma cells and MRC-5 non cancer cells. Upper panels: a
representative immunoblot showing PARP (A, C, E and G) or Caspase 8 (B, D, F and H) expression in every cell line after exposure to 2OHOA (H) or
palmitate (P: 150 mM; 72 hours). Lower panels: Bar diagram showing the mean6SEM values of PARP expression in MRC-5 (A), 1321N1 (C), SF-767 (E)
and U118 (G) cells or Caspase 8 in MRC-5 (B), 1321N1 (D), SF-767 (F) and U118 (H) cells after exposure to 2OHOA (H) or palmitate (P: 150 mM; 72 h)
when compared with untreated controls (C, *p,0.05, **p,0.01, ***p,0.001; n = 6). doi:10.1371/journal.pone.0048235.g007
2OHOA Induces UPR and Autophagy in Glioma Cells 2OHOA Induces UPR and Autophagy in Glioma Cells 2OHOA Induces UPR and Autophagy in Glioma Cells Figure 7. Expression of PARP and Caspase 8 in 1321N1, SF-767, U118 human glioma cells and non cancer MRC-5 cells after
treatment with 2OHOA. PARP and Caspase 8 proteins in 1321N1, SF-767, U118 human glioma cells and MRC-5 non cancer cells. Upper panels: a
representative immunoblot showing PARP (A, C, E and G) or Caspase 8 (B, D, F and H) expression in every cell line after exposure to 2OHOA (H) or
palmitate (P: 150 mM; 72 hours). Discussion and Conclusions Exposure of 1321N1 (B), SF-767 (C)
and U118 (D) cells to 2OHOA or palmitate (150 mM, 72 h) induced a significant increase in LC3BI and LC3BII protein expression while in MRC-5 (A)
only palmitate induced significant increases of these proteins. Exposure of 1321N1 cells to 2OHOA or palmitate (150 mM, 72 h) induced a significant
increase in ATG7 (F) protein expression while in MRC-5 (E), and U118 (H) did not induce significant changes. Finally exposure of SF-767 cells to
2OHOA or palmitate (150 mM, 72 h) induced a significant increase in ATG5 (G) protein expression (*p,0.05, **p,0.01, ***p,0.001; n = 6). doi:10.1371/journal.pone.0048235.g009 Figure 9. Expression of ATG 7, ATG5 and LC3BI, LC3BII in 1321N1, SF-767, U118 and MRC-5 cells after treatment with 2OHOA. The
effects of 2OHOA and palmitate on the levels of ATG 7, ATG5, LC3BI and LC3BII were determined by immunoblots. Exposure of 1321N1 (B), SF-767 (C)
and U118 (D) cells to 2OHOA or palmitate (150 mM, 72 h) induced a significant increase in LC3BI and LC3BII protein expression while in MRC-5 (A)
only palmitate induced significant increases of these proteins. Exposure of 1321N1 cells to 2OHOA or palmitate (150 mM, 72 h) induced a significant
increase in ATG7 (F) protein expression while in MRC-5 (E), and U118 (H) did not induce significant changes. Finally exposure of SF-767 cells to
2OHOA or palmitate (150 mM, 72 h) induced a significant increase in ATG5 (G) protein expression (*p,0.05, **p,0.01, ***p,0.001; n = 6). doi:10.1371/journal.pone.0048235.g009 Increasing our understanding of the molecular basis of cell
death induced by activating ER stress/UPR signaling is of
considerable interest, since many proteins in these pathways
constitute important potential drug targets [26]. In a previous
study [35], we showed that cancer cells have very low membrane
sphingomyelin and high phosphatidylethanolamine levels. In
glioma and other types of cancer cells but not normal cells,
2OHOA induces changes in these lipids to reach values found in
healthy tissues. The present study sheds light on the signaling
events that follow the activation of this molecular switch. Here, we
demonstrate the selective induction of several key effectors of ER
stress/UPR cell death (P-eIF2a, ATF4 and CHOP) by 2OHOA in
three human glioma cells. Moreover, we provide cellular and
molecular evidence that 2OHOA induces autophagy in these cells,
which may constitute a novel therapeutic strategy to combat
glioma, when the cells are reluctant to enter apoptosis. Discussion and Conclusions Scale bar = 10 mm (8A, 8B, 8E, 8F, 8I, 8J); 15 mm (8C, 8G and 8K). doi:10.1371/journal.pone.0048235.g008
PLOS ONE | www plosone org
11
October 2012 | Volume 7 | Issue 10 | e48235 Figure 8. 2OHOA induction of acidic vesicles in 1321N1 but not in MRC-5 cells. Analysis of acidic vesicles in cells stained with Hoechst and
LysoSensor Green to visualize nuclei and lysosomes, respectively. The images were captured by live cell imaging and they all represent merged
images of Hoechst (blue) and LysoSensor Green (green). The acidic vesicles in photomicrographs were analyzed with Image J 1.38x software. Neither
the vehicle (FBS, A) nor 2OHOA (150 mM; 48 h, B) resulted in the formation of acidic vesicular organelles (lysosomes and autophagosomes) in non-
cancer MRC-5 human fibroblast cells, as detected by the LysoSensor fluorescence probe, whereas palmitate (150 mM, 48 h: C) induced acidic vesicle
formation. Graphs show the integrated fluorescence density of the lysosomes (56104 cells per experiment) in MRC-5 cells (D): Control, 11.5463.36%;
2OHOA (150 mM), 16.8862.45%; palmitate (150 mM), 100*63.65%; *p,0.05. No vesicular organelles accumulated in 1321N1 human astrocytoma cells
treated with the vehicle alone (control, E), while exposure to 2OHOA (F) or palmitate (G) (150 mM; 48 h) resulted in the appearance of acidic vesicular
organelles. Integrated fluorescence density of lysosomes in 1321N1 cells (56104 cells per experiment) (H): Control, 3.160.37%; 2OHOA (150 mM)
81**66.18%; P (150 mM) 100**66.12%; **p,0.01 (n = 6 experiments). Scale bar = 10 mm (8A, 8B, 8E, 8F, 8I, 8J); 15 mm (8C, 8G and 8K). doi:10.1371/journal.pone.0048235.g008 October 2012 | Volume 7 | Issue 10 | e48235 PLOS ONE | www.plosone.org 11 2OHOA Induces UPR and Autophagy in Glioma Cells 2OHOA Induces UPR and Autophagy in Glioma C PLOS ONE | www.plosone.org
12
October 2012 | Volume 7 | Issue 10 | e48235 October 2012 | Volume 7 | Issue 10 | e48235 12 October 2012 | Volume 7 | Issue 10 | e48235 PLOS ONE | www.plosone.org 12 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 2OHOA Induces UPR and Autophagy in Glioma Cells 2OHOA Induces UPR and Autophagy in Glioma Cells Figure 9. Expression of ATG 7, ATG5 and LC3BI, LC3BII in 1321N1, SF-767, U118 and MRC-5 cells after treatment with 2OHOA. The
effects of 2OHOA and palmitate on the levels of ATG 7, ATG5, LC3BI and LC3BII were determined by immunoblots. Cell Culture Human glial cells from 1321N1 brain astrocytoma, U118
glioblastoma and human fetal lung fibroblast-like MRC-5 cells
were obtained from the European Collection of Cell Cultures and
SF-767 cells were obtained from the Brain Tumor Research
Center Tissue Bank (University of California-San Francisco, Cell proliferation (MTT) assay Cell proliferation was determined using the MTT (methylthia-
zolyl diphenyl tetrazolium bromide) method [37]. 1321N1, SF-
767, U118 and MRC-5 cells were plated in 96-well plates at
densities of 36103 cells/well (glioma cells) and 66103 cells/well
(MRC-5), respectively, and with 150 ml culture medium (5% FBS)
per well. After incubating overnight to allow cell attachment, the
cells were treated with 50–1000 mM of 2OHOA or palmitate for
24 h, 48 h or 72 h, and 10% of MTT (5 mg/ml in PBS 1X)
reagent was then added for 4 h. The medium was removed,
200 ml of DMSO was added to the cells for 5 min and they were
gently shaken. Absorbance at 550 nm was measured using a Micro
Plate Reader. 2-Hydroxyoleic Acid (2OHOA) 2OHOA was obtained from Lipopharma and its purity (99.7%)
was confirmed by HPLC and gas chromatography. Discussion and Conclusions As a matter
of fact, we have demonstrated that 2OHOA has greater efficacy
than the reference drug for the treatment of glioma, temozolo-
mide, in subcutaneous and orthotopic xenograft models of human
glioma in nude mice [36]. In conclusion, the design of new lipid
molecules like 2OHOA that can modulate ER stress/UPR,
constitutes a promising and novel approach to treat gliomas and
other neoplasias. Department of Neurological Surgery). They were cultured in
Dulbecco’s
Modified
Eagle
Medium
(DMEM)
low
glucose
medium, supplemented with L-glutamine (2 mM), Non Essential
Amino Acids (NEAA, 1%), Fetal Bovine Serum (FBS, 10%),
penicillin (100 U/ml) and streptomycin (0.1 mg/ml), at 37uC in a
humidified atmosphere of 5% CO2. The cell culture medium and
supplements were all purchased from Sigma-Aldrich (Madrid,
Spain). Electrophoresis, immunobloting and protein
quantification 1321N1, SF-767, U118 were plated at densities of 16104 cells/
cm2 and MRC-5 cells at 36104 cells/cm2, respectively, in 10 cm2
plates containing 8 ml of culture medium (5% FBS). After
incubation overnight, the cells were treated with 150 mM of
2OHOA or palmitate for 12 h, 24 h, 48 h and 72 h. Although
2OHOA was diluted in FBS (50 mM) and palmitate in DMSO
(100 mM), DMSO was always present at a final concentration of
0.1–1%. After incubating in the presence or absence of 2OHOA
or palmitate at the indicated concentrations and times (see the
Results section), the cells were washed twice with PBS and
harvested with a rubber policeman in 300 ml of protein extraction
buffer (10 mM Tris-HCl [pH 7.4], containing 50 mM NaCl,
1 mM MgCl2, 2 mM EDTA, 1% SDS, 5 mM iodoacetamide,
1 mM PMSF, 2% cantaridin and 0.1% sodium orthovanadate). Cell suspensions were twice subjected to ultrasonication for 10 s at
50 W using a Braun Labsonic U sonicator and 30 ml aliquots were
removed for protein quantification using the BCA method
(bicinchoninic acid) [39] (Pierce - Thermo Fisher Scientific Inc,
Roskilde, Denmark). The remaining suspension (about 270 ml)
was mixed with 30 ml of 10X electrophoresis loading buffer
(120 mM Tris-HCl [pH 6.8], containing 4% SDS, 50% glycerol,
0.1% bromophenol blue, 10% mercaptoethanol) and boiled for
5 mins. Proteins were fractionated on 8% polyacrylamide gels
(SDS-PAGE: 15-well and 1.5 mm thick) and transferred to
nitrocellulose membranes (WhatmanH protranH, Dassel, Ger-
many). The nitrocellulose membranes were then blocked for 1 h at
room temperature in Tris-buffered saline (TBS 1X) containing 5%
non-fat dry milk and 0.1% Tween 20 (blocking solution), and the
membranes were incubated overnight at 4uC with one of the
following primary anti-human antibodies diluted in TBS contain-
ing 0.5% bovine serum albumin and 0.1% Tween 20: monoclonal
anti-IRE1a, anti-CHOP, anti-P-eIF2a, anti-caspase 8, anti-
ATG7, ATG5 and anti-LC3B (1:1,000, Cell Signaling Technology
Inc., Beverly, MA) or polyclonal anti-Cyclin B, anti-Cdk1/Cdc2
(1:1,000, BD Transduction LaboratoriesTM Heidelberg, Germany)
and anti-PARP (1:2,000, Sant Cruz Biotechnology, Santa Cruz,
CA). Cell DNA content Trypan blue staining was done as previously described [38]. Briefly, 10 ml of sample (cell suspension) was mixed with 10 ml of
trypan blue (Invitrgen), and pipeted into CountessH chamber slide
(Invitrogen) that was inserted in the CountessH Automated Cell
Counter (Invitrogen). To determine cell growth and the cell cycle phase of the cells,
the cellular DNA content was determined by staining cells with
ethidium bromide followed by single-cell fluorescence flow
cytometry. 1321N1 and MRC-5 cells were seeded in 6-well plates
containing 2 ml of culture medium (5% FBS) per well at densities
of 16104 cells/cm2 and 36104 cells/cm2, respectively, and they
were incubated for 72 h in the presence or absence of of 2OHOA
or palmitate (150 mM). The cells were then washed twice with
phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM potassi-
um
chloride,
12 mM
dibasic
sodium
phosphate,
1.38 mM
monobasic
potassium
phosphate
[pH 7.4]),
resuspended
in
500 ml of methanol and vortexed. The cells were subsequently
incubated at 4uC for 1 h, and then for 30 min at room
temperature with 100 mg/ml ethidium bromide and 100 mg/ml
RNAse A (Sigma-Aldrich) in PBS. Single-cell ethidium bromide
fluorescence (25,000 events) was measured on a Coulter Epics XL
flow cytometer using EXPO 32 flow cytometry software (Beckman
Coulter, Inc.) with the gates set to differentiate between G0/G1, S
and G2/M phases. Electrophoresis, immunobloting and protein
quantification After removing the primary antibody, the membranes were
washed three times for 10 min with 1X TBS and incubated for 1 h
at room temperature in fresh blocking solution with a horseradish
peroxidase-linked goat anti-mouse IgG antibody (against mono-
clonal primary antibodies, 1:2,000; Amersham Pharmacia) or a
horseradish peroxidase-linked goat anti-rabbit IgG antibody
(against polyclonal primary antisera, 1:2,000; Cell Signaling
Technology Inc., Beverly, MA). Immunoreactivity was detected
using the Enhanced Chemiluminescence Western Blot Detection
system (ECL; Amersham Pharmacia) and by exposure to ECL
hyperfilm (Amersham Pharmacia). The films were scanned at a
resolution of 600 dpi for quantification using the Foto Look 32
software (Agfa Gevaert, Leverkusen, Germany) and the images Cell viability assay (Trypan Blue exclusion) Cell viability was determined using the Trypan blue staining
method [38]. 1321N1, U118, SF-767 and MRC-5 cells were
plated
in
6-well
plates
at
densities
of
26104 cells/cm2 Figure 10. Electron microscopy of 1321N1 cells treated with 2OHOA: induction of Autophagosomes. Electron microscopy of 1321N1
cells maintained for 48 h in the absence (control: 10A and 10E) or presence of 2OHOA (150 mM: 10B and 10F; 250 mM: 10C, 10G and 10I; 500 mM:
10D and 10H). N: Nuclei; A: Autophagosomes; LP: Lipid Droplets; ER: Rough Endoplasmatic Reticulum; M: Mitochondria. Scale bar = 10 mm (10A–
10D); 1 mm (10E–10H) and 500 nm (10I). doi:10.1371/journal.pone.0048235.g010 Figure 10. Electron microscopy of 1321N1 cells treated with 2OHOA: induction of Autophagosomes. Electron microscopy of 1321N1
cells maintained for 48 h in the absence (control: 10A and 10E) or presence of 2OHOA (150 mM: 10B and 10F; 250 mM: 10C, 10G and 10I; 500 mM:
10D and 10H). N: Nuclei; A: Autophagosomes; LP: Lipid Droplets; ER: Rough Endoplasmatic Reticulum; M: Mitochondria. Scale bar = 10 mm (10A–
10D); 1 mm (10E–10H) and 500 nm (10I). doi:10.1371/journal.pone.0048235.g010 October 2012 | Volume 7 | Issue 10 | e48235 October 2012 | Volume 7 | Issue 10 | e48235 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 13 2OHOA Induces UPR and Autophagy in Glioma Cells 2OHOA Induces UPR and Autophagy in Glioma Cells (1.866105 cells/well)
for
MRC-5
cells;
66104 cells/cm2
(66105 cells/well)
for
1321N1
cells
and
36104 cells/cm2
(36105 cells/well) for SF-767 and U118 cells. Cells were plated
at 50% confluence and cultured with 2 ml of culture medium (5%
FBS) per well. After incubating overnight to allow cell attachment,
the cells were treated with 50–1000 mM of 2OHOA or palmitate
for 24 h, 48 h or 72 h. After 48 h confluence was reached. were analyzed with TotalLab v2005 (Nonlinear Dynamics, All Saints,
UK) to obtain the integrated optical density (IOD) of each band. The a-tubulin content of each sample was determined by the same
procedure
and
the
concentration
of
a
given
protein
was
normalized to the a-tubulin content of the same sample. References 1. Barcelo´ F, Prades J, Funari SS, Frau J, Alemany R, et al. (2004) The hypotensive
drug 2-hydroxyoleic acid modifies the structural properties of model mem-
branes. Mol Membr Biol 21: 261–8. 1. Barcelo´ F, Prades J, Funari SS, Frau J, Alemany R, et al. (2004) The hypotensive
drug 2-hydroxyoleic acid modifies the structural properties of model mem-
branes. Mol Membr Biol 21: 261–8. 10. Karaskov E, Scott C, Zhang L, Teodoro T, Ravazzola M, et al. (2006) Chronic
Palmitate But Not Oleate Exposure Induces Endoplasmic Reticulum Stress,
Which May Contribute to INS-1 Pancreatic-Cell Apoptosis. Endocrinology 147:
3398–3407. 2. Martı´nez J, Vo¨gler O, Casas J, Barcelo´ F, Alemany R, et al. (2005b) Membrane
structure modulation, protein kinase C alpha activation, and anticancer activity
of Minerval. Mol Pharmacol 67: 531–40. 11. Kim I, Xu W, Reed JC (2008) Cell death and endoplasmic reticulum stress:
disease relevance and therapeutic opportunities. Nat. Revs. Drug Discov 7: 1013–
1030. 3. Cordomi A, Prades J, Frau J, Vo¨gler O, Funari SS, et al. (2010) Interactions of
fatty acids with phosphatidylethanolamine membranes: X-ray diffraction and
molecular dynamics studies. J. Lipid Res 51: 1113–1124. 12. Oyadomari S, Mori M (2004) Roles of CHOP/GADD153 in endoplasmic
reticulum stress. Cell Death Differ 11: 381–389. 4. Martı´nez J, Gutie´rrez A, Casas J, Llado´ V, Lo´pez-Bellan A, et al. (2005a) The
repression of E2F-1 is critical for the activity of Minerval against cancer. J Pharmacol Exp Ther 315: 466–474. 13. Lu PD, Harding HP, Ron D (2004) Translation reinitiation at alternative open
reading frames regulates gene expression in an integrated stress response. J Cell
Biol 167: 27–33. 5. Llado´ V, Gutierrez A, Martı´nez J, Casas J, Tere´s S, et al. (2010) Minerval
induces apoptosis in Jurkat and other cancer cells. J. Cell Mol Med 13: 1–12. 14. Kouroku Y, Fujita E, Tanida I, Ueno T, Isoai A, et al. (2007) ER stress (PERK/
eIFa phosphorylation) mediates the polyglutamine-induced LC3 conversion, an
essential step for autophagy formation. Cell Death Differ 14: 230–239. 6. Llado´ V, Tere´s S, Higuera M, Alvarez R, Noguera-Salva MA, et al. (2009)
Pivotal role of dihydrofolate reductase knockdown in the anticancer activity of 2-
hydroxyoleic acid. Proc. Natl. Acad. Sci. U.S.A 106: 13754–13758. 15. Fujita E, Kouroku Y, Isoai A, Kumagai H, Misutani A, et al. (2007) Two
endoplasmic reticulum-associated degradation (ERAD) systems for the novel
variant of the mutant dysferlin: ubiquitin/proteasome ERAD(I) and autophagy/
lysosome ERAD(II). Acknowledgments The authors thank the anonymous reviewers for their useful comments and
suggestions that greatly helped to improve an earlier version of this paper. We thank Dr. Juana Barcelo´ for her contribution during preliminary
research on this work. Fluorescence Microscopy y
1321N1, SF-767 and MRC-5 cells were seeded in 4-well
(1.7 cm2) plates containing 750 ml of culture medium (5% FBS)
per well at densities of 16104 cells/cm2 (glioma cells) and
36104 cells/cm2 (MRC-5), respectively. After incubating over-
night to allow cell attachment, cells were treated with 2OHOA or
palmitate (150 mM) for 48 h and they were then incubated for 1 h
with LysoSensor Green DND-189 pH Indicator (2 mM, pH 4.5–6:
Invitrogen/Molecular probes). During the last 5 minutes of this
incubation, Hoechst (trihydrochloride trihydrate) stain (40 mg/ml,
Invitrogen/Molecular probes) was added to each well. The cells
were examined on a Nikon Eclipse TE2000-S Fluorescence
microscope (400X) and the photomicrographs of the acidic vesicles
were analyzed using Image J 1.38x software (Wayne Rasband,
National Institutes of Health; rsb.info.nih.gov). 2OHOA Induces UPR and Autophagy in Glioma Cells 39 (reverse). RT-qPCR amplifications were carried out on a Step
One v 2.0 thermal cycler (Applied Biosystems) using the SYBRH
Premix Ex TaqTM (Perfect Real Time, Takara) containing TaKaRa
Ex TaqTM HS, dNTP’s, Mg2+, and the SYBRH Green I and
ROXTM Reference Dye. Thermal cycling was preceded by an
initial denaturation step at 95uC for 5 min. DNA amplification
and fluorescence quantification was performed over 35 cycles, with
a denaturation step at 95uC for 5 s, and a 34 s annealing and
extension step at 60uC. The melting curve was determined by one
denaturation step at 95uC for 5 s followed by an annealing step for
34 s (55uC). Fluorescence quantification was performed after each
DNA extension step (60uC), and the data was analyzed using Step
One v 2.0 software. The ratio between the expression of CHOP,
IRE1a, ATF6 or XBP1 and that of b-actin (for 1321N1 cells whose
expression is not modulated by 2OHOA), was determined as
described by Pfaffl et al., 2005 [40]. The results were expressed as
ddCt values (as a percentage) using the following formula:
ddCt = E
X(Ctc-Ctx)/E
Bact(Ctc-Ctx). Efficiency
(E) = 10(21/m). (m) = slope of the graph formed by Ct values of mRNA vs the
logarithm (log) of its concentration (ng/ml). This value was used to
calculate the relative expression in 2OHOA or palmitate-treated
cells with respect to untreated (control) cells. The PCR products
were further characterized by melting curve analysis and agarose
gel electrophoresis. Electron microscopy Cells were seeded at 16104 cells/cm2 in a Lab-Tek chamber
slides of 4 wells (Nalge Nunc International, Naperville, IL) and
were fixed in 3.5% glutaraldehyde for 1 hour at 37uC. Cells were
postfixed in 2% OsO4 for 1 h at room temperature and stained in
2% uranyl acetate in the dark for 2 h at 4uC. Finally, cells were
rinsed in sodium phosphate buffer (0.1 M, pH 7.2), dehydrated in
ethanol, and infiltrated overnight in Araldite (Durcupan, Fluka,
Buchs SG, Switzerland). Following polymerization, embedded
cultures were detached from the chamber slide and glued to
Araldite blocks. Serial semi-thin (1.5 mm) sections were cut with a
diamond knife in a Leica ultramicrotome Ultracut UC-6 (Leica,
Heidelberg, Germany) and mounted onto slides and stained with
1% toluidine blue. Selected semi-thin sections were glued (Super
Glue, Loctite) to araldite blocks and detached from the glass slide
by repeated freezing (in liquid nitrogen) and thawing. Ultrathin
(0.07 mm) sections were prepared with a diamond knife ultracut
and stained with lead citrate. Finally, photomicrographs were
obtained under a transmission electron microscope (FEI Tecnai
G2 Spirit Biotwin) using a digital camera (Morada, Soft Imaging
System, Olympus). Author Contributions Conceived and designed the experiments: AM-E XB. Performed the
experiments: AM-E MLM MAN-S. Analyzed the data: AM-E XB. Contributed reagents/materials/analysis tools: JMG-V MS-N ID. Wrote
the paper: AM-E PVE XB. Statistics The results were expressed as the mean6SEM of at least three
independent experiments, and the level of significance was set at
P,0.05 (Student’s t-test). Quantitative Reverse Transcription-Polymerase Chain
Reaction (qRT-PCR) (q
)
1321N1 cells were seeded in 6-well plates containing 2 ml of
culture medium (5% FBS) per well at density of 16104 cells/cm2. After incubating overnight, the cells were treated with 2OHOA or
palmitate (150 mM) for 24 or 48 h and the regulatory effects of
2OHOA on CHOP, IRE1a, ATF4, ATF6 and sXBP1 mRNA
expression was assessed by Real-time quantitative PCR (RT-
qPCR). Total RNA was extracted from 1321N1 cells using the
RNeasy Mini kit in combination with the RNase-free DNase kit
(Qiagen, Hilden, Germany). Reverse transcription of total RNA
(1 mg) was carried out in a final volume of 20 ml, containing the
following reagents (Roche, Mannheim, Germany): anchored-
oligo(dT) primer (2.5 mM); random hexamer primer (60 mM);
dNTP mix (dGTP, dCTP, dATP, and dTTP, each at 1 mM);
reverse transcriptase reaction buffer (8 mM MgCl2); RNase
inhibitor (20 U); reverse transcriptase (10 U), and RNase-free
water. The reaction mixtures were then incubated at 65uC
(5 min), 37uC (50 min), and 70uC (15 min), and the cDNA
samples
obtained
were
then
stored
at
220uC. For
PCR
amplification, primers were designed based on the CHOP, IRE1a,
ATF4, ATF6 and XBP1 sequences obtained from GenBank: 59-
CCG CAG CAG GTG CAG G-39 (XBP1 spliced forward primer)
and 59-GAG TCA ATA CCG CCA GAA TCC A-39 (XBP1
spliced reverse primer); 59-GCC AAA ATC AGA GCT GGA
ACC T-39 (CHOP forward primer) and 59-ACA GTG TCC CGA
AGG AGA AAG G-39 (CHOP reverse primer); 59-TGT ACC
ATT GAG GGA GAG GC-39 (IRE1a forward primer) and 59-
GAG ACC CTG CGC TAT CTG AC-39 (IRE1a reverse primer);
59-TTC CTG AGC AGC GAG GTG TTG-39 (ATF4 forward
primer) and 59-TCC AAT CTG TCC CGG AGA AGG-39 (ATF4
reverse primer); 59-TGA CAA AGC CCT GAT GGT GCT A-39
(ATF6 forward primer) and 59-TGT TCC AGA GCA CCC TGA
AGA A-39 (ATF6 reverse primer). As an endogenous control, b-
actin expression of was determined in 1321N1 cells using the
following primers: 59-GCG GGA AAT CGT GCG TGA CAT T-
39 (forward) and 59-CTA CCT CAA CTT CCA TCA AAG CAC- October 2012 | Volume 7 | Issue 10 | e48235 PLOS ONE | www.plosone.org 14 2OHOA Induces UPR and Autophagy in Glioma Cells 19. Ogata M, Hino SI, Saito A, Morikawa K, Kondo S, et al. (2006) Autophagy is
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in cancer. Autophagy 6: 322–329. 20. Kondo Y, Kanzawa T, Sawaya R, Kondo S (2005) The role of autophagy in
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5-androstene 3b,17a diol; induces endoplasmic reticulum stress and autophagy
through PERK/eIF2a signaling in malignant glioma cells and transformed
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endoplasmic reticulum load to secretory capacity by processing the XBP-1
mRNA. Nature 415: 93–96. g
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fibroblasts. Int J Biochem Cell Biol 42; 2019–2029. 35. Barcelo´-Coblijn G, Martin ML, de Almeida RFM, Noguera-Salva` MA,
Marcilla-Etxenike A, et al. (2011) Sphingomyelin and sphingomyelin synthase
(SMS) in the malignant transformation of glioma cells and in 2-hydroxyoleic acid
therapy. Proc Natl Acad Sci USA 108; 19596–19574. 25. Yoshida H, Matsui T, Yamamot A, Okada T, Mori K (2001) XBP1 mRNA Is
Induced by ATF6 and Spliced by IRE1 in Response to ER Stress to Produce a
Highly Active Transcription Factor. Cell 107: 881–891. g y
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26. References Hum Mol Genet 16: 618–629. 7. Jakobsen CH, Størvold GL, Bremseth H, Follestad T, Sand K, et al. (2008)
DHA induces ER stress and growth arrest in human colon cancer cells:
associations with cholesterol and calcium homeostasis. Journal of Lipid Research 49:
2089–2100. lysosome ERAD(II). Hum Mol Genet 16: 618–629. 16. Boyce M, Bryant KF, Jousse C, Long K, Harding HP, et al. (2005) A selective
inhibitor of eIF2a dephosphorylation protects cells from ER stress. Science 307:
935–939. 8. Klausner RD, Donaldson JG, Lippincott-Schwartz J (1992) Brefeldin A: insights
into the control of membrane traffic and organelle structure. J Cell Biol 116:
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1197–1202. 18. Mizushima N, Levine B, Cuervo AM, Klionsky DJ (2008) Autophagy fights
disease through cellular self-digestion. Nature 451: 1069–1075. October 2012 | Volume 7 | Issue 10 | e48235 PLOS ONE | www.plosone.org 15 2OHOA Induces UPR and Autophagy in Glioma Cells Johnson GG, White MC, Grimaldi M (2011) Stressed to death: Targeting
Endoplasmatic Reticulum Stress Response Induced Apoptosis in Gliomas. Curr
Pharm Des 17(3):284–292 36. Tere´s S, Llado´ V, Higuera M, Barcelo´-Coblijn G, Martin ML, et al. (2012) 2-
Hydroxyoleate, a nontoxic membrane binding anticancer drug, induces glioma
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Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance. Plant Cell 9, 1573–84. 28. Hansen K, Wagner B, Hamel W, Schweizer M, Haag F, et al. (2007)
Autophagic cell death induced by TrkA receptor activation in human
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Measurement of protein using bicinchoninic acid. Anal Biochem 150: 76–85. 29. Chen S, Rehman SK, Zhang W, Wen A, Yao L, et al. (2010) Autophagy is a
therapeutic target in anticancer drug resistance. Biochim Biophys Acta 1806: 220–
229. 40. Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool
(REST) for group-wise comparison and statistical analysis of relative expression
results in realtime PCR. Nucleic Acids Res 30: 1–10. 30. Scho¨nthal AH (2009) Endoplasmic reticulum stress and autophagy as targets for
cancer therapy. Cancer Lett 275: 163–169. PLOS ONE | www.plosone.org October 2012 | Volume 7 | Issue 10 | e48235 PLOS ONE | www.plosone.org 16
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English
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Numerical Simulation of Methane Under Phase Transfer Conditions with its Real Properties
|
EPJ web of conferences
| 2,016
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cc-by
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NUMERICAL SIMULATION OF METHANE UNDER PHASE
TRANSFER CONDITIONS WITH ITS REAL PROPERTIES
Boris V. Borisov 1, a, Sergey S. Bondarchuk 2, Dimitrii O. Skryabinsky1, Victorya S. Striha1
1National Research Tomsk Polytechnic University, 634050 Tomsk, Russia
2 National Research Tomsk State University, 634050 Tomsk, Russia g
y
The parameters are determined by the current system of equations [2]: The parameters are determined by the current system of equations [2]: ρ
ρ
τ
condensate
u
G
x
,
(1)
2
(ρ
)
ρ
u
p
w ρ
ρ
τ
condensate
u
G
x
,
(1)
2
(ρ
)
ρ
friction
condensate
u
p
w
F
G
w
x
,
(2) ρ
ρ
τ
condensate
u
G
x
,
(1)
2
(ρ
)
ρ
friction
condensate
u
p
w
F
G
w
x
,
(2)
2
2
ρ(ε
0.5
)
ρ (
0.5
)
τ
out
condensate
condensate
u
u h
u
Q
G
h
x
,
(3) (1) 2
(ρ
)
ρ
friction
condensate
u
p
w
F
G
w
x
,
(2) (2) 2
2
ρ(ε
0.5
)
ρ (
0.5
)
τ
out
condensate
condensate
u
u h
u
Q
G
h
x
,
(3) 2
2
ρ(ε
0.5
)
ρ (
0.5
)
τ
out
condensate
condensate
u
u h
u
Q
G
h
x
,
(3) (3) Where τ, , ρ, ,
, ε, ,
,
,
condensate
friction
out
x
u p
h G
F
Q
, respectively, time, coordinate, density, velocity, Where τ, , ρ, ,
, ε, ,
,
,
condensate
friction
out
x
u p
h G
F
Q
, respectively, time, coordinate, density, velocity,
pressure, specific internal energy, specific enthalpy, mass flow rate of the phase transition from the
condensation on the walls, or evaporation from the surface of the film of condensation on the walls of
the channel, the force of friction and heat flux. In this case
,
,
condensate
friction
out
G
F
Q
given on unit
volume. DOI: 10.1051/
C
⃝Owned by the authors, published by EDP Sciences, 201
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0 0
(201 )
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epjconf
EPJ Web of Conferences
,
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11 DOI: 10.1051/
C
⃝Owned by the authors, published by EDP Sciences, 201
/
0 0
(201 )
201
epjconf
EPJ Web of Conferences
,
0 01
1
61
6
6
10
10
1
1
1
11 DOI: 10.1051/
C
⃝Owned by the authors, published by EDP Sciences, 201
/
0 0
(201 )
201
epjconf
EPJ Web of Conferences
,
0 01
1
61
6
6
10
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1
1
1
11 NUMERICAL SIMULATION OF METHANE UNDER PHASE
TRANSFER CONDITIONS WITH ITS REAL PROPERTIES
Boris V. Borisov 1, a, Sergey S. Bondarchuk 2, Dimitrii O. Skryabinsky1, Victorya S. Striha1
1National Research Tomsk Polytechnic University, 634050 Tomsk, Russia
2 National Research Tomsk State University, 634050 Tomsk, Russia Abstract. The technique and results of calculation of parameters of the flow of methane in
single and two-phase state is discussed. The flow is described by the one-dimensional non-
stationary equations of a continuous medium. Is taken into account the processes of friction,
heat transfer and phase transitions. The results of the application of various schemes of
numerical integration of the equations are discussed. In this paper the parameters of the flow of metal having generally two phases investigates. For a
description of the thermodynamic properties of the working wide range of parameters an
approximation of the experimental data suggested in the article [1] in a fluid equation of state is used. The essence of this approach lies in the fact that the experimental data for methane were approximated
by the authors for the molar Helmholtz free energy, as well as approximating were chosen easily
integrable and is easily differentiable functions. a Corresponding author: bvborisov@tpu.ru
4
Article available at http://www.epj-conferences.org or http://dx.doi.org/10.1051/epjconf/201611001011 EPJ Web of Conferences EPJ Web of Conferences The initial conditions are given a uniform initial distribution of density, velocity and internal
energy as the rest gas, and for uniformly moving stream for whole canal. Border conditions:
0
0
0
0,
0 ; 0,
0 ;
,
. (
)
(
)
(
)
( )
G
G
h
h
p l
p
,
(4) (4) System (1) - (4) together with the state equation is solved numerically. Using of equation of state
is implemented as a procedure, an algorithm which allows for the input parameters of the density and
specific internal energy, found from the system of equations (1) - (3), determine the values of the
appropriate temperature and then all the other thermodynamic parameters [1], used for numerical
integration of (1) - (3). Simulation is performed with taking into account of the hydraulic and thermal interaction with the
walls of the tube. For this the numerical implementation of the control (finite) volume method has
been selected [2]. To determine the greatness of the original stream was used TVD scheme with
approximation of derivatives against the flow and using the principle of the minimum of the
derivative. The use of this approach was followed by the appearance of significant instability that is a
consequence of a very strong dependence of the pressure and specific internal energy according to the
equation of state. The introduction of artificial viscosity in the flow of the total momentum and the
total enthalpy in the form proposed in 1944 and von Neumann J. Richtmyer R.D. [2] only partly
solved the problem. Scheme proposed by Lax P.D. in 1954 [2] for numerical integration helped fully
repay nonphysical oscillation of solutions. The very limited number of Courant was used. This scheme
has a substantial viscosity, which allows it to avoid significant fluctuations nonphysical solutions. a Corresponding author: bvborisov@tpu.ru But
some embodiments, the calculation results included an unjustified increase in the pressure in the
steady stream through the channel of constant cross section with increasing density and fall of the
speed, in some cases, when a two-phase flow simulation included on hydraulic losses and heat
exchange with the wall of the channel for providing condensation conditions. g
p
g
Example of a detailed calculation for the steady flow is shown in Figure 1. Figure 1. Parameters in the two-phase flow field of methane produced by the scheme Lax PD. Figure 1. Parameters in the two-phase flow field of methane produced by the scheme Lax PD. This the pressure behavior is explained due to on the one hand the strong diffusion properties of
Lax's schemes, and on the other hand, the special features determining the pressure according to of the
obtained values of density and internal energy [1]. Authors have the experience the use for determine
of gas-dynamic parameters in difficult areas and in conditions of high gradients of pressure due to
monotone SK Godunov scheme of decay of a discontinuity [3, 4]. This method of determining the
values of flows calculated on the channel border and at the borders of cells is well physically justified. For an ideal gas, this method is deeply studied and widely used. As for implementation for a real gas of such an algorithm is difficult. Based on the proposal of SK
Godunov [3] trusts that the flow does not contain strong discontinuities parameters. In this case the
shock waves and the waves underpressure converted to sound waves. Dynamic relations on the 01011-p.2 Thermophysical Basis of Energy Technologies 2015 Thermophysical Basis of Energy Technologies 2015 discontinuities derived from the equations of continuity, momentum and energy (1-3) are determined
by [3]: [ρ]D [ρ ]
0
u
,
(5)
2
[ρ ]D [
ρ
]
0
u
p
u
,
(6)
2
2
[ρ(ε
0.5
)]
[ρ (ε
/ ρ
0.5
]
0
u
D
u
p
u
. (7) (5) (6) (7) It is believed that the mass flow rate of gas through the discontinuity
ia is determined by the
relation
ρ
i
i
i
a
c , and the speed of propagation of perturbations
i
D (for “right” and “left”
characteristics, respectively) is given by:
i
i
i
D
w
c . To determine the flow variables are considered four options presented schematically in Figure 2. In these schemes shows propagating discontinuities and contact discontinuity according change in the
x and in the time relative to the boundary (x = 0) between the cells named Latin numbers. A)
B) A)
B)
C)
D)
Figure 2. Schemes options decay of an arbitrary discontinuity. C) D) C) Figure 2. Schemes options decay of an arbitrary discontinuity. Figure 2. Schemes options decay of an arbitrary discontinuity. Figure 2. Schemes options decay of an arbitrary discontinuity. Density Ro , pressure P , velocity U and specific energy E at the interface between the cell is
determined according to the following analysis of the decay of an arbitrary discontinuity options:
Option (A):
1
1
(
)
0
u
c
. There is the supersonic flow “to right”. Parameters at the cell border
assumed equal the parameters of the left cell:
1
1
1
1
,
,
,
ρ
Ro
U
u P
p E
. Option (B):
2
2
(
)
0
u
c
. There is the supersonic flow “to left”. Parameters at the cell border assumed
equal the parameters of the cell right:
2
2
2
2
,
,
,
ρ
Ro
U
u P
p E
. Density Ro , pressure P , velocity U and specific energy E at the interface between the cell is
determined according to the following analysis of the decay of an arbitrary discontinuity options: Option (B):
2
2
(
)
0
u
c
. 01011-p.2 The solutions of elementary waves are used to implement the boundary conditions in accordance
with the sign of the velocity at the boundary. It is believed that the channel is finished at borders of the
large volumes, which are having a complete set of relevant thermodynamic parameters. For definiteness we consider the ratio of parameters on the left border. It is believed that the flow is
carried out in the subsonic mode, and in accordance with the pressure at the corresponding pressure in
the volume (
0
P
p ).Speed and density determined by the decision on the appropriate wave: with the sign of the velocity at the boundary. It is believed that the channel is finished at borders of the
large volumes, which are having a complete set of relevant thermodynamic parameters. For definiteness we consider the ratio of parameters on the left border. It is believed that the flow is
carried out in the subsonic mode, and in accordance with the pressure at the corresponding pressure in
the volume (
0
P
p ).Speed and density determined by the decision on the appropriate wave: 2
2
2ρ
P
p
U
u
and
2
2
2
2
1
a
Ro
a c
P
p
. In case inflow (
0
U
) considered that the specific enthalpy 2
2
2ρ
P
p
U
u
and
2
2
2
2
1
a
Ro
a c
P
p
. In case inflow (
0
U
) considered that the specific enthalpy corresponds to a value in the adjacent volume and in case outflow (
0
U
) the specific internal
energy's value is determined according to the following (5) - (7) the relation: corresponds to a value in the adjacent volume and in case outflow (
0
U
) the specific internal
energy's value is determined according to the following (5) - (7) the relation:
2
2
2
2
2
2
2
2
2
ε
0.5
2
a
u
PU
p u
U
E
Ro U
u
c
. Thanks to the SK Godunov circuit application and the appropriate implementation of the boundary
conditions is received significant impact to the calculation of the parameters (Fig. 01011-p.2 There is the supersonic flow “to left”. Parameters at the cell border assumed
equal the parameters of the cell right:
2
2
2
2
,
,
,
ρ
Ro
U
u P
p E
. To analyze of the two other options the speed and pressure of the contact discontinuity is
determined according to by SK Godunov proposal [3]: 1
1
2
2
1
2
1
2
CD
a u
a u
p
p
U
a
a
, 1
1
2
2
1
2
1
2
CD
a u
a u
p
p
U
a
a
, and 01011-p.3 EPJ Web of Conferences EPJ Web of Conferences 1
2
2
1
1
2
1
2
1
2
(
)
CD
p a
p a
u
u a a
P
a
a
. Option (C):
1
1
(
)
0
u
c
,
2
2
(
)
0
u
c
and
0
CD
U
. The density and internal energy is determined
from (5) - (7) according to the “left” elementary wave:
2
1
1
1
1
1
1
1
1
1
1
2
ε
0.5
,
2
CD
CD
CD
CD
CD
a
u
p u
P U
U
a
Ro
E
c
u
U
Ro
u
c
U
. Option (D):
1
1
(
)
0
u
c
,
2
2
(
)
0
u
c
and
0
CD
U
. The density and internal energy is determined
from (5) - (7) according to the “right” elementary wave:
2
2
2
2
2
2
2
2
2
2
2
2
ε
0.5
,
2
CD
CD
CD
CD
CD
a
u
p u
P U
U
a
Ro
E
c
u
U
Ro
u
c
U
. The solutions of elementary waves are used to implement the boundary conditions in accordance
with the sign of the velocity at the boundary. It is believed that the channel is finished at borders of the
large volumes, which are having a complete set of relevant thermodynamic parameters. Thermophysical Basis of Energy Technologies 2015 The presented method of calculation can be recommended for use in the process design of the
special devices. 01011-p.2 3) obtained in the
same option of that the respective calculation of Lax PD (Fig. 1). Figure 3. Parameters in the two-phase flow field of methane produced by the scheme Godunov SK. Figure 3. Parameters in the two-phase flow field of methane produced by the scheme Godunov SK 01011-p.4 01011-p.4 Thermophysical Basis of Energy Technologies 2015 References 1. Daniel G. Friend, James F. Ely, and Hepburn Ingharn, Thermophysical
properties of methane. – National Institute of standards and technology
(Boulder, Colorado 1988) 2. P. J. Roache. Computational fluid dynamics. Albuquerque (Hermosa Publs,
1976) 3. S.K. Godunov,
A.V. Zabrodin,
M.Ya. Ivanov,
A.N. Kraiko,
G.P. Prokopov. Chislennoe
reshenie
mnogomernykh
zadach
gazovoi dinamiki (Numerical Solution of Multidimensional Problems of Gas
Dynamics, Moscow: Nauka, 1976) 4. S.S. Bondarchuk,
A.B. Vorozhtsov,
A.S. Zhukov,
B.V. Borisov,
Russian
Physics Journal, 557 (12), 1796 (2015) 01011-p.5
|
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|
de
|
Erratum zu: Entwicklung des Lebensstilprogramms „Gemeinsam Gesund“
|
Prävention und Gesundheitsförderung
| 2,021
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cc-by
| 385
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Erratum
Präv Gesundheitsf 2021 · 16:269
https://doi.org/10.1007/s11553-021-00859-4
Online publiziert: 3. Mai 2021
© Der/die Autor(en) 2021
Ragna-Marie Kranz · Heike Englert
Fachbereich Oecotrophologie, Fachhochschule Münster, Münster, Deutschland
Erratum zu: Entwicklung des
Lebensstilprogramms
„Gemeinsam Gesund“
Erratum zu:
Präv Gesundheitsf 2020 15:256
https://doi.org/10.1007/s11553-020-007
59-z
Der Artikel „Entwicklung des Lebensstilprogramms ,Gemeinsam Gesund‘.
Planung und Strukturierung eines community-basierten Projekts unter Berücksichtigung des Intervention-MappingAnsatzes“ von Ragna-Marie Kranz und
Heike Englert wurde ursprünglich Online First ohne „Open Access“ auf der
Internetplattform des Verlags publiziert.
Nach der Veröffentlichung in Band 15
Heft 3 pp. 256–262 hatten sich die Autoren für eine „Open Access“-Veröffentlichung entschieden. Das Urheberrecht
des Artikels wurde deshalb in © Der/die
Autor(en) 2021 geändert. Dieser Artikel
ist jetzt unter der Creative Commons Namensnennung 4.0 International Lizenz
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Weitere Details zur Lizenz entnehmen
Sie bitte der Lizenzinformation auf http://
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Fachbereich Oecotrophologie, Fachhochschule
Münster
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ragna.kranz@fh-muenster.de
Open Access. Dieser Artikel wird unter der Creative
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Die in diesem Artikel enthaltenen Bilder und sonstiges
Drittmaterial unterliegen ebenfalls der genannten
Creative Commons Lizenz, sofern sich aus der Abbildungslegende nichts anderes ergibt. Sofern das betreffende Material nicht unter der genannten Creative
Commons Lizenz steht und die betreffende Handlung
nicht nach gesetzlichen Vorschriften erlaubt ist, ist für
die oben aufgeführten Weiterverwendungen des Materials die Einwilligung des jeweiligen Rechteinhabers
einzuholen.
Weitere Details zur Lizenz entnehmen Sie bitte der
Lizenzinformation auf http://creativecommons.org/
licenses/by/4.0/deed.de.
Die Online-Version des Originalartikels ist unter
https://doi.org/10.1007/s11553-020-00759-z
zu finden.
Prävention und Gesundheitsförderung 3 · 2021
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